Abstract
The participation of NMDA and non-NMDA receptors in domoic acid-induced neurotoxicity was investigated in cultured rat cerebellar granule cells (CGCs). Neurons were exposed to 300 uM L- glutamate or 10 uM domoate for two hours in physiologic buffer at 22C followed by a 22 hr incubation in 37C conditioned growth media. Excitotoxic injury was monitored as a function of time by measurement of lactate dehydrogenase (LDH) activity in both the exposure buffer and conditioned media. Glutamate and domoate evoked respectively, 50% and 65% of the total 24 hr increment in LDH efflux after 2 hrs. Hyperosmolar conditions prevented this early response, but did not significantly alter the extent of neuronal injury observed at 24 hrs. The competitive NMDA receptor antagonist, D(-)-2- amino-5-phosphonopentanoic acid (D-AP5), and the non-NMDA receptor antagonist, 2,3-dihydroxy-6- nitro- 7-sulfamoyl-benzo(F)quinoxaline (NBQX), reduced glutamate-induced LDH efflux totals by 73% and 27%, respectively, whereas together, these glutamate receptor antagonists completely prevented neuronal injury. Domoate toxicity was reduced 65-77% when CGCs were treated with competitive and non-competitive NMDA receptor antagonists. Unlike the effect on glutamate toxicity, NBQX completely prevented domoate-mediated injury. HPLC analysis of the exposure buffer revealed that domoate stimulates the release of excitatory amino acids (EAAs) and adenosine from neurons. Domoate-stimulated EAA release occured almost exclusively through mechanisms related to cell swelling and reversal of the glutamate transporter. Thus, while glutamate-induced injury is mediated primarily through NMDA receptors, the full extent of neurodegeneration is produced by the coactivation of both NMDA and non-NMDA receptors. Domoate-induced neuronal injury is also mediated primarily through NMDA receptors, which are activated secondarily as a consequence of AMP Nkainate receptor-mediated stimulation of EAA efflux.