Glutamate has been implicated as mediator of injury in the developing brain. It was previously demonstrated that, after 6 days in vitro(DIV), cultured neurons from the forebrain of 14-day-old rat embryo possess various functional receptor subtypes for glutamate. In other respects, exposure of these neurons to hypoxia for 6h has been shown to induce programmed apoptotic cell death. In order to correlate these findings with excitotoxicity, effects of a 6-h exposure to 100 μM glutamate or its analogues (NMDA, AMPA, kainate, trans-ACPD) were studied over a period of 72h in cultured neurons at 6 DIV and at a more mature stage, i.e. 13 DIV. Neuronal viability was measured by the MTT method, functional activity of living cells was assessed by 2-[3H]deoxyglucose uptake and synthesis of total proteins was monitored by [3H]leucine incorporation. In parallel, necrosis and apoptosis, two distinct modes of cell death, were scored under fluorescence microscopy following nuclear staining with DAPI. Using its specific inhibitor, staurosporine (30 nM), the role of protein kinase C in neuronal response to glutamate was also investigated. At 6 DIV, glutamate and derivatives were without toxic effects, but neurons became vulnerable when coexposed to staurosporine. In these conditions, cell viability decreased by 33% (n=15, p<0.01), and 17% of the neurons became necrotic (n=9, p<0.01). Exposure of 13 DIV neurons, especially to glutamate or NMDA, induced cell death; glutamate reduced viability by 29% and deoxyglucose uptake by 26% (n=15, p<0.01). The final rate of necrotic neurons reached 41%, while the percentage of apoptotic neurons remained similar to controls (3%). At this developmental stage, staurosporine was without significant effect on subsequent cell outcome. Glutamate-induced persistent inhibition of protein synthesis (12 to 26%, p<0.01) as well as lack of beneficial effect from a protein synthesis inhibitor argue against a programmed phenomenon. Additional studies performed with antagonists of NMDA or non-NMDA receptors (5-10 μM MK-801 or NBQX, respectively) suggest the participation of both categories of receptors, since each compound was able to protect neurons. In conclusion, glutamate, by contrast to hypoxia, may induce neuronal injury in the developing but not very immature brain and its action would reflect necrosis but not apoptosis. The inability of immature neurons to trigger proteine kinase C inhibition may account for their higher resistance to excitotoxicity.
