Key pointsr Anoxia induces hyper-excitability and cell death in mammalian brain but in the western painted turtle (Chrysemys picta bellii) enhanced GABA transmission prevents injury.r The mechanism responsible for increased GABA transmission is unknown; however, reactive oxygen species (ROS) generated by mitochondria may play a role because this is an oxygen-sensitive process.r In this study, we show that inhibition of mitochondrial ROS production is sufficient to initiate a redox-sensitive GABA signalling cascade that suppresses pyramidal neuron action potential frequency.r These results further our understanding of the turtle's unique strategy for reducing ATP consumption during anoxia and highlights a natural mechanism in which to explore therapies to protect mammalian brain from low-oxygen insults (e.g. cerebral stroke).Abstract Anoxia induces hyper-excitability and cell death in mammalian brain but in the anoxia-tolerant western painted turtle (Chrysemys picta bellii) neuronal electrical activity is suppressed (i.e. spike arrest), adenosine triphosphate (ATP) consumption is reduced, and cell death does not occur. Electrical suppression is primarily the result of enhanced γ-aminobutyric acid (GABA) transmission; however, the underlying mechanism responsible for initiating oxygen-sensitive GABAergic spike arrest is unknown. In turtle cortical pyramidal neurons there are three types of GABA A receptor-mediated currents: spontaneous inhibitory postsynaptic currents (IPSCs), giant IPSCs and tonic currents. The aim of this study was to assess the effects of reactive oxygen species (ROS) scavenging on these three currents since ROS levels naturally decrease with anoxia and may serve as a redox signal to initiate spike arrest. We found that anoxia, pharmacological ROS scavenging, or inhibition of mitochondrial ROS generation enhanced all three types of GABA currents, with tonic currents comprising ß50% of the total current. Application of hydrogen peroxide inhibited all three GABA currents, demonstrating a reversible redox-sensitive signalling mechanism. We conclude that anoxia-mediated decreases in mitochondrial ROS production are sufficient to initiate a redox-sensitive inhibitory GABA signalling cascade that suppresses electrical activity when oxygen is limited. This unique strategy for reducing neuronal ATP consumption during anoxia represents a natural mechanism in which to explore therapies to protect mammalian brain from low-oxygen insults.

Oxygen deprivation triggers excitotoxic cell death in mammal neurons through excessive calcium loading via over-activation of N-methyl-Daspartate (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 Ca 2+ concentrations ([Ca 2+ ] 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 Nacetylcysteine (NAC). Unlike anoxia, ROS scavengers increased NMDA receptor whole-cell currents by 100%, while hydrogen peroxide decreased currents. AMPA receptor currents and [Ca 2+ ] i concentrations were unaffected by ROS manipulation. Because decreases in [ROS] increased NMDA receptor currents, we next asked whether mitochondrial Ca 2+ release prevents receptor potentiation during anoxia. Normoxic activation of mitochondrial ATPsensitive potassium (mK ATP ) 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. INTRODUCTIONAerobic organisms use diatomic oxygen (O 2 ) as the terminal electron acceptor of the mitochondrial electron transport chain. As a result of inconsistencies in electron flux, a portion of all oxygen consumed (~3%) is left partially reduced as the superoxide anion (Chen et al., 2003;Liu et al., 2002). This highly reactive molecule reacts rapidly with water, leading to the formation of other reactive oxygen species (ROS), the most prevalent and stable of which is (Starkov, 2008). Recently, changes in ROS levels have been identified to play roles in feedback systems and cellular signalling processes through reversible oxidation of critical cysteine residues on target proteins that can alter protein conformation and levels of activity (Cross and Templeton, 2006;D'Autréaux and Toledano, 2007;Rhee et al., 2003). In the absence of O 2 (anoxia) ROS production ceases and it is not known what effects this may have on cellular metabolism or health. For the most part it is a non-issue as most vertebrate species are unable to survive under anoxic conditions and are deleteriously affected by more than a few minutes of O 2 deprivation. Damage is most rapidly incurred within the central nervous system, where the loss of oxidative phosphorylation reduces ATP production to levels that cannot sustain the high energetic demands of neural tissue. Na + /K + -ATPase activity decreases and membrane ion gradients are lost, leading to membrane potential depolarization, increased action potenti...