Reactive Oxygen Species in the Regulation of the GABA Mediated Inhibitory Neurotransmission

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Reactive Oxygen Species in the Regulation of the GABA Mediated Inhibitory Neurotransmission Andrea N. Beltrán González, Manuel I. López Pazos and Daniel J. Calvo* IFIBYNE-UBA-CONICET, Instituto de Fisiología, Biología Molecular y Neurociencias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas

Summary—Reactive oxygen species (ROS) are best known for being involved in cellular metabolism and oxidative stress, but also play important roles in cell communication. ROS signaling has become increasingly recognized as a mechanism implicated in the regulation of synaptic neurotransmission, under both physiological and pathological conditions. Hydrogen peroxide (H2O2) and superoxide anion are the main biologically relevant endogenous ROS in the nervous system. They are predominantly produced in the mitochondria of neurons and glial cells and their levels are tightly regulated by the antioxidant cell machinery, which allows for dynamic signaling through these agents. Physicochemical and biological properties of H2O2 enable it to effectively play an important role in signaling. This review brings up some or the most significant evidence supporting ROS as signaling agents in the nervous system and summarizes data showing that ROS modulate γ-aminobutyric acid (GABA)-mediated neurotransmission by pre- and postsynaptic mechanisms. ROS induce changes on both, the activity of phasic and tonic GABAA receptors and GABA release from presynaptic terminals. Based on these facts, ROS signaling is discussed as a possible selective mechanism linking cellular metabolism to inhibitory neurotransmission through the direct or indirect modulation of the GABAA receptor function.This article is part of a Special Issue entitled: SI: Miledi’s contributions. Item Group Code: IG005160. © 2019 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: inhibitory neurotransmission, GABAA receptor, reactive oxygen species, redox signaling, hydrogen peroxide.

INTRODUCTION

ROS can be physiologically relevant signaling mediators in different cell types and tissues (Rhee, 2006; Toledano et al., 2010; Woo et al., 2010) including the nervous system (Klann, 1998; Bao et al., 2009; Stowe and Camara, 2009; Rice, 2011; Oswald et al., 2018a; Oswald et al., 2018b). The mechanisms underlying ROS production, signaling and removal are extremely sophisticated and can involve multiple players (Egea et al., 2017). ROS participate in several forms of synaptic and non-synaptic signaling to regulate nervous system function (Rice, 2011). For example, they contribute to neuron–glia communication and to the regulation of the excitatory and inhibitory neurotransmission, eventually impacting on synaptic plasticity and the finetuning of neuronal networks (Aizenman et al., 1990; Atkins and Sweatt, 1999; Thiels et al., 2000; Kamsler and Segal, 2003; Safiulina et al., 2006; Campanucci et al., 2008; Lee et al., 2010; Massaad and Klann, 2011; Ohashi et al., 2016a; Giniatullin et al., 2019). ROS can also play important roles during development, for instance, in the establishment of neuronal polarity, growth cone pathfinding and neuronal connectivity and in the control of proliferation of stem/progenitor cells (Oswald et al., 2018a; Oswald et al., 2018b).

ROS are known for inducing noxious actions on cells and were generally involved in oxidative stress, aging and diverse pathological processes (Sies et al., 2017; Wang and Hekimi, 2015). However, under physiological conditions they can operate as signaling molecules without causing cellular damage. This concept remained controversial for a long time (Dröge, 2002; Giorgio et al., 2007; Rhee, 2006; Demaurex and Scorrano, 2009), but growing evidence indicates that *Corresponding author at: Ciudad Universitaria-Pabellón IFIBYNE, (C1428EHA) Ciudad Autónoma de Buenos Aires, Argentina. Tel.: +54 11 4576 3368x4576-3386x248x215 (Office, Lab) E-mail address: [email protected] (Daniel J. Calvo). Abbreviations: CNS, central nervous system; GABA, γ-aminobutyric acid; GABAAR, γ-aminobutyric acid receptor type A; GABABR, γaminobutyric acid receptor type B; ROS, reactive oxygen species; H2O2, hydrogen peroxide; ATP, adenosine triphosphate; SOD, superoxide dismutase; nNOS, neural nitric oxide synthase; NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; Nox, nicotinamide adenine dinucleotide phosphate oxidase; MAO, monoamino oxidase; NMDA, N-methyl-D-aspartic acid; GIRK, G protein-coupled inwardly-rectifying potassium channel. https://doi.org/10.1016/j.neuroscience.2019.05.064 0306-4522/© 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 1

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Over the last few years, the importance of ROS in the physiological regulation of inhibitory neurotransmission mediated by GABA was increasingly recognized and insight into the molecular mechanisms of modulation is emerging (Penna et al., 2014; Accardi et al., 2014; Accardi et al., 2015; Hogg et al., 2015; Calvo and Beltrán González, 2016). This review summarizes the more significant advances in this subject.

