AMPA Neurotoxicity in Cultured Cerebellar Granule Neurons: Mode of Cell Death

Various forms of cell death induced by the glutamate receptor agonist,

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-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), were analyzed by determining the capacity of cultured cerebellar granule cells to metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan, by measuring the leakage of lactate dehydrogenase (LDH), by using confocal microscopy to visualize propidium iodide staining of apoptotic nuclei, and by using field inversion gel electrophoresis (FIGE) for the detection of AMPA-produced cleavage of DNA into high molecular-weight fragments (50 kbp). All these measures indicated that stimulation of AMPA receptors may be involved in the neurotoxic effects of glutamate, and that AMPA-induced neurotoxicity in cerebellar granule cells display morphologically distinct features of both necrotic and apoptotic modes of cell death. In agreement with previous observations, a blockade of AMPA receptor desensitization was necessary to unmask AMPA-induced functional responses in cultured cerebellar granule neurons in vitro. Microfluorimetric measurements of free cytoplasmic calcium concentrations ([Ca²⁺]_i) in single cerebellar neurons revealed that AMPA neurotoxicity was accompanied by a pronounced elevation of [Ca²⁺]_i. Our current results add further evidence to the notion that glutamate-induced neurotoxicity in cerebellar granule cells is mediated not only through NMDA receptors but also through a direct activation of AMPA receptor-regulated cation channels.