EFFECTS OF INJECTING CALCIUM-BUFFER SOLUTIONS ON [CA2-CLAMPED SNAIL NEURONS(](I) IN VOLTAGE)

We have investigated why fura-2 and Ca2+-sensitive microelectrodes rep ort different values for the intracellular free calcium ion concentrat ion ([Ca2+](i) or its negative log, pCa(i)) of snail neurons voltage-c lamped to -50 or -60 mV. Both techniques were initially calibrated in vitro, using calcium calibration solutions that had ionic concentratio ns similar to those of snail neuron cytoplasm. Pressure injections of the same solutions at resting and elevated [Ca2+](i) were used to cali brate both methods in vivo. In fura-2-loaded cells these pressure inje ctions generated changes in [Ca2+](i) that agreed well with those expe cted from the in vitro calibration. Thus, using fura-2 calibrated in v itro, the average resting [Ca2+](i) was found to be similar to 38 nM ( pCa(i) 7.42 +/- 0.05). With Ca2+-sensitive microelectrodes, the first injection of calibration solutions always caused a negative shift in t he recorded microelectrode potential, as if the injection lowered [Ca2 +](i). No such effects were seen on the fura-2 ratio. When calibrated in vivo the Ca2+-sensitive microelectrode gave an average resting [Ca2 +](i) of similar to 25 nM (pCa(i) 7.6 +/- 0.1), much lower than when c alibrated in vitro, We conclude that [Ca2+](i) in snail neurons is sim ilar to 40 nM and that Ca2+-sensitive microelectrodes usually cause a leak at the point of insertion. The effects of the leak were minimized by injection of a mobile calcium buffer.