ROS as signaling molecules in the nervous system Mitochondrial and extra-mitochondrial production of ROS Brain energy requirements are very high compared to other organs (Bélanger et al., 2011). In effect, excitable cells maintain extremely elevated metabolic rates in order to work, for example, to preserve ionic gradients which are critical for action potential firing, synaptic activity and cell communication. Consequently, oxygen consumption is very high and huge amounts of ROS are produced as a function of respiration during oxidative metabolism in the central nervous system (CNS) (Halliwell, 1992; Stowe and Camara, 2009; Angelova and Abramov, 2016; Devine and Kittler, 2018). Mitochondria, the main generators of energy in the cells, are also one of the most important sources of ROS (Adam-Vizi, 2005). ROS derive from adenosine triphosphate (ATP) synthesis, a mitochondrial process that eventually involves the transfer of electrons from nicotinamide adenine dinucleotide (NADH) to molecular oxygen by a series of electron carriers, during oxidative phosphorylation in the respiratory chain. The major by-product is the superoxide anion, which is quickly converted to hydrogen peroxide (H2O2) by the mitochondrial enzyme superoxide dismutase (SOD2) or by spontaneous dismutation in aqueous solution (Woo et al., 2010; Forman et al., 2014; Devine and Kittler, 2018). Mitochondrial ROS generation is related to neuronal activity, since ROS levels increase if N-Methyl-D-aspartic acid (NMDA) receptor-mediated excitatory neurotransmission is enhanced, or by intracellular calcium concentration increments induced through other means (Dykens, 1994; Dugan et al., 1995; Reynolds and Hastings, 1995; Bindokas et al., 1996; Hongpaisan et al., 2004; Hidalgo and AriasCavieres, 2016). Significant amounts of ROS can also be enzymatically generated outside the mitochondria, either by the nicotinamide adenine dinucleotide phosphate oxidase (Nox), the monoamino oxidases (MAOs) or the neural nitric oxide synthase (nNOS) (Forman et al., 2014; Nayernia et al., 2014; Beckhauser et al., 2016). Nox is widely distributed in the CNS and its presence in the cell bodies and dendrites of neurons was confirmed, as well as its localization to synaptic sites. The catalytic subunits of Nox (Nox1 to Nox5, Duox1, and Duox2) are transmembrane proteins that faces the extracellular space and can assemble with other membrane components (e.g., proteins, lipid rafts, endosomes) and cytosolic proteins (Woo et al., 2010). Nox stays latent during the cell resting state, but during respiratory bursts can be rapidly induced to the active state by forming multi-subunit complexes in the plasma membrane that

catalyze the oxidation of NADPH and produce superoxide anion. Nox is also an important target for regulating cellular ROS production through many signaling pathways, including calcium transients induced by activation of voltagedependent calcium channels, NMDA receptor or other stimuli (Brennan et al., 2009; Girouard et al., 2009). H2O2 can also be generated as subproduct of MAO enzymatic activity in monoaminergic neurons. In the brain, MAOs (MAO-A and MAO-B) inactivate the neurotransmitters dopamine, serotonin and noradrenaline by oxidative catalysis. MAO-A is mainly located in neurons and MAO-B is preferentially expressed in glial cells (Beckhauser et al., 2016). Finally, nNOS can contribute to the generation of superoxide anion and H2O2 in the brain in a direct manner, through the Ca 2+ /calmodulin-dependent synthesis of ROS in the presence of L-arginine, or indirectly, by producing nitric oxide, which reacts with superoxide anion to form strong oxidant agents such as peroxynitrite and eventually interferes with global ROS dynamics (Heinzel et al., 1992).

