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

Abstract

We studied N-methyl- d-aspartate-induced cell death in organotypic hippocampal slices from seven-day-old Wistar rat pups cultured for 12–14 days 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 2 h to l-glutamate (1–500 μM) prior to the N-methyl- d-aspartate challenge significantly reduced N-methyl- d-aspartate-induced cell death. Glutamate at 10 and 500 μM concentrations was highly protective against N-methyl- d-aspartate-induced cell death, but was less protective at the 1 μM concentration. The protection was not blocked by the Na + channel blocker tetrodotoxin (1 μM), the N-methyl- d-aspartate receptor antagonist d-2-amino-5-phosphonopentanoic acid (20 μM) or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 μM). 1 S,3 R-1-Aminocyclopentane- trans-1,3-dicarboxylic acid, an agonist at metabotropic glutamate receptor types 1, 2/3 and 5, was protective at 100 μM but not at 50 μM. In contrast, the ionotropic glutamate receptor agonist aspartate (250 μM) facilitated N-methyl- d-aspartate toxicity. Treatment of slices with the protein kinase C inhibitor staurosporine (0.2 μM) or antisense oligonucleotide (10 nM, 72 h) that selectively inhibits metabotropic glutamate receptor type 5 synthesis significantly reduced glutamate protection. These results suggest that ambient glutamate may reduce nerve cell susceptibility to injury caused by excessive N-methyl- d-aspartate receptor activation by acting at metabotropic glutamate receptors linked to protein kinase C.