Free calcium ions in neurones of Helix aspersa measured with ion‐selective micro‐electrodes.

Abstract

Intracellular free Ca concentration, [Ca2+]i, was measured in giant neurons of the sub-esophageal ganglia of H. aspersa, using Ca-selective micro-electrodes containing a PVC[polyvinyl chloride]-gelled, neutral-ligand sensor. In calibration solutions the electrodes had a virtually ideal, Nernstian response down to 1 .mu.M-Ca2+ in the presence of 0.125 M-K+, 18-24 mV from 1 to 0.1 .mu.M-Ca2+ and 8-14 mV from 0.1 to 0.01 .mu.M-Ca2+. Interference from H+ and Mg2+ was negligible. The small response to Na+ at sub-micromolar Ca2+ was taken into account, when necessary, in measurement of [Ca2+]i. Measurements of basal [Ca2+]i were made in ganglia from animals kept only a few weeks in captivity, in a bathing solution equilibrated with air and containing 2 mM-Ca2+. In 13 measurements from impalements which met stringent criteria for electrode performance and cell viability, the mean basal pCa (-log10[Ca2+]) was 6.77 .+-. 0.07 (SE), corresponding to a mean free Ca2+ concentration of 0.17 .mu.M. The basal [Ca2+]i in neurons from snails kept hibernating for several months was higher, mean pCa 6.15, for ganglia handled in 2 mM-Ca2+ solution. Intracellular injections of Ca2+ or EGTA [ethylene glycol bis(.beta.-aminoethyl ether) tetraacetate] raised and lowered, respectively, the indicated basal [Ca2+]i, showing that the electrodes responded appropriately inside the cells and that unknown or untested components of cytoplasm were not significantly interfering with the Ca-sensor. Altering the external Ca2+ concentration between 0.1-10 mM usually produced only small, .+-. 0.1 pCa units, changes in basal [Ca2+]i of satisfactorily impaled, quiescent cells. In cell 1F, which has repetitive spikes with a substantial Ca current, changes in Ca gradient or blockade of voltage-dependent Ca channels sometimes markedly altered [Ca2+]i, showing that Ca entry with the spikes was elevating [Ca2+]i. Replacing external Na+ with Li+ or bis(2-hydroxyethyl)dimethylammonium had little effect on [Ca2+]i. Elevating CO2 to 5% or 79% lowered [Ca2+]i by an average of 0.16 and 0.26 pCa units, respectively.