ROS signaling vs. oxidative stress Since ROS are highly reactive oxidizing molecules, imbalances or failures of the cellular defense mechanisms against oxidation normally lead to high levels of oxidative stress, deregulation of redox-sensitive signaling pathways, damage to biomolecules, cellular dysfunction, development of pathologies and/or aging and eventually neuronal death (Dröge 2002; Sies et al., 2017). Excessive production of ROS was found in a variety of neurological disorders, such as ischemic-reperfusion injury, stroke, diabetes and neurodegenerative diseases (Halliwell, 1992; Andersen, 2004; Allen and Bayraktutan, 2009; Kapogiannis and Mattson, 2011; Smith et al., 2017). However, normal cells can effectively manage to selectively direct these agents to their participation in physiological redox signaling, more than in uncontrolled oxidative pathways. The concentration of ROS in the nervous system, as in other tissues, is kept under tight control by the antioxidant machinery, an intricate metabolic network constituted by diverse enzymes, molecules and minerals, in which glial cells play a fundamental role (Desagher et al., 1996; Maher, 2006; Harrison and May, 2009; Allaman et al., 2011; Baxter et al., 2015; Baxter and Hardingham, 2016). Cells have sophisticated control mechanisms that allow, for example, an extraordinarily selective and localized regulation of the activity of key detoxifying enzymes. Fine-tuning of the physiological redox signaling is ensured by the concerted actions of Nox, catalase, glutathione peroxidases, peroxiredoxins and other powerful back-up systems that include enzymes, scavenging agents such as ascorbic acid and glutathione, as well as interactive thiols, metabolically controlled mitochondrial sources of ROS and diffusion (Maher, 2006; Papadia et al., 2008; Yankner et al., 2008; Harrison and May, 2009; Stowe and Camara, 2009; Woo et al., 2010; Baxter and Hardingham, 2016). As a result, transient and controlled increases in ROS levels can build up around critical cell signaling components, while preventing their toxic accumulation somewhere else (Toledano et al., 2010; Woo et al., 2010; Breckwoldt et al., 2016; Scialò et

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al., 2016). Thus, a key aspect to consider is the site or subcellular location of both the source of ROS production and the potential targets, because occurrence of redox reactions in confined microenvironments that highly condition the spatial and temporal course of ROS actions (Woo et al., 2010; Scialò et al., 2016). Due to their complexity, the incidence of many of the above mentioned regulatory mechanisms, which have been revealed using a variety of experimental models, still needs to be corroborated in nervous system cells.

Physiologically relevant ROS in the nervous system H2O2 and superoxide anion are the more physiologically relevant ROS involved in signaling in neuronal and glial cells (Rice, 2011). Other ROS are improbable, because they are produced exclusively during oxidative stress (e.g., lipid peroxides) or only generated outside the nervous system and at very low levels (e.g., hydroxyl radical, singlet oxygen, hypochlorous acid) (Dickinson and Chang, 2011). The less damaging and membrane permeable agent H2O2 is more probably suited to play second messenger functions, rather than a short-lived and highly reactive chemical intermediate such as the free-radical superoxide anion (Rice, 2011). Thus, dynamically generated H2O2 could operate as a diffusible (extra and intracellular) signaling mediator and act as a possible link between neuronal activity and metabolism. The relative contribution of mitochondrial oxidative phosphorylation and Nox activity to shape H2O2 and superoxide anion transient signals still needs to be clearly elucidated in each particular cell type (Bao et al., 2009; Rice, 2011).

Modulation of the GABA-mediated inhibitory neurotransmission by ROS GABA-mediated inhibitory neurotransmission GABA is a neurotransmitter with a pivotal role in neuronal inhibition in the CNS and its actions are mediated via ionotropic GABAA (GABAAR) and metabotropic GABAB receptors (GABABR) (Bowery et al., 2002; Farrant and Nusser, 2005; Olsen, 2018). GABAAR mediates classic neurotransmission, whereas GABABR are principally involved in neuromodulation, normally associated with the activation of a wide variety of intracellular effectors. GABAAR are pentameric proteins members of the Cysloop ligand-gated ion channel superfamily (Miller and Aricescu, 2014). Diversity in subunit composition confers variation in the physiologic and pharmacological properties of GABAAR. To date, 19 GABAAR subunits have been cloned in the mammalian CNS and classified into classes based on the following sequence identity: α(1–6), β(1–3), γ(1–3), δ, ε, π, θ and ρ (1–3) (Sigel, 2018). They are arranged forming an integral chloride-selective channel and contain specific binding sites for typical anxiolytic, anticonvulsant and sedative/hypnotic drugs, such as benzodiazepines, barbiturates and neurosteroids, features that make these receptors critical targets for therapeutic interventions (Sieghart, 2015; Engin et al., 2018; Olsen, 2018). Meanwhile, GABABR are G-protein coupled receptors (Gi/o

