Caspase-3 activation induced by glutamate in cultured rat hippocampal neurons

Abstract

Excitotoxic neuronal death induced by glutamate is generally divided into two types; one is acute and the other is delayed. The mechanisms underlying the delayed neuronal death involve apoptotic process. Since caspase-3 is a central downstream effector of caspase cascade executing the apoptosis program, glutamate may activate caspase-3. However, the time-course of activation remains unclear. In the present study, we examined the effects of brief (20 min) and continuous exposure to glutamate on the activation of caspase-3 in cultured rat hippocampal neurons. Neurons were prepared from 1-day-old rats. Necrotic and apoptotic cells were detected with Annexin-V-FITC and propidium iodide, respectively. Morphological changes in the neurons were observed with a video-enhanced contrast-differential interference contrast microscope. The activations of caspase-3 were analyzed by fluorescence imaging with a confocal laser microscope using a cell-permeable caspase-3 substrate FAM-DEVD-FMK. Our results showed that 20-min-exposure to glutamate induced delayed neuronal death and activated caspase-3 significantly in 6 h in a dose-dependent manner. Interestingly, the continuous exposure for 1 h, which induced necrosis, also significantly activated caspase-3. Our data indicated that caspase-3 pathway is activated in the process of acute as well as delayed neuronal death induced by glutamate. Even in the process of necrosis, apoptotic signal is already activated but covered with the rapid cell death. [Jpn J Physiol 54 Suppl:S249 (2004)]