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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...

Unlike anoxia-intolerant mammals, painted turtles can survive extended periods without oxygen. This is partly accomplished by an anoxia-mediated increase in gamma-aminobutyric acid (GABA) release, which activates GABA receptors and mediates spike arrest in turtle neurons via shunting inhibition. Extracellular taurine levels also increase during anoxia; why this occurs is unknown but it is speculated that glycine and/or GABA receptors are involved. Given the general importance of inhibitory neurotransmission in the anoxia-tolerant painted turtle brain, we investigated the function of taurine as an inhibitory neuromodulator in turtle pyramidal neurons. Using whole-cell patch-clamp electrophysiological methods to record from neurons within a cortical brain sheet, we found that taurine depolarized membrane potential by ∼8 mV, increased whole-cell conductance ∼2-fold, and induced an inward current that possessed characteristics similar to GABA- and glycine-evoked currents. These effects were mitigated following glycine receptor antagonism with strychnine and GABA receptor antagonism with gabazine, bicuculine or picrotoxin, but were unchanged following GABA or glutamatergic receptor inhibition. These data indicate that a high concentration of taurine mediates its effects through both glycine and GABA receptors, and suggests that taurine, in addition to GABA, inhibits neuronal activity during anoxia in the turtle cortex.

The western painted turtle (Chrysemys picta bellii) can survive extended periods of anoxia via a series of mechanisms that serve to reduce its energetic needs. Central to these mechanisms is the response of mitochondria, which depolarize in response to anoxia in turtle pyramidal neurons due to an influx of K+. It is currently unknown how mitochondrial matrix pH is affected by this response and we hypothesized that matrix pH acidifies during anoxia due to increased K+/H+ exchanger activity. Inhibition of K+/H+ exchange via quinine led to a collapse of mitochondrial membrane potential (Ψm) during oxygenated conditions in turtle cortical neurons, as indicated by rhodamine‐123 fluorescence, and this occurred twice as quickly during anoxia which indicates an elevation in K+ conductance. Mitochondrial matrix pH acidified during anoxia, as indicated by SNARF‐1 fluorescence imaged via confocal microscopy, and further acidification occurred during anoxia when the F1Fo‐ATPase was inhibited with oligomycin‐A, indicating that ΔpH collapse is prevented during anoxic conditions. Collectively, these results indicate that the mitochondrial proton electrochemical gradient is actively preserved during anoxia to prevent a collapse of Ψm and ΔpH.

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The western painted turtle ( Chrysemys picta bellii) has the unique ability of surviving several months in the absence of oxygen, which is termed anoxia. One major protective strategy that the turtle employs during anoxia is a reduction in neuronal electrical activity, which may result from a natural reduction in reactive oxygen species (ROS). We previously linked a reduction in ROS levels to an increase in γ-amino butyric acid (GABA) receptor currents. The purpose of this study is to understand how fast-spiking, GABA-releasing neurons respond to reductions in ROS and how this affects GABA release. Using a fluorescence-coupled enzymatic microplate assay for GABA, we found that anoxia, the ROS scavenger N-(2-mercaptopriopionyl)glycine (MPG), or the mitochondria-specific ROS scavenger MitoTEMPO resulted in a 2.5-, 2.0-, and 2.5-fold increase in extracellular GABA concentration, respectively. This phenomenon could be blocked with TTX, indicating that it is activity dependent. Using whole cell patch-clamping techniques, we found that fast-spiking, burst-firing GABAergic turtle neurons increase the duration and number of action potentials per burst by 26% and 42%, respectively, in response to ROS scavenging via MPG. These results suggest that the reduction in mitochondrially produced ROS that occurs during anoxia leads to increased GABA release, which promotes postsynaptic inhibitory activity through activation of GABA receptors. NEW & NOTEWORTHY This is a novel study examining the response of cerebral cortical stellate interneurons to anoxia and mitochondrial reactive oxygen species (ROS) scavenging with MitoTEMPO. Under both conditions burst firing increases in these cells, and we show that extracellular GABA release increases in the presence of the ROS scavenger. We conclude that in the anoxia-tolerant painted turtle brain, a decrease in ROS levels is an important low oxygen signal.

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