Maintenance of Fura-2 fluorescence in glial cells and neurons of the leech central nervous system.

Identified glial cells and neurones of the leech central nervous system (CNS) were injected iontophoretically with the calcium indicator dye Fura-2 to measure intracellular Ca2+, while simultaneously recording the membrane potential using a double-barrelled theta-type microelectrode. Both glial cells and neurones responded with Ni(2+)-sensitive Ca2+ transients upon membrane depolarization, indicating Ca2+ influx through voltage-gated Ca2+ channels. In contrast to neurones, the glial cells showed a rapid loss of fluorescence with a half-time of 6.3 +/- 1.8 min (n = 6) after dye injection. Both kinetics and amplitudes of the stimulus-induced Ca2+ transients were affected by this rapid dye loss. The anion exchange inhibitor probenicid (2 mM) significantly reduced, but did not prevent, the loss of Fura-2 fluorescence, suggesting that some dye left the glial cell via an anion exchanger. In order to compensate this fluorescence loss, we injected Fura-2 throughout the experiment. Under this condition, similar Ca2+ transients could be elicited repeatedly for more than 1 h. In Retzius neurones single injections of Fura-2 yielded enough intracellularly trapped dye to allow measurement of intracellular Ca2+ for up to 30 min after the end of injection without large decrease in absolute fluorescence.