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 microM 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 microM), and cellular injury was analysed 2 and 24 h after insult using morphology (phase-contrast microscopy), a 3-(4,5-dimethylthiazol-2-yl)-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 microM), whilst CNQX (20 microM) attenuated injury by 20%. Exposure to intensive insults (100 and 500 microM 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 microM 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 microg/ml). Our findings indicate for the first time that physiological concentrations of Glu produce neuronal injury across a continuum involving apoptosis (< 20 microM) and increasingly necrosis(> 20 microM), dependent on the severity of the initial insult.
Cheung
N S
NS
Department of Pharmacology, Monash University, Clayton, Victoria, Australia.
Pascoe
C J
CJ
Giardina
S F
SF
John
C A
CA
Beart
P M
PM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects* / Q000473:pathology / Q000502:physiology; D053938:DNA Fragmentation / Q000187:drug effects; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity*; D018698:Glutamic Acid / Q000633:toxicity*; D051379:Mice; D009336:Necrosis
D018690:Excitatory Amino Acid Agonists; D018698:Glutamic Acid