Electrically evoked [3H]GABA release from cerebral cortical cultures: An in vitro approach for studying glutamate-induced neurotoxicity

In the present study the [3H]GABA release in the rat cerebral cortex primary cultures, kept at rest or electrically stimulated, was measured. In addition, the development of excitotoxic cell damage caused by pretreating the cells for 10 min with increasing glutamate concentrations (10-300 microM) was examined 2 and 24 h after the insult. Cellular injury was quantitatively assessed by measuring the electrically-evoked [3H] GABA release, the [3H] GABA uptake, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide staining. Trains of electrical pulses at different frequencies (2, 5, 10, and 20 Hz) applied to the cultures elicited a [3H]GABA release which was frequency related, Ca++-dependent, and tetrodotoxin sensitive. Either 2 or 24 h after glutamate exposure, the electrically evoked [3H]GABA release was reduced by glutamate in a concentration dependent manner, while [3H]GABA uptake and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining appeared less sensitive. The N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and metabotropic receptor antagonists were tested on 100 microM glutamate-exposed cells and a prominent N-methyl-D-aspartate receptor-mediated component was observed. The present findings indicate that the electrically-evoked [3H]GABA release from cerebral cortical cells could represent a useful approach not only to study the spike-triggered neurosecretion but also to the neuronal damage caused by glutamate, as well as to test potential neuroprotective compounds.