Glutamate-mediated neuroprotection against N-methyl-d-aspartate toxicity: a role for metabotropic glutamate receptors

We studied N-methyl-d-aspartate-induced cell death in organotypic hippocampal slices from seven-day-old Wistar rat pups cultured for 12–14days in a medium containing no added glutamate. Propidium iodide fluorescence intensity was used as an indicator of cell death measured with the help of confocal microscopy. Exposure of slices for 2h to l-glutamate (1–500μM) prior to the N-methyl-d-aspartate challenge significantly reduced N-methyl-d-aspartate-induced cell death. Glutamate at 10 and 500μM concentrations was highly protective against N-methyl-d-aspartate-induced cell death, but was less protective at the 1μM concentration. The protection was not blocked by the Na+ channel blocker tetrodotoxin (1μM), the N-methyl-d-aspartate receptor antagonist d-2-amino-5-phosphonopentanoic acid (20μM) or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20μM). 1S,3R-1-Aminocyclopentane-trans-1,3-dicarboxylic acid, an agonist at metabotropic glutamate receptor types 1, 2/3 and 5, was protective at 100μM but not at 50μM. In contrast, the ionotropic glutamate receptor agonist aspartate (250μM) facilitated N-methyl-d-aspartate toxicity. Treatment of slices with the protein kinase C inhibitor staurosporine (0.2μM) or antisense oligonucleotide (10nM, 72h) that selectively inhibits metabotropic glutamate receptor type 5 synthesis significantly reduced glutamate protection.