Detection of high molecular weight DNA fragments characteristic of early stage apoptosis in cerebellar granule cells exposed to glutamate

In cerebellar granule cells a rapid necrotic cell death has been observed d uring and immediately after glutamate exposure, followed by a delayed apopt otic type of neuronal death in a subpopulation of the surviving neurons. In some experimental models the DNA fragmentation characteristic of apoptosis is readily detected. In other systems apoptosis may occur only in a limite d number of cells, rendering DNA fragmentation undetectable using conventio nal; DNA-staining techniques (e.g., ethidium bromide). We have used a sensi tive and non-radioactive method for labeling, detection, and quantification of high molecular weight (HMW) DNA fragments. This method is based on the introduction of thymine dimers into DNA after separation by pulse field gel electrophoresis, followed by detection with thymine dimer specific antibod ies. Applying this method to cerebellar granule cells in culture, we detect ed an increase in the amount of HMW DNA fragments characteristic of apoptos is as early as 4 h after glutamate exposure. The N-methyl-D-aspartic acid ( NMDA)receptor antagonist MK801 protected against the fragmentation, whereas no protection was observed using the non-NMDA-receptor antagonist CNQX.