Real‐time, two‐dimensional visualization of ischaemia‐induced glutamate release from hippocampal slices

Abstract

The involvement of excitatory amino acid (EAA) toxicity in ischaemia-induced neuronal cell death has long been suggested. However, in the hippocampus, the brain site most vulnerable to ischaemia, the detailed spatial and temporal patterns of EAA release are not yet known. To address this issue, we have developed a novel strategy for the continuous, real-time, two-dimensional monitoring of EAA release from brain slices. As EAA detector, we used a cell line transformed with the N-methyl-d-aspartate (NMDA) receptor, which is exclusively activated by EAAs, leading to an increase in the intracellular Ca²⁺ level. Combined with a calcium imaging technique, the use of this cell line allowed the temporal and regional analysis of EAA release from a brain slice placed directly on top of the clonal cells in a culture dish. Using this strategy, we demonstrated ischaemia-induced EAA release in rat hippocampal slices. Increased EAA release was seen initially in the CA1 region, about 3 min after the beginning of ischaemia, then in the CA3 region and dentate gyrus, and, finally, throughout the hippocampal slice. Regional differences in extracellular EAA levels were also seen, with more EAA being released from the CA1 region than from the middle dentate gyrus. The present results are especially interesting as neurons in the CA1 region are more vulnerable to ischaemia than those in the CA3 region and dentate gyrus.