Separation of carrier mediated and vesicular release of GABA from rat brain slices

In this study the temperature dependence of [H-3]GABA release from brain slices evoked by electrical field stimulation and the Na+/K(+)ATPase inhibitor ouabain was investigated. [3H]GABA has been taken up and released from hippocampal slices at rest and in response to electrical field stimulation (20 V, 10 Hz, 3 msec, 180 pulses) at 37 degrees C. When the bath temperature was cooled to 7 degrees C, during the sample collection period, the tissue uptake and the resting outflow of [H-3]GABA were not significantly changed. In contrast, the stimulation-induced tritium outflow increased both in absolute amount (Bq/g) and in fractional release and the S-2/S-1 ratio was also higher at 7 degrees C. Perfusion of the slices with tetrodotoxin (TTX, 1 mu M) inhibited stimulation-induced [H-3]GABA efflux indicating that exocytotic release of vesicular origin is maintained under these conditions. 15 min perfusion with ouabain (10-20 mu M) induced massive tritium release both in hippocampal and in striatal slices. However, the fraction of [H-3]GABA outflow evoked by ouabain was much higher in the hippocampus than in the striatum. Sequential lowering the bath temperature from 37 degrees C to 17 degrees C completely abolished ouabain-induced [H-3]GABA release in both brain regions, indicating that it is a temperature-dependent, carrier-mediated process. When the same experiments were repeated under Ca2+ free conditions, cooling the bath temperature to 17 degrees C, although substantially decreased the release but failed to completely abolish the tritium outflow evoked by ouabain, a significant part was maintained. Our results show that vesicular (field stimulation-evoked) and carrier-mediated (ouabain-induced) release of GABA is differentially affected by low temperature: while vesicular release is unaffected, carrier-mediated release is abolished at low bath temperature. Therefore, lowering the temperature offers a reliable tool to separate these two kinds of release and makes possible to study exclusively the pure neuronal release of GABA of vesicular origin. (C) 1999 Elsevier Science Ltd. All rights reserved.