Intracellular calcium measured with calcium-sensitive micro-electrodes and Arsenazo III in voltage-clamped Aplysia neurones.

Selected neurones of the abdominal ganglion of Aplysia californica were voltage clamped, injected with the Ca2+-indicator dye Arsenazo III, and impaled with Ca2+-selective micro-electrodes. Measurements of the absorbance signal (Arsenazo III) and Ca2+ micro-electrode potential during and following voltage-dependent Ca2+ influx (induced by voltage-clamp pulses) were simultaneously recorded. In neurones held at -50 mV, the mean intracellular free Ca2+ concentration [( Ca]i) measured by the Ca2+ micro-electrode was 0.18 microM, S.D. = 0.22 microM, n = 13. Bathing the cell in 0 Ca2+ artificial sea water (ASW) or intracellularly injecting EGTA decreased the resting [Ca]i. Voltage-clamp pulses, which maximally activated Ca2+ channels (from -50 to +30 mV), transiently increased both the Arsenazo III absorbance and the Ca2+ micro-electrode signals, indicating a rise in [Ca]i. Given the Ca2+ micro-electrode's limited band width, the peak of the Ca2+ signal during the pulse train could not be resolved; however, there was a net deflexion of this signal following the last pulse which slowly decayed to base line. Bathing the cells in 0 Ca2+ ASW, or reducing the driving force for Ca2+ entry (by stepping the voltage-clamp pulses to much higher membrane potentials) dramatically reduced both the absorbance and the Ca2+ micro-electrode signal increases. On the other hand, bathing the cells in 100 mM-Ca2+ ASW increased both signals. The intracellular Ca2+ gradient within the cytoplasm following voltage-clamp pulses was investigated by moving the Ca2+-selective micro-electrode tip in a step-wise manner relative to the membrane surface. The measured rise in [Ca]i was greatest near the membrane and not measurable within 40-50 microns of the membrane surface. The amplitude of the [Ca]i rise at different distances from the membrane could be fitted by a model based on a simple diffusion of Ca2+ from a plane source.