Spatial and dye correlation analysis of intracellular Ca2+ distribution

Intracellular Ca2+ is an important regulator of many cellular processes. Besides ion channels and transporters in the plasmalemma, changes in [Ca]i can be mediated by uptake and release mechanisms of internal organelles. Theoretical and experimental procedures are developed aiming to reveal the distribution of internal Ca2+ pools and their role in generating complicated spatial patterns of [Ca]i gradients. Cultured pyramidal neurons from rat hippocampus were loaded with Ca(2+)-sensitive fluorescent dyes, fura-2 and fluo-3. Cell images were partitioned according to pixel amplitude and highlighted pictures were characterized by their intensity, relative area and connectivity. This approach facilitates the localization of the sites of Ca2+ release from internal stores induced by application of different agents. After each trial, neurons were stained with dyes, acridine orange or DiOC6, which bind preferentially to nucleus and endoplasmic reticulum. A correlation between images confirmed the spatial localization of Ca2+ release sites. Application of the partition procedure also gave a clear evidence for the importance of Ca2+ influx in the mechanism of [Ca]i oscillations.