Factors influencing the release of labelled γ-aminobutyric acid and acetylcholine evoked by electrical stimulation with alternating polarity from rat cortical slices

Abstract

To establish conditions under which electrical stimulation causes Ca2+-dependent and Ca2+-independent release of 3H-labelled γ-aminobutyric acid ([3H]GABA), rat cortical slices were incubated with [3H]GABA and were superfused at either 32 or 37 °C. To release [3H]GABA the slices were stimulated with 2-ms pulses of alternating polarity at either 16 or 64 Hz. For comparison, the release of [3H] acetylcholine ([3H]ACh), after incubation with [3H]choline, was followed under identical conditions. In the presence of Ca2+ maximal release of [3H]GABA was obtained with 64-Hz stimulation at 32 °C whereas maximal release of [3H]ACh was obtained with 16 Hz at 37 °C. In the presence of Ca2+ the release of both [3H]GABA and of [3H]ACh evoked by 64-Hz stimulation was less at 37 than at 32 °C. A second period of 64-Hz stimulation at 32 °C released less [3H]ACh after stimulation at 37 than after stimulation at 32 °C, indicating that 64-Hz stimulation at 37 °C causes an irreversible heat inactivation of release. In the absence of Ca2+ the release of [3H]ACh evoked by 2-ms pulses was much reduced under all conditions but that of [3H]GABA was eliminated only at 32 °C. Tetrodotoxin suppressed the evoked release of both transmitters. Intermittent stimulation with bursts of 128 Hz (average frequency 16 Hz) at 32 °C was as effective in releasing [3H]GABA as 64-Hz continuous stimulation. Stimulation with long (5 ms) pulses at 64 Hz and 32 °C evoked a large release of [3H]GABA both in the presence and absence of Ca2+. However, in the presence of 0.5 mM Ca2+ this release was reduced. Results can be explained by the following. Major differences in the release of [3H]GABA and [3H]ACh induced by low-frequency stimulation, in the presence of Ca2+, may be due to differences in the properties of voltage-dependent Ca2+ channels in the two types of terminals. The Na+-dependent release of [3H]GABA in the absence of Ca2+ is inhibited by small concentrations of Ca2+ by reducing Na+ influx. The effect of temperature on [3H]GABA release can be explained by the temperature dependence of the voltage-dependent Ca2+ channels and of the sodium pump, both of which determine [Na+]i which in turn governs the Na+-dependent efflux of [3H]GABA.