Intracellular calcium recordings from isolated cells of the mammalian central nervous system

Abstract

Measurements made with two different techniques of intracellular calcium levels from small isolated cells of the mammalian central nervous system are described and compared. Recordings in cultured mouse embryo spinal cord and dorsal root ganglion neurons, made with double-barrelled borosilicate Ca2+-selective microelectrodes yielded a mean Ca2+ level of 2.3 (SE ± 0.54) μM for the lowest values recorded in 24 out of 46 cells. Intracellular Ca2+ dependence on membrane potential was apparent with levels of calcium ≥4 μM (r = 0.371, n = 29). Both cyclic fluctuations induced by tetraethylammonium and an apparent increase in Ca2+ evoked by the depolarizing excitatory amino acid, L-aspartate, were observed. In contrast, estimates of intracellular Ca2+ obtained by spectrofluorimetry of suspensions of mouse embryo brain cells, loaded with the intracellular Ca-binding fluorescent probe, quin2 provided a [Formula: see text]-fold lower value, 152 (SE ± 7) nM. This more closely resembles levels reported for large neurons where large-tip microelectrodes with greater sensitivity were used, and in spite of the heterogeneity of the cells this value is presumed to be a more accurate estimate of intraneuronal Ca2+ concentration. In these fluorescence studies KCl readily evoked increases in intracellular Ca2+ which could be blocked by verapamil and Cd2+ and were not induced in the absence of Ca2+. Increases were also produced by N-methyl-D-aspartate, but not by the kainate-like Lathyrus neurotoxin, L-3-oxalylamino-2-aminopropionic acid. These results provide preliminary evidence for both voltage-sensitive and receptor-activated Ca channels in embryonic brain cells. Although the recording of intraneuronal Ca2+ with conventional ion-selective microelectrodes in small cells has problems with respect to accuracy, stability, and time constant, recent advances in the design of Ca2+ sensors and electrodes are promising. These, as well as developments in techniques of single cell fluorescence analysis, now offer methods with improved and powerful capacity for accurate and simultaneous measurements of intracellular Ca2+ and membrane electrophysiological parameters.