CHARACTERIZATION OF K+-EVOKED [H-3] D-ASPARTATE OUTFLOW IN THE RAT HIPPOCAMPUS IN-VITRO

The characteristics of K+-evoked outflow of [H-3]D-aspartate, a glutamate release marker, were systematically investigated in the rat hippocampus, using 35 mM K+-evoked [H-3]noradrenaline outflow as a reference. Elevation of external K+ concentrations increased [H-3]D-aspartate outflow in a concentration-dependent manner both in slices and synaptosomes. In the absence of external Ca2+, K+-evoked [H-3]D-aspartate outflow was decreased by approx 60% in synaptosomes and 80% in slices. However, elimination of external Ca2+ in the presence of 2 mM EGTA significantly reduced only 100 mM K+-evoked outflow, both in slices and synaptosomes. In the absence of external Ca2+, 35 mM K+-evoked [H-3]noradrenaline outflow was abolished even when EGTA was present in the solution. Furthermore, the Ca2+-channel blockers omega-conotoxin (10 nM) and nifedipine (0.5 muM) did not significantly reduce K+-evoked [H-3]D-aspartate outflow; [H-3]noradrenaline outflow, however, was reduced by more than one third by omega-conotoxin. Finally [H-3]D-aspartate overflow was insensitive to tetrodotoxin (0.5 muM) both in synaptosomes and in slices; while that of [H-3]noradrenaline was significantly reduced in slices. It is concluded that (1) [H-3]D-aspartate outflow is partly Ca2+-dependent; (2) differences between K+-evoked [H-3]D-aspartate and [H-3]noradrenaline outflow include sensitivity to stimulation by EGTA, to Ca2+-channel blockers and to tetrodotoxin. Some of these discrepancies may be ascribed to the existence of a cytosolic, Ca2+-independent pool of releasable glutamate and [H-3]D-aspartate. These observations pose some problems as to the experimental approach for the study of Ca2+-dependent [H-3]D-aspartate release.