Gorman A L, Levy S, Nasi E, Tillotson D
J Physiol. 1984 Aug;353:127-42. doi: 10.1113/jphysiol.1984.sp015327.
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.
选取加利福尼亚海兔腹神经节的特定神经元，对其进行电压钳制，注入Ca2+指示剂染料偶氮胂III，并插入Ca2+选择性微电极。在电压依赖性Ca2+内流期间及之后（由电压钳脉冲诱导），同时记录偶氮胂III吸光度信号和Ca2+微电极电位。在保持于-50 mV的神经元中，通过Ca2+微电极测得的平均细胞内游离Ca2+浓度[(Ca)i]为0.18微摩尔，标准差=0.22微摩尔，n=13。将细胞置于无Ca2+的人工海水（ASW）中或细胞内注射乙二醇双四乙酸（EGTA）可降低静息[(Ca)i]。使Ca2+通道最大程度激活的电压钳脉冲（从-50 mV到+30 mV）会使偶氮胂III吸光度和Ca2+微电极信号瞬时增加，表明[(Ca)i]升高。鉴于Ca2+微电极的带宽有限，脉冲序列期间Ca2+信号的峰值无法分辨；然而，最后一个脉冲后该信号有净偏转，并缓慢衰减至基线。将细胞置于无Ca2+的ASW中，或降低Ca2+内流的驱动力（通过将电压钳脉冲调节到更高的膜电位），可显著降低吸光度和Ca2+微电极信号的增加。另一方面，将细胞置于100 mM - Ca2+的ASW中会使两个信号均增加。通过相对于膜表面逐步移动Ca2+选择性微电极尖端，研究了电压钳脉冲后细胞质内的细胞内Ca2+梯度。测得的[(Ca)i]升高在膜附近最大，在距膜表面40 - 50微米范围内无法测量。距膜不同距离处[(Ca)i]升高的幅度可用基于Ca2+从平面源简单扩散的模型拟合。
