Domoic Acid-Induced Neurotoxicity Is Mainly Mediated by the AMPA/KA Receptor: Comparison between Immature andMature Primary Cultures of Neurons and Glial Cells from Rat Cerebellum

Domoic acid is a naturally occurring shellfish toxin that can induce brain damage in mammalians. The toxic effects is thought to be mediated through activation of the AMPA/KA receptor, which induces increased levels of intracellular Ca2+ which in turn cause glutamate release and activates the NMDA receptor. Most studies have been performed in adult animals but neonates have been shown to be more sensitivity to domoic acid per body weight than adults. Prenatal exposure to domoic acid has been associated with damage to neurons in different brain regions, decreased brain GABA levels and increased glutamate levels. In this study we evaluated domoic acid induced toxicity in immature and mature cultures of primary rat cerebellar granule cells (CGCs) by measuring the mRNA levels of selected genes identified as specific glial and neuronal markers. Moreover, we assessed if the induced effects were mediated by activation of the AMPA/KA and/or the NMDA receptor. In addition the influence of the neurotransmitter GABA on domoic acid toxicity was evaluated. The mRNA levels of all the neuronal markers (NF-68, NF-200, NMDA receptor and GABAA receptor) were down-regulated after domoic acid exposure in both immature and mature cultures. However, the mature cultures seemed to be more sensitive to the treatment as the effects were observed at lower concentrations and at an earlier time point than for the immature ones. This could be due to lower expression in young cultures of the receptors that are mediating the toxicity. Indeed, the domoic acid effect could be prevented by the antagonist of the AMPA/KA receptor (NBQX) indicating that this receptor is involved, in contrast to the antagonist for the NMDA receptor (APV) that did not induced any effects. Interestingly, the astrocytic markers (GFAP and S100ß) and the neural precursor marker (nestin) were only affected in the mature cultures. These effects could partly be prevented by NBQX, APV and the neurotransmitter GABA, indicating that domoic acid induced toxicity by different mechanisms in astrocytes compared to neurons.JRC.I.3-Validation of Alternative Method