Characterization of glutamate and [3H]D-aspartate outflow from various in vitro preparations of the rat hippocampus

The characteristics of high-K+ and electrically evoked endogenous glutamate and [3H]D-aspartate release have been studied in multiple in vitro preparations of the rat hippocampus (transverse slices, granule cells cultures, synaptosomes and mossy fibre synaptosomes) under similar experimental conditions. High external K+ concentrations evoked [3H]D-aspartate and endogenous glutamate overflow in a concentration-dependent manner in all preparations (except it was not possible to measure endogenous glutamate outflow from granule cells). This effect was tetrodotoxin-insensitive but partially calcium-dependent. In slices, field electrical stimulation evoked an overflow of endogenous glutamate, but not of [3H]D-aspartate, in a frequency-dependent manner. This effect was concentration-dependently amplified by the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC). The electrically evoked glutamate overflow in the presence of t-PDC was tetrodotoxin-sensitive and calcium-dependent. In primary dentate gyrus cell cultures, electrical stimulation evoked an overflow of [3H]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 pharmacological modulability of [3H]D-aspartate and endogenous glutamate stimulation-induced overflow. Adenosine was found to inhibit 35 mM K(+)- and 20 Hz electrical stimulation-induced [3H]D-aspartate and endogenous glutamate overflow. These effects were all prevented by the A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT). These data are in line with the hypothesis that reuptake plays a role in regulating glutamate release, and that [3H]D-aspartate represents a valid marker of endogenous glutamate under most (but not all) experimental conditions.