CHARACTERIZATION OF GLUTAMATE AND [H-3] D-ASPARTATE OUTFLOW FROM VARIOUS IN-VITRO PREPARATIONS OF THE RAT HIPPOCAMPUS

The characteristics of high-K+ and electrically evoked endogenous glut amate and [H-3]D-aspartate release have been studied in multiple in vi tro preparations of the rat hippocampus (transverse slices, granule ce lls cultures, synaptosomes and messy fibre synaptosomes) under similar experimental conditions. High external K+ concentrations evoked [H-3] D-aspartate and endogenous glutamate overflow in a concentration-depen dent manner in all preparations (except it was not possible to measure endogenous glutamate outflow from granule cells). This effect was tet rodotoxin-insensitive but partially calcium-dependent. In slices, fiel d electrical stimulation evoked an overflow of endogenous glutamate, b ut not of [H-3]D-aspartate, in a frequency-dependent manner. This effe ct was concentration-dependently amplified by the glutamate uptake inh ibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC). The electric ally evoked glutamate overflow in the presence of t-PDC was tetrodotox in-sensitive and calcium-dependent. In primary dentate gyrus cell cult ures, electrical stimulation evoked an overflow of [H-3]D-aspartate in a frequency-dependent manner, while endogenous glutamate outflow was not detectable. This effect could be inhibited by tetrodotoxin and by the N-type calcium channel blocker omega-conotoxin GVIA. Finally, the effect of adenosine has been studied in order to assess the pharmacolo gical modulability of [H-3]D-aspartate and endogenous glutamate stimul ation-induced overflow. Adenosine was found to inhibit 35 mM K+- and 2 0 Hz electrical stimulation-induced [H-3]D-aspartate and endogenous gl utamate overflow. These effects were all prevented by the A(1) recepto r antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT). These data are in line with the hypothesis that reuptake plays a role in regulating g lutamate release, and that [H-3]D-aspartate represents a valid marker of endogenous glutamate under most (but not all) experimental conditio ns. (C) 1997 Elsevier Science Ltd.