Scavenging ROS dramatically increases NMDA receptor whole cell currents in painted turtle cortical neurons

Abstract

Abstract Oxygen deprivation triggers excitotoxic cell death in mammal neurons through excessive calcium loading via over-activation of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. This does not occur in the western painted turtle which overwinters for months without oxygen. Neurological damage is avoided through anoxia-mediated decreases in NMDA and AMPA receptor currents that are dependent upon a modest rise in intracellular Ca2+ concentrations ([Ca2+]i) originating from mitochondria. Anoxia also blocks mitochondrial reactive oxygen species (ROS) generation which is another potential signaling mechanism to regulate glutamate receptors. To assess the effects of decreased intracellular [ROS] on NMDA and AMPA receptor currents we scavenged ROS with N-2-mercaptopropionylglycine (MPG) or N-acetylcysteine (NAC). Unlike anoxia, ROS scavengers increased NMDA receptor whole-cell currents by 100% while hydrogen peroxide decreased currents. AMPA receptor currents and [Ca2+]i concentrations were unaffected by ROS manipulation. Since decreases in [ROS] increased NMDA receptor currents we next asked if mitochondrial Ca2+ release prevents receptor potentiation during anoxia. Normoxic activation of mitochondrial ATP-sensitive potassium (mKATP) channels with diazoxide decreased NMDA receptor currents and was unaffected by subsequent ROS scavenging. Diazoxide application following ROS scavenging did not rescue scavenger-mediated increases in NMDA receptor currents. Fluorescent measurement of [Ca2+]i and ROS levels demonstrated that [Ca2+]i increases before ROS decreases. We conclude that decreases in ROS concentration are not linked to anoxia-mediated decreases in NMDA / AMPA receptor currents but are rather associated with an increase in NMDA receptor currents that is prevented during anoxia by mitochondrial Ca2+ release.