3

type) that can exert inhibitory actions via the inhibition of voltage-gated calcium channels and activation of G protein-coupled inwardly-rectifying potassium channels (GIRK) potassium channels (Bowery et al., 2002). Typical inhibitory GABAergic neurotransmission in the nervous system occurs in two modes, denominated “phasic” and “tonic” (Farrant and Nusser, 2005). Phasic GABA transmission operates point to point, at high GABA concentrations, to produce fast and transient postsynaptic responses mediated by different subtypes of heteromeric GABAAR which show a relatively low affinity for GABA (e.g., GABAARα1β2γ2) (Ferando and Mody, 2014; Knoflach et al., 2016). In contrast, tonic GABA transmission involves the activation of GABAAR by low concentrations of ambient GABA built up during spillover, to produce responses which are slower to develop, persistent and less spatially and temporally restricted. Tonic GABA responses are mediated by extrasynaptic receptors with a relatively high affinity for GABA (e.g., α5βγ2 and α4βδ in the hippocampus; α6βδ in the cerebellum; ρ1 in the retina), which can be located at presynaptic terminals, beyond the synaptic cleft, or at neighboring synapses on the same or adjacent neurons (Farrant and Nusser, 2005; Brickley and Mody, 2012). In both modalities, GABAAR activation leads to increase membrane permeability to chloride and bicarbonate ions, with a net inward flow of anions that generally hyperpolarizes the cells, decreasing excitability (Kuffler and Edwards, 1958; Obata, 2013). A different scenario occurs during development (e.g., in radial glial progenitors and immature neurons) where GABA exerts depolarizing excitatory actions because, due to a differential expression of the corresponding anion transporters in the membrane, the driving force for anions is outward (Ben-Ari, 2014).

Effects of ROS on inhibitory GABA neurotransmission Pioneering research, analyzing the global impact of oxidative stress and damage in neurons and its effects on synaptic neurotransmission and neuronal excitability, described alterations in GABA neurotransmission by ROS. In these studies depressing actions of ROS, induced by H2O2 applications at particularly high concentrations, were observed on GABA neurotransmission (Colton et al., 1986; Pellmar, 1987). Later work using diverse experimental approacheso yielded dissimilar results. Table 1 summarizes the main findings of the literature review. ROS production was more commonly induced by exposure to H2O2, ascorbate/Fe 2+ or glucose-free deoxygenated buffers, circulatory occlusion or experimental diabetes. The reported effects included increase in [ 3 H]-GABA release, decrease in [ 3 H]-GABA uptake, enhancement of GABA levels and changes in the binding of GABAAR agonists and allosteric modulators in different brain areas. ROS also produced a number of changes in phasic and/or tonic GABAAR-mediated responses in diverse neuronal types (Table 1). The actions of ROS on GABA neurotransmission depended on the cell type analyzed and the CNS area under study, as well as on the biological preparation and technique utilized and the concentration of the agent employed, if any. Based on these results, pre and/or postsynaptic effects of ROS have

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Table 1. . Effects of ROS on inhibitory GABA neurotransmission in different experimental models.

ROS effect ↑[ H]-GABA release 3

↓ [ 3H]-GABA release ↓ [ 3H]-GABA uptake

↑ GABA levels

↑[ 3H]-GABA binding ↓[ 3H]-SR95531 binding ↓ [ 3H]-muscimol binding ↓ [ 3H]-muscimol binding ↓[ 35S]-TBPS binding

↓[ 35S]-TBPS binding

↓ MEQ fluorescence (prevented by PTX) ↑ GABAergic sPSCs frequency ↑ GABAergic sIPSCs frequency ↑ GABAergic mIPSCs frequency

↑ GABAergic mIPSCs frequency (absent in α3-KO mice) ↑ GABAergic mIPSCs frequency (prevented by furosemide) ↓ GABAergic mIPSCs frequency ↓ Inhibitory synaptic GABA conductances ↓ IPSPs amplitude

