César Mattei
email: cesar.mattei@univ-angers.fr

Antoine Taly

Zineb Soualah

Ophélie Saulais

Daniel Henrion

Nathalie C Guérineau

Marc Verleye

Christian Legros
email: christian.legros@univ-angers.fr

Involvement of the GABA A receptor a subunit in the mode of action of etifoxine

Keywords: 171544), bromazepam etifoxine, GABAA receptors, a subunit, anxiolysis, behavioral pharmacology, EFXbinding mode Highlights

Etifoxine (EFX) is a non-benzodiazepine psychoactive drug which exhibits anxiolytic effects through a dual mechanism, by directly binding to GABAA receptors (GABAARs) and to the mitochondrial 18-kDa translocator protein, resulting in the potentiation of the GABAergic function. The b subunit subtype plays a key role in the EFX-GABAAR interaction, however this does not explain the anxiolytic effects of this drug. Here, we combined behavioral and electrophysiological experiments to challenge the role of the GABAAR a subunit in the EFX mode of action. After single administrations of anxiolytic doses (25-50 mg/kg, intraperitoneal), EFX did not induce any neurological nor locomotor impairments, unlike the benzodiazepine bromazepam (0.5-1 mg/kg, intraperitoneal). We established the EFX pharmacological profile on heteropentameric GABAARs constructed with α1 to α6 subunit expressed in Xenopus oocyte. Unlike what is known for benzodiazepines, neither the γ nor δ subunits influenced EFX-mediated potentiation of GABA-evoked currents. EFX acted first as a partial agonist on α2b3γ2S, α3b3γ2S, α6b3γ2S and α6b3d GABAARs, but not on α1b3γ2S, α4b3γ2S, α4b3d nor α5b3γ2S GABAARs. Moreover, EFX exhibited much higher positive allosteric modulation towards α2b3γ2S, α3b3γ2S and α6b3γ2S than for α1b3γ2S, α4b3γ2S and α5b3γ2S GABAARs. At 20 µM, corresponding to brain concentration at anxiolytic doses, EFX increased GABA potency to the highest extent for α3b3γ2S GABAARs. We built a docking model of EFX on α3β3γ2S GABAARs, which is consistent with a binding site located between α and β subunits in the extracellular domain. In conclusion, EFX preferentially potentiates α2b3γ2S and α3b3γ2S GABAARs, which might support its advantageous anxiolytic/sedative balance.

Introduction

GABAARs are heteropentameric membrane proteins that belong to the cys-loop ligand-gated ion channel superfamily [START_REF] Olsen | International Union of Pharmacology. LXX. Subtypes of gammaaminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update[END_REF]. They are permeant to chloride ions in response to GABA and decrease neuronal excitability through membrane hyperpolarization. To date, 19 mammalian GABAAR subunits have been described and cloned (α1-6, β1-3, γ1-3, δ, ε, π, θ, ρ1-3) [START_REF] Olsen | International Union of Pharmacology. LXX. Subtypes of gammaaminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update[END_REF]. The putative combination of these subunits provides a large heterogeneity of GABAARs, with a stoichiometry of 2α, 2β and a complementary subunit (mainly γ or δ). The contribution of GABAARs to fast or slow neuronal inhibition depends on their stoichiometry, their tissue distribution and their synaptic or extrasynaptic location. The most frequent assembly of synaptic receptors is α(1-3)β(1-3)γ(1-3), whereas extrasynaptic receptors dominantly contain α4 or α6 with β(1-3) and δ, or α5, β(1-3) and γ2 [START_REF] Olsen | International Union of Pharmacology. LXