[Experimental techniques for developing new drugs acting on dementia (11)--Experimental methods on glutamate...

Glutamate-induced neurotoxicity was examined in cultured rat cortical cells. Primary cultures were obtained from the cerebral cortex of fetal rats (17-19 days of gestation). Single cells dissociated from the cerebral cortex were plated on plastic coverslips placed in 35- or 60-mm culture dishes. Cultures were incubated in Eagle's minimal essential medium supplemented with 10% fetal calf serum or 10% horse serum at 37 degrees C in a humidified 5% CO2 atmosphere for 10-14 days. The neurotoxicity induced by glutamate was quantified by trypan blue exclusion. The viability of cultures was markedly reduced by a 10-min exposure to glutamate followed by incubation with glutamate-free medium for 1-24 hr. Glutamate neurotoxicity was prevented by the N-methyl-D-aspartate (NMDA) receptor antagonists, MK-801, 3-[(+/-)-2-carboxypiperazin-4-yl] propyl-1-phosphoric acid (CPP), ifenprodil and 7-Cl-kinurenate. Glutamate neurotoxicity was augmented by phorbol dibutyrate, that activates protein kinase C (PKC), but reduced by H-7, that inhibits PKC. These results suggest that PKC plays an important role in NMDA receptor-mediated glutamate neurotoxicity in the cerebral cortex.