Experimental model

Treatment 2+

Chick retina, cultured cells Mouse hippocampus, slices

Ascorbate/Fe O2-free buffer, O2and glucose-free buffer, Ascorbate/Fe 2+, H2O2/Fe 2+, H2O2 Rat cerebral cortex, synaptosomes H2O2 Ship cerebral cortex, synaptosomes Ascorbate/Fe 2+ Ship cerebral cortex, synaptosomes Ascorbate/Fe 2+ Rat cerebral cortex, synaptosomes, Ascorbate/Fe 2+ diabetic GK Rat cerebral cortex, hippocampus, Circulatory occlusion and systemic striatum and thalamus hypotension Rat cerebral cortex Circulatory occlusion Chick retina, cultured cells Ascorbate/Fe 2+ Rat hippocampus, slices O2 and glucose-free buffer

Reference Agostinho et al., 1994 Saraansari & Oja, 1997 and 1998 Tarasenko et al., 2012 Palmeira et al., 1993 Palmeira et al., 1993 Duarte et al., 2004 Globus et al., 1991

Rat hippocampus, slices Rat striatum, homogenates Gerbil hippocampus, slices, CA1 and CA3 and dentate gyrus Gerbil brain, frontal cortex and caudate putamen Mouse brain, motor and somatosensory cortex Rat cerebral cortex, hippocampus CA1 and striatum, homogenates Rat cerebral cortex, hippocampus and striatum homogenates Rat hippocampus, slices

H2O2 Circulatory occlusion Circulatory occlusion

Phillis et al., 1994 Rego et al., 1991 Inglefield and Schwartz-Bloom, 1998 Sah and Schwartz-Bloom, 1999 Francis and Pulsinelli, 1983 Alicke & Schwartz-Bloom, 1995

Circulatory occlusion

Onodera et al., 1987

Circulatory occlusion

Qü et al., 1998

Circulatory occlusion

Mileson et al., 1992

H2O2

Sah et al., 2002

O2 and glucose-free buffer

Inglefield & Schwartz-Bloom, 1998

Rat hippocampus, slices Rat hippocampus, slices, CA3 pyramidal neurons Rat spinal cord, slices, substantia gelatinosa neurons Rat brain, slices, CA1 pyramidal neurons Mouse spinal cord, slices, substantia gelatinosa neurons Rat cerebral cortex, mechanically-isolated neurons Turtle cerebral cortex, slices, pyramidal neurons Rat spinal cord, slices, ventral horn neurons Mouse cerebellum, slices, stellate cells Mouse cerebellum, slices, granule cells Mouse spinal cord, slices, substantia gelatinosa neurons, neuropathic pain model Lobster neuromuscular junction

H2O2 H2O2

Sah& Schwartz-Bloom, 1999 Safiulina et al., 2006

O2 and glucose-free buffer

Kawasaki et al., 2004

O2-free buffer

Katchman et al., 1994

H2O2

Takahashi et al., 2007

H2O2

Hahm et al., 2010

MPG, MitoTEMPO or Sodium cyanide H2O2

Hogg et al., 2015

Antimycin-A

Accardi et al., 2014

Antimycin-A Insulin Tert-butyl hydroperoxide

Accardi et al., 2015

H2O2

Colton et al., 1986

H2O2

Pellmar, 1987

O2-free buffer

Katchman et al., 1994

MPG, MitoTEMPO or Sodium cyanide

Hogg et al., 2015

O2-free buffer

Katchman et al., 1994

H2O2

Takahashi et al., 2007

H2O2

Hahm et al., 2010

H2O2

Frantseva et al., 1998

Guinea pig hippocampus, slices, CA1 pyramidal neurons ↓ GABAergic IPSCs amplitude Rat brain, slices, CA1 pyramidal neurons ↑ GABAergic sIPSC and gIPSC Turtle cerebral cortex, slices, amplitude pyramidal neurons ↑ GABAergic mIPSCs amplitude Rat brain, slices, CA1 pyramidal neurons Mouse spinal cord, slices, substantia gelatinosa neurons Rat cerebral cortex, mechanically-isolated neurons

Ohashi et al., 2016 (a)

Yowtak et al., 2011

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Table 1 (continued)

