Apoptosis Induced via AMPA‐Selective Glutamate Receptors in Cultured Murine Cortical Neurons

• Published in: Journal of neurochemistry Vol. 69; no. 2; pp. 617 - 622
• Main Authors: Larm, Jari A., Cheung, Nam S., Beart, Philip M.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.08.1997; Blackwell
• Subjects: 6-Cyano-7-nitroquinoxaline-2,3-dione - pharmacology; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid - pharmacology; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid - toxicity; AMPA; Apoptosis; Biochemistry and metabolism; Biological and medical sciences; Cells, Cultured; Central nervous system; Cerebral Cortex - cytology; Chromatin - ultrastructure; Cortical neurons; DNA Fragmentation; Electrophoresis, Agar Gel; Embryo, Mammalian; Embryo, Nonmammalian; Fundamental and applied biological sciences. Psychology; Isoquinolines - pharmacology; Neurites - ultrastructure; Neurons - ultrastructure; Receptors, AMPA - antagonists & inhibitors; Receptors, AMPA - physiology; Tetrazoles - pharmacology; Vertebrates: nervous system and sense organs; Agonist; Cerebral cortex; Rodentia; Central nervous system; Glutamate receptor; Fragmentation; Vertebrata; AMPA receptor; Mammalia; Mouse; DNA; Brain (vertebrata); Apoptosis
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.1997.69020617.x

: We have investigated the mechanisms of cell death induced by long‐term exposure to the glutamate receptor agonist (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate [(S)‐AMPA]. Using primary cultures of pure neurons (95%) grown in serum‐free conditions, we found that 24‐h exposure to (S)‐AMPA (0.01–1,000 µM) induced concentration‐dependent neuronal cell death (EC50 = 3 ± 0.5 µM) with cellular changes including neurite blebbing, chromatin condensation, and DNA fragmentation, indicative of apoptosis. (S)‐AMPA induced a delayed cell death with DNA fragmentation occurring in ∼50% of cells at concentrations between 100 and 300 µM detected using terminal transferase‐mediated dUTP nick end‐labeling (TUNEL) and agarose gel electrophoresis. Apoptotic chromatin condensation was detected using 4,6‐diamidino‐2‐phenylindole, a fluorescent DNA binding dye. Cell death induced by (S)‐AMPA was attenuated by the AMPA receptor‐selective antagonist LY293558 (10 µM) and the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 50 µM), yielding EC50 values of 73 ± 5 and 265 ± 8 µM, respectively, and was unaffected by the NMDA receptor antagonist MK‐801 (10 µM). The number of apoptotic nuclei induced by 300 µM (S)‐AMPA (57%) was also reduced substantially by the antagonists LY293558 and CNQX, with only 20% and 18% of neurons, respectively, staining TUNEL‐positive at 24 h. In addition, cycloheximide (0.5 µg/ml) also inhibited (S)‐AMPA‐induced DNA fragmentation and cell death. Our results show that long‐term exposure to AMPA can induce substantial neuronal death involving apoptosis in cultured cortical neurons, suggesting a wide involvement of AMPA‐sensitive glutamate receptors in excitotoxic injury and neurodegenerative pathologies.