Recent studies on ischemic
brain disease in vivo and
glutamate excitotoxicity in vitro suggest that apoptosis may play a role in excitotoxic neuronal death. To examine the possible involvement of apoptosis in
glutamate excitotoxicity, we studied an early process of morphological changes in rat cortical neurons exposed to 1 mM
glutamate. Observations under Nomarski optics combined with a digital image processor revealed a rapid change in the nucleus followed by a cellular swelling. The nucleus increased in granularity and swelled in 5 min, then became liquefied in 30 min. The cell body swelled slowly in 15-45 min. These changes could be prevented by treatment of the neuron with
MK-801 (
dizocilpine maleate), a blocker of
N-methyl-D-aspartate (
NMDA) receptor-coupled
ion channel. However, treatment of the neurons with N(G)-nitro-
L-arginine (N-NORG), a
nitric oxide synthase inhibitor, had no significant effect. Use of the in situ end-labeling technique for the demonstration of free 3'-hydroxyl ends revealed that DNA fragmentation took place within 1 hr after
glutamate exposure. A change in intracellular Ca(2+) concentration was examined with
fluo-3 under a confocal
laser microscope. Application of 1 mM
glutamate induced rapid Ca transients in the nucleus as well as in the cytoplasm. Both of these Ca responses were blocked by
MK-801. These results indicate that
glutamate excitotoxicity in the brain neuron does not fulfill morphological criteria of apoptosis, but suggest that the nuclear disintegration associated with DNA fragmentation is involved as a leading step in
glutamate excitotoxicity.