ROS effect

Run-down of whole-cell IGABA ↑ GABAAρ1-R currents amplitude ↑ GABAA tonic currents amplitude

↑ GABAA tonic currents amplitude ↓ GABAA tonic currents amplitude

Experimental model

Treatment

Rat thalamus and cortex, slices, pyramidal neurons and ventrobasal thalamic neurons Rat hippocampus, O2-free buffer CA1 pyramidal dissociated neurons O2 and glucose-free buffer Xenopus laevis oocytes H2O2 application Mouse hippocampus, cultured neurons and slices CA1 pyramidal neurons Rat spinal cord slices, ventral horn neurons Turtle cerebral cortex, slices, pyramidal neurons Turtle cerebral cortex, slices, pyramidal neurons

been accordingly proposed, but in most of the studies the mechanisms underlying ROS actions were not tackled. Nevertheless, some of the more recent work (also referenced in Table 1) shed light on the cellular and molecular mechanisms by which ROS can exert modulatory actions on GABAAR, so we will center our discussion on them.

Modulation of the activity of diverse GABAA receptor subtypes by ROS The role of redox signaling in the regulation of the GABAAR function in normal physiology and pathological states is still far from being completely understood at present. However, a number of evidence indicate that mitochondrial-derived ROS might couple cell metabolism to the control of neuronal inhibition, by participating in diverse forms of modulation and plasticity of synaptic and extrasynaptic GABAAR. Based on the fact that retinal GABAAR showed susceptibility to sulfhydryl reagents, Stuart Lipton and colleagues earlier predicted that “endogenous redox agents may constitute a novel modulatory system for the differential regulation of inhibitory neurotransmission” (Pan et al., 1995). Their observations were further corroborated and extended by different authors studying the sensitivity of GABAAR to different pharmacological and endogenous redox agents, including ROS, in diverse areas of the CNS and in heterologous systems (Amato et al., 1999; Pan et al., 2000; Sah et al., 2002; Wilkins and Smart, 2002; Calero and Calvo, 2008; Calero et al., 2011; Accardi et al., 2014; Beltrán González et al., 2014; Penna et al., 2014; Accardi et al., 2015). As a result, the idea that ROS can physiologically regulate the GABAergic inhibitory tone in neurons started to grow. A good step in this direction was given by Schwartz-Bloom and colleagues showing that moderate concentrations of H2O2 (300 μM) induced picrotoxin sensitive increases in the intracellular concentration of chloride of hippocampal cells, which were compatible to an enhanced GABAAR activity (Sah and Schwartz-Bloom, 1999). These results contrasted to previous studies, where the amplitude of GABAergic IPSPs recorded in the hippocampus, cerebral cortex, and thalamus was reduced by very high concentrations of H2O2 (1.5–3.5 mM) (Pellmar, 1987; Frantseva et

Reference

Harata et al., 1997 Beltrán González et al., 2014

H2O2 application O2 and glucose-free buffer

Penna et al., 2014

H2O2

Ohashi et al., 2016 (b)

MPG, MitoTEMPO or Sodium cyanide H2O2

Hogg et al., 2015 Hogg et al., 2015

al., 1998) (Table 1). However, experimental designs were not equivalent and none of all these data were conclusive, taking into account the possible effects of H2O2 on both GABA release from presynaptic terminals and direct actions on GABAAR. The first compelling evidence supporting a selective mechanism of modulation of the inhibitory GABAergic neurotransmission by ROS came from studies in which pharmacological and genetic approaches were combined to analyze the effects of H2O2 on tonic and synaptic responses mediated by GABAAR in hippocampal and cerebellar neurons (Penna et al., 2014; Accardi et al., 2014; Accardi et al., 2015). Meanwhile, the molecular mechanisms by which ROS alter synaptic GABA release will need to be addressed in equivalent experiments. Besides, the effects of ROS on GABAB receptor-mediated neuromodulation are less documented (Huang et al., 2015). Exposure of brain slices to H2O2 caused a significant and reversible potentiation of the tonic GABA responses recorded in hippocampal CA1 pyramidal cells, leaving the synaptic GABAergic responses unaffected (Penna et al., 2014). This potentiation was abolished by extracellular, but not intracellular, applications of an endogenous antioxidant such as glutathione. Thus, authors proposed a direct action of H2O2 on GABAAR that produced an extracellular oxidative reaction increasing the potency of GABA for eliciting tonic currents in the low concentration range. Oxygen–glucose deprivation, a model to induce ischemia–reperfusion injury that generates high endogenous levels of H2O2, potentiated the tonic GABA currents in a similar manner. Surprisingly, ROS actions do not appear to be exerted through the extrasynaptic GABAAR subtypes formed by α5 and δ subunits, which are commonly involved in typical hippocampal tonic GABAA responses, but rather by multiple subtypes containing αβ, α1βδ, α3βγ2, α4δ, α6δ, and αβε subunits, as suggested by experiments performed in α5 knockout transgenic mice (Penna et al., 2014). Endogenous ROS were also shown to enhance GABAR-mediated inhibitory neurotransmission in cerebellar neuA rons (Accardi et al., 2014; Accardi et al., 2015). The intracellular perfusion of antimycin A, a mitochondrial

