Pulsed laser imaging of Ca2+ signals in adrenal chromaffin cells

FERNANDO D. MARENGO

Pucón, Chile.

Simposio; 13th Internacional Symposium of Chromaffin Cell Biology. Workshop of Microscopic and Spectroscopic Techniques; 2006

Internacional Symposium of Chromaffin Cell Biology

The trigger of exocytosis in adrenal chromaffin cells is a localized Ca2+ increase near the plasma membrane that develops after activation of voltage dependent Ca2+ channels. Characterization of the temporal and spatial properties of Ca2+ gradients generated as consequence of membrane depolarization is, therefore, necessary to understand the regulation of stimulus induced exocytosis. We used pulsed laser Ca2+ imaging to follow the development and dissipation of Ca2+ gradients. The imaging system consist of an inverted epifluorescence microscope, a Peltier-cooled CCD camera, a microcomputer and a high-intensity pulsed coaxial flash lamp dye laser which provides 350 ns illumination pulses. A patch-clamp setup, comprising amplifier and data adquisition interface, was used to apply membrane depolarization pulses and recording the associated Ca2+ currents. We used this technique to characterize various aspects of Ca2+ signaling, for example: (a) the development of Ca2+ gradients from very early times (<5 ms) during short depolarizing stimulus, and the dissipation of these gradients occurring during several hundred of milliseconds (1); (b) the pattern of Ca2+ gradient development, dissipation and clearance in response to repetitive stimuli (2); (c) the correlation of these spatially localize Ca2+ signals with exocytosis (3). This imaging technique provide high resolution spatial information of whole cell Ca2+ distribution at a single time, so it is possible to have an instantaneous mapping of Ca2+ signal during a controlled triggered process. The time evolution of the signal can be obtained by successive, independent, measurements at different times. 1.        Marengo and Monck (2000). Biophys J 79: 1800-1820. 2.        Marengo and Monck (2003). Biophys J 85: 3397-3417. 3.        Marengo (2005). Cell Calcium 38: 87-99.