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.