• Domoic acid;
  • Glutamate;
  • Aspartate;
  • NMDA receptor;
  • α-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor;
  • Excitotoxicity


  1. Top of page
  2. Abstract

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 µMl-glutamate or 10 µM domoate for 2 h in physiologic buffer at 22°C followed by a 22-h incubation in 37°C 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 the conditioned media. Glutamate and domoate evoked, respectively, 50 and 65% of the total 24-h increment in LDH efflux after 2 h. Hyperosmolar conditions prevented this early response but did not significantly alter the extent of neuronal injury observed at 24 h. The competitive NMDA receptor antagonist d(−)-2-amino-5-phosphonopentanoic acid and the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(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 noncompetitive 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 occurred almost exclusively through mechanisms related to cell swelling and reversal of the glutamate transporter. Thus, whereas 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 α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor-mediated stimulation of EAA efflux.

Abbreviations used: AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionate; d-AP5, d(−)-2-amino-5-phosphonopentanoic acid; BME, basal medium Eagle; CGC, cerebellar granule cell; CPA, N6-cyclopentyladenosine; CPP, 3-[(R)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid; DIC, days in culture; DX, dextrorphan; DXM, dextromethorphan; EAA, excitatory amino acid; LDH, lactate dehydrogenase; MK-801, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline; NMDA, N-methyl-d-aspartate; OPT, o-phthaldialdehyde; PDA, l-trans-pyrrolidine-2, 4-dicarboxylic acid; 8-pSPT, 8-(p-sulfophenyl)theophylline; TCP, N-[1-(2-thienyl)cyclohexyl]piperidine; TTX, tetrodotoxin.