AMPA NEUROTOXICITY IN CULTURED CEREBELLAR GRANULE NEURONS - MODE OF CELL-DEATH

Various forms of cell death induced by the glutamate receptor agonist, pha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), were analy zed by determining the capacity of cultured cerebellar granule cells t o metabolize [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromid e (MTT) into formazan, by measuring the leakage of lactate dehydrogena se (LDH), by using confocal microscopy to visualize propidium iodide s taining of apoptotic nuclei, and by using field inversion gel electrop horesis (FIGE) for the detection of AMPA-produced cleavage of DNA into high molecular-weight fragments (50 kbp), All these measures indicate d that stimulation of AMPA receptors may be involved in the neurotoxic effects of glutamate, and that AMPA-induced neurotoxicity in cerebell ar granule cells display morphologically distinct features of both nec rotic and apoptotic modes of cell death, In agreement with previous ob servations, a blockade of AMPA receptor desensitization was necessary to unmask AMPA-induced functional responses in cultured cerebellar gra nule neurons in vitro. Microfluorimetric measurements of free cytoplas mic calcium concentrations ([Ca2+](i)) in single cerebellar neurons re vealed that AMPA neurotoxicity was accompanied by a pronounced elevati on of [Ca2+](i). Our current results add further evidence to the notio n that glutamate-induced neurotoxicity in cerebellar granule cells is mediated not only through NMDA receptors but also through a direct act ivation of AMPA receptor-regulated cation channels. (C) 1997 Elsevier Science Inc.