Release of endogenous glutamate from rat cortical slices in presence of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid

Summary

The effect of the new glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), on the electrically evoked release or, rather, overflow of endogenous glutamate in superfusates from rat cortical slices was compared with that of dihydrokainate. In the absence of these presumed uptake inhibitors, electrical stimulation for 4 min at 1 Hz did not elicit a measurable glutamate overflow over baseline at all. Basal overflow increased concentration-dependently in the presence of 10–100 μM L-trans-PDC, about 5-fold at 100 μM. Also, electrical stimulation caused increases of glutamate overflow over basal levels progressive with increasing concentrations of trans-PDC; a stimulated overflow corresponding to about 50% of basal overflow was obtained at 100 μM. Basal as well as evoked release in the presence of dihydrokainate did not exceed ca. 60% of that obtained with 100 μM L-trans-PDC. In synaptosomes, L-trans-PDC much more than dihydrokainate caused a transient increase of spontaneous glutamate release which was diminished in the absence of Na⁺, indicating that it is transported into the cytoplasm by the glutamate carrier and induces some efflux of the amino acid from this compartment. Moreover, trans-PDC caused a weak to moderate inhibition of K⁺-evoked glutamate release from synaptosomes at 10–300 μM, without obvious concentration-dependence.

Glutamate overflow elicited from rat cortical slices by electrical field stimulation at 1 Hz was Ca²⁺-dependent to about 80%. Tetrodotoxin (0.3 μM) reduced it by about 90%. Lowering the temperature from 37°C to 22°C increased the ratio between evoked and basal overflow.

As an application for L-trans-PDC as a glutamate uptake inhibitor in release studies, the regulation of glutamate release by GABA_B receptors was investigated. At 1 Hz, (−)-baclofen reduced evoked glutamate overflow at and above 3 μM by maximally 40% at 30 μM. This maximal effect was not increased when higher or lower stimulation frequencies were used nor when the Ca²⁺ concentration in the medium was increased or lowered, nor when the slices were prepared from other brain areas (hippocampus or striatum). The GABA uptake inhibitor, SK&F 89976, had no significant effect on evoked glutamate overflow, and the potent GABA_B antagonist, CGP 55845, induced only a small increase, indicating that tonic inhibition of glutamate by GABA via GABA_B receptors was not marked. On the other hand, the GABA_B antagonist was able to prevent the inhibitory effect of (−)-baclofen when applied before it and to abolish it when applied afterwards. The conclusion is that L-trans-PDC is a useful tool in glutamate release studies in brain slices for many purposes, with the reservation that its inhibitory effect on evoked glutamate release in synaptosomes is not yet understood.