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uncoupler that increases ROS levels, produced increases in the frequency synaptic GABAA responses recorded from stellate and granule cells, with no effects on tonic GABAAR responses in both cases. In stellate cells, the enhancement of ROS levels promoted the appearance of postsynaptic events compatible with a recruitment of α3-containing GABAAR into discrete postsynaptic sites, without affecting resident α1-containing GABAAR (Accardi et al., 2014). In granule cells, the rise in ROS levels enhanced inhibitory synaptic transmission in a similar way, but was associated to the recruitment of α6-containing GABAAR instead. Effects induced by ROS on GABAAR in cerebellar neurons were also sensitive to antioxidant thiol agents (Accardi et al., 2015). Taking together, these data suggest that ROS could regulate the degree of inhibition mediated by tonic and phasic GABAAR in the hippocampus and cerebellum. It will be interesting to know if this kind of regulation might also take place in other CNS areas. Tonic and phasic GABAARmediated responses in retinal bipolar neurons were shown to be also modulated by redox mechanisms (Calero et al., 2011). Restoration of the extra and intracellular levels of ascorbic acid, to their corresponding physiological values, induced increases in the amplitude of GABA responses mediated by tonic and phasic GABAAR and prevented the run-down of tonic currents mediated by homomeric GABAAρ1 receptors in these neurons. These effects could be particularly relevant in a tissue like the retina, where ROS production is elevated, but the participation of ROS in these events was not eventually substantiated. ROS might be also involved in the modulation of GABAAR by diabetesinduced oxidative stress (Duarte et al., 2004; Ramsey et al., 2007; Okumichi et al., 2008). The fact that ROS-induced plasticity of the diverse GABAR subtypes was repeatedly found susceptible to thiol A agents suggests that one of the possible mechanisms underlying ROS actions would be the oxidation of different cysteine residues in the GABAAR subunits, which might be critical for ion channel gating, as observed for other ligand-gated ion channels (Aizenman et al., 1990; Pan et al., 1995; Amato et al., 1999; Pan and Lipton, 2000; Chu et al., 2006; Campanucci et al., 2008; Calero and Calvo, 2008; Coddou et al., 2009; Beltrán González et al., 2014). Remarkably, homomeric GABAAρ1 receptors carry an intracellular cysteine (C364) at the M3–M4 linker, conserved in many other GABAAR subtypes, which is a possible target of endogenous ROS (Beltrán González et al., 2014). However, more insight will be required to explain the molecular mechanisms underlying homeostatic changes induced by ROS on GABAAR. Mutational analysis of different recombinant GABAAR subtypes expressed in heterologous systems will help to clarify the differential contribution of discrete cysteine residues to ROS modulation (Calvo and Beltrán González, 2016). An interesting matter to look at in future works will be to assess both the actual intra and extracellular concentration of ROS and the time course of ROS effects on GABAAR, through simultaneous recording. This will allow to establish more realistic correlations, but there are still technical problems in the implementation of new tools such as imaging dyes and fluorescent protein-based

probes for monitoring ROS, both in vitro and in vivo (Rice, 2011). Another key aspect to consider will be to distinguish physiological from pathological processes in the modulation of the inhibitory GABAergic neurotransmission by ROS, for example with the help of animal models for pathologies.

ACKNOWLEDGMENTS Supported by CONICET and FONCYT PICT 2015-2417.

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(Received 3 April 2019, Accepted 31 May 2019) (Available online xxxx)