Low-affinity kainate receptor agonists induce insult-dependent apoptosis and necrosis in cultured murine cortical neurons

Overstimulation of ionotropic glutamate receptors leads to excitotoxic neuronal death, which has been implicated in the neurodegeneration of neurological diseases. The present study examined the role of putative low-affinity kainate receptor subtype. (GluR5-7) agonists in excitotoxicity in cultured murine cortical neurons. The concentration-dependent decrease in cell viability induced by the agonists kainate (1-1,000 μM) and (RS)-2-amino-3-(hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA; 1-1,000 μM) was only attenuated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM) and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466; 20 μM). (S)-5-iodowillardiine (1-1,000 μM)-induced toxicity was attenuated by CNQX (20 μM), GYKI 52466 (20 μM) and MK-801 (10 μM); however, (2S,4R)-4-methylglutamate (1-120 μM)-induced toxicity was not attenuated by the antagonists. None of the agonists possessed selective actions at GluR5-7. Morphological observations (phase-contrast and fluorescence microscopy) revealed that the agonists induced two distinct patterns of neuronal injury. After 24 hr of treatment, low concentrations of agonists (1-30 μM) produced cellular shrinkage and nuclear granulation consistent with slow, apoptotic-like neuronal death. Pyknotic labeling with the DNA binding dye Sytox green confirmed these apoptotic characteristics, which significantly decreased with increasing concentrations. After 4 hr, increasing concentrations of agonists (100-1,000 μM) induced cellular swelling, with subsequent extracellular debris; labeling with propidium iodide revealed isolated nuclei consistent with the increased involvement of rapid necrosis. Thus, all putative GluR5-7 agonists produced excitotoxicity across a necrotic-apoptotic continuum in murine cortical neuron cultures. (C) 2000 Wiley-Liss, Inc.