Micromolar L-glutamate induces extensive apoptosis in an apoptotic-necrotic continuum of insult-dependent, excitotoxic injury in cultured cortical neurones

Excitotoxicity induced by L-glutamate (Glu), when examined in a pure neuronal cortical culture, involved widespread apoptosis at concentrations of 1-10 mu M as part of a continuum of injury, which at its most servere was purely necrotic. Cells, maintained in chemically defined neurobasal/B27 medium, were exposed at d7 for 2 h to Glu (1-500 mu M), and cellular injury was analysed 2 and 24 h after insult using morphology (phase-contrast microscopy), a 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) viability assay, nuclear staining with 4,6-diamidino-2-phenylindole (DAPI), terminal transferase-mediated dUTP nick end-labelling (TUNEL) and DNA fragmentation by gel electrophoresis. Glu-mediated neurotoxicity was prevented by MK-801 (5 mu M), whilst CNQX (20 mu M) attenuated injury by 20%. Exposure to intensive insults (100 and 500 mu M Glu) induced necrosis characterized by rapid cell swelling (< 2 h) and lack of chromatin condensation, confirmed by DAPI nuclear staining. In contrast, mild insults (< 20 mu M Glu) failed to produce acute neuronal swelling at < 2 h, but 24 h after injury resulted in a large number of apoptotic nuclei as confirmed by TUNEL and electrophoretic evidence of DNA fragmentation; which was attenuated by cycloheximide (0.1 mu g/ml). Our findings indicate for the first time that physiological concentrations of Glu produce neuronal injury across a continuum involving apoptosis ( < 20 mu M) and increasingly necrosis( > 20 mu M), dependent on the severity of the initial insult. (C) 1998 Elsevier Science Ltd. All rights reserved.