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 Ni2+-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.
