Involvement of three glutamate receptor epsilon subunits in the formation of N-methyl-D-aspartate receptors mediating excitotoxicity in primary cultures of mouse cerebellar granule cells.

The N-methyl-D-aspartate receptors have been implicated in neuronal plasticity and their overactivation leads to neurotoxicity. Molecular cloning and co-expression of various glutamate receptor zeta and epsilon complementary DNAs support a heteromeric structural organization for N-methyl-D-aspartate receptors. In this study, we show that cerebellar granular neurons in primary culture of mouse express glutamate receptor zeta1 and at least three glutamate receptor epsilon (epsilon1, epsilon2, and epsilon3) protein subunits. In vitro, the temporal patterns of glutamate receptor epsilon1, epsilon2, and epsilon3 subunit expression depend on culture stages. By day 9, a somatic and neuritic immunolocalization for all N-methyl-D-aspartate subunits was clearly identified in most neuronal, but not glial cells. The role of particular subunits in N-methyl-D-aspartate-mediated excitotoxicity was probed by exposing the cerebellar granule cells to antisense oligodeoxynucleotides generated against specific N-methyl-D-aspartate receptor subunits. Antisense oligodeoxynucleotide treatments significantly down-regulated the amounts of the corresponding N-methyl-D-aspartate subunits. The decrease in N-methyl-D-aspartate subunit protein correlated with a reduction in N-methyl-D-aspartate-induced calcium influx and N-methyl-D-aspartate-mediated excitotoxicity in cerebellar cultures. In contrast, antisense oligodeoxynucleotide treatment failed to protect neurons from 1-methyl-4-phenylpyridinium-induced metabolic cell toxicity. Antisense oligodeoxynucleotide treatment targeted at N-methyl-D-aspartate glutamate receptor epsilon subunits demonstrate that glutamate receptor epsilon1, epsilon2, and epsilon3 proteins form N-methyl-D-aspartate receptors responsible for neurotoxic effects on cerebellar neurons. This study provides direct evidence for the existence of distinct N-methyl-D-aspartate receptor subunit proteins in cerebellar granule cells developing in vitro that may trigger N-methyl-D-aspartate-dependent excitotoxicity.