Simultaneous measurement of cytosolic and mitochondrial Ca(2+) during ischemia in mice whole-brain slice preparation and its application to drug evaluation.

We developed a conventional imaging method to measure Ca(2+) concentration in cytosol (using FuraRed as an indicator) and mitochondria (using Rhod-2 as an indicator), simultaneously, by alternative excitation with specific wave length. After confirming the availability of the method in Hela cells, we applied it to mouse whole-brain slice -preparation, which was exposed to oxygen- and glucose-deprived artificial cerebrospinal fluid (ischemic ACSF) for 12 min. The fluorescence (>570 nm) at the cerebral cortex and hippocampus due to FuraRed (excited by 480 ± 10 nm) decreased (indicating the increase in cytosolic Ca(2+)-concentration), while the fluorescence due to Rhod-2 (excited by 560 ± 10 nm) increased (indicating the increase in mitochondrial Ca(2+) concentration) during exposure to ischemic conditions.