Calcium dependency of n-methyl- d-aspartate toxicity in slices from the immature rat hippocampus

Abstract

The literature on ionic requirements for excitotoxity is largely contradictory. Depending on the experimental paradigms, it has been concluded that either Ca 2+ or Na + and Cl − mediate excitotoxicity. In the present study, the dependency on Ca 2+ of N-methyl- d-aspartate-induced damage to neurons in immature rat hippocampal slices was investigated with light microscopy. In addition N-methyl- d-aspartate-induced cell damage was followed by measurement of release of lactate dehydrogenase from slices. When incubated in N-methyl- d-aspartate-containing (100 μM) buffer for 30 min, hippocampal neurons displayed fine chromatin aggregation and swelling of neuronal nuclei and neuropil. Slices incubated in standard medium for 90 min after exposure to N-methyl- d-aspartate-contained a large number of neurons that failed to recover from the initial lesion. The acute edema was at least as severe in slices incubated in N-methyl- d-aspartate-containing, Ca 2+-free buffer. In contrast, clumping of the chromatin could not be observed. CA1 neurons recovered completely from the acute changes, and granule cells recovered to some extent. While omission of Ca 2+ had no obvious morphological effects on the tissue in its own right, the efflux of lactate dehydrogenase was significantly increased after incubation in Ca 2+-free medium. Slices exposed to N-methyl- d-aspartate released approximately twice as much lactate dehydrogenase as controls 1–5 h after the exposure, and the same rate of release was seen if Ca 2+ was absent during N-methyl- d-aspartate treatment. The morphological results suggest that N-methyl- d-aspartate toxicity is Ca 2+dependent in pyramidal cells whereas the toxicity in granule cells is partly Ca 2+ -independent. As the effects on lactate dehydrogenase release of Ca 2+-free medium and N-methyl- d-aspartate in Ca 2+-free medium were not additive, the biochemical data indicate the N-methyl- d-aspartate toxicity is partially Ca 2+-dependent. However, the relation between pathological changes and lactate dehydrogenase release is not entirely clear since slices exposed to N-methyl- d-aspartate in Ca 2+-free buffer release more lactate dehydrogenase than could be expected from the light microscopic observations. Therefore, there is a certain divergence between the morphological and biochemical data.