Decreases in mitochondrial reactive oxygen species initiate GABA(A) receptor-mediated electrical suppression in anoxia-tolerant turtle neurons

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 ∼50% 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.

Figure 1

Fluorescent assessment of mitochondrial ROS generation and intracellular redox state following ROS-modulating treatments in cortical neurons A, summary of changes in CM-DCF fluorescence. B, sample CM-DCF fluorescence recordings from A. Note: when trace is horizontal there is zero [ROS]_i generation. Horizontal scale bar: 5 min in a–c, and 10 min in d. C, summary of changes in MitoSOX fluorescence. D, sample MitoSOX fluorescence recordings from C. Arrow indicates onset of H₂O₂ application. Bars represent duration of treatment. Arbitrary fluorescence units (AFU). Treatments: normoxia (95% O₂/5% CO₂-bubbled aCSF), anoxia (95% N₂/5% CO₂-bubbled aCSF), CN (0.5 mm), MPG (0.5 mm), MitoTEMPO (20 μm) and H₂O₂ (50 μm). Data are means ± SEM, n = 4–5 replicates per treatment. *Significant difference of treatment from normoxic controls (P < 0.05).

Figure 2

Pharmacological or anoxia-mediated decreases in [ROS]_i shift pyramidal neuron V_m to E_GABA by activating a GZ-sensitive increase in G_w; ROS scavenging decreases AP_f in a subset of spontaneously active cortical pyramidal neurons A, summary graph showing V_m depolarization following a 30 min ROS-depleting treatment, anoxia, ROS scavenger MPG and mitochondrial cytochrome c oxidase inhibitor CN. Addition of the GABA_A receptor inhibitor GZ prevents V_m changes, and the oxidant H₂O₂ reverses V_m changes. Note: the dashed line represents E_GABA. B, sample raw traces showing treatment-induced changes in V_m. C, summary of changes in G_w following treatments as outlined in A. D, sample I–V relationships used to determine G_w in C. E, summary of change in AP_f following treatment with MPG or MitoTEMPO. F, sample raw traces used to generate E. Horizontal bars represent duration of treatment. Treatments: normoxia (95% O₂/5% CO₂-bubbled aCSF), anoxia (95% N₂/5% CO₂-bubbled aCSF), MPG (0.5 mm), MitoTEMPO (20 μm), GZ (25 μm), CN (0.5 mm) and H₂O₂ (50 μm). Data are means ± SEM, n = 4–11 replicates per treatment. *Significant difference from normoxic controls. ^#Significant difference from E_GABA in A or anoxia in C. The letters ‘a’ and ‘b’ indicate a significant within-treatment different due to GZ (normoxia and anoxia, respectively) (P < 0.05).

Figure 3

Pharmacological and anoxia-mediated decreases in [ROS]_i increase mIPSC frequency but not amplitude in cortical pyramidal neurons A, summary of mIPSC amplitude following a 30 min treatment with anoxia or the ROS scavenger MPG. B, summary of normalized mIPSC frequency under the same conditions as A. C, sample raw traces used to generate A and B. Note: normoxic pre-treatment (10 min) and treatment (30 min). GABA_A receptor-mediated mIPSCs were enhanced with high [Cl⁻] pipette solution (130 mm) and isolated with NMDA and AMPA receptor antagonists (AP5 and CNQX, respectively; 25 μm each), a voltage-gated Na⁺ channel inhibitor (TTX; 1 μm), and a glycine receptor antagonist (strychnine; 2 μm). Treatments: normoxia (95% O₂/5% CO₂-bubbled aCSF), anoxia (95% N₂/5% CO₂-bubbled aCSF), MPG (0.5 mm). Data are means ± SEM, n = 6 replicates per treatment. *Significant difference from normoxic controls (P < 0.05).

Figure 4

GABA_A receptor-mediated spontaneous IPSCs and giant IPSCs are sensitive to pharmacological and anoxia-mediated decreases in [ROS]_i in cortical pyramidal neurons A, summary of changes in sIPSC amplitude in response to a 30 min normoxic or anoxic treatment with the ROS scavenger MPG, mitochondrial cytochrome c oxidase inhibitor CN, or mitochondrial O₂^•− scavenger MitoTEMPO. Note: data were normalized to a preceding 2 min normoxic time point. B, sample raw spontaneous voltage-clamp recording demonstrating the effect of MitoTEMPO on GABAergic IPSCs. C, summary of gIPSC amplitude in response to the same 30 min treatment as in A. D, sample raw spontaneous voltage-clamp recording demonstrating the change in GABA currents during a transition to anoxia. a–c expanded trace showing the effect of anoxia and GZ on gIPSCs. d–f, expanded trace showing the effect of anoxia and GZ on sIPSCs. The scale bars correspond, from top to bottom, to: 200 pA/75 s, 200 pA/7.5 s, 40 pA/750 ms. E, sample raw spontaneous voltage-clamp recording demonstrating MPG-mediated changes in GABA currents. Note: GZ application inhibits sIPSCs and gIPSCs demonstrating ROS scavenger-mediated increase in GABA release and activation of GABA_A receptors. F, superimposed spontaneous gIPSC recordings demonstrating that gIPSC amplitude increases following application of CN and these are GABA_A receptor mediated because GZ inhibits them. Note: traces are averaged from 4 recordings each, 10 gIPSC events per recording. Neurons were voltage clamped at −100 mV and GABA_A receptor currents were enhanced with high [Cl⁻] pipette solution (130 mm) and isolated with NMDA and AMPA receptor antagonists (AP5 and CNQX, respectively; 25 μm each). Note: due to high pipette chloride, anoxia and ROS scavenging occasionally caused action currents. Bars indicate treatment duration. Treatments: normoxia (95% O₂/5% CO₂-bubbled aCSF), anoxia (95% N₂/5% CO₂-bubbled aCSF), MPG (0.5 mm), GZ (25 μm), CN (0.5 mm). Data are means ± SEM, n = 5–7 replicates per treatment. *Significant difference from normoxic controls (P < 0.05).

Figure 5

Tonic GABA_A receptor currents in cortical pyramidal neurons are inhibited by H₂O₂ A, summary of the effects of bicuculline on tonic GABA_A receptor currents following normoxia, anoxia, MPG or CN. B, summary of the effects of H₂O₂ on tonic GABA_A receptor currents following the same treatments as in A. Note: no treatment (grey bars) in A and B represent the average change in the holding current prior to application of BIC or H₂O₂, respectively. C, sample raw traces used to generate A. D, sample raw trace used to generate B. Bars denote treatment duration. Dashed lines highlight the GABA-mediated tonic current. Pyramidal neurons were voltage clamped at −100 mV and GABA_A receptor currents were enhanced with high [Cl⁻] pipette solution (130 mm) and isolated with NMDA and AMPA receptor antagonists (AP5 and CNQX, respectively; 25 μm each). Treatments: normoxia (95% O₂/5% CO₂-bubbled aCSF), anoxia (95% N₂/5% CO₂-bubbled aCSF), MPG (0.5 mm), CN (0.5 mm), BIC (100 μm) and H₂O₂ (50 μm). Data are means ± SEM, n = 5–7 replicates per treatment. ^§Significant difference from baseline. *Significant difference from normoxic controls (P < 0.05).

Figure 6

Comparison of the pharmacological and anoxia-mediated charge transfer associated with GABA_A receptor-mediated sIPSCs, gIPSCs and tonic currents A, summary of the cumulated charge transfer resulting from GABA_A receptor sIPSCs, gIPSCs and tonic currents after a 30 min treatment with anoxia, MPG or CN. Note: charge transfer is calculated over a duration of 2 min at the end of the treatment period. B, schematic drawing and equations detail the methods used to calculate charge transfer. Note: grey shading indicates charge transfer associated with GABA_A receptor currents in pyramidal neurons. Bar denotes treatment duration. Pyramidal neurons were voltage clamped at −100 mV and GABA_A receptor currents were enhanced with high [Cl⁻] pipette solution (130 mm) and isolated with NMDA and AMPA receptor antagonists (AP5 and CNQX, respectively; 25 μm each). Treatments: normoxia (95% O₂/5% CO₂-bubbled aCSF), anoxia (95% N₂/5% CO₂-bubbled aCSF), MPG (0.5 mm) and CN (0.5 mm). Data are means ± SEM, n = 5–7 replicates per treatment. *Significant difference from normoxic sIPSCs. ^†Significant difference from normoxic gIPSCs. ^‡Significant difference from normoxic gIPSCs (P < 0.05).