Abstract
A system for real-time quantitative monitoring of intracellular free calcium ion concentration ([Ca2+]i) on a single cell basis was developed by the combination of a fluorescent Ca2+ indicator fura-2, a fluorescence microscope, a video-camera and photometrical devices. It was applied to rat individual hippocampal neurones under culture for detection of L-glutamate-induced alterations in the [Ca2+]i level. L-Glutamate (0.01-100 microM) induced a dose-dependent elevation of the [Ca2+]i. The [Ca2+]i in the rat hippocampal neurone was found to be around 30 nM in the resting state, and was increased up to 500 nM by the application of 100 microM L-glutamate. N-methyl-D-aspartate, kainate and quisqualate in a concentration of 10 microM also increased the [Ca2+]i level in the same single neurone, but their efficacy varied between individual cells. The L-glutamate-induced [Ca2+]i elevation was abolished after removal of extracellular Ca2+ and was much reduced by Mg2+ (3 mM). The increase was, however, still observed in a Na+-free medium. The L-glutamate-induced [Ca2+]i elevation was not affected substantially after treatment with nitrendipine (10 microM) which blocked the increase in [Ca2+]i induced by an isotonic high KCl-medium (50 mM). The present results suggest that the L-glutamate-induced [Ca2+]i elevation in the hippocampal neurone is due to an influx of Ca2+ through both L-glutamate receptor-coupled and voltage-sensitive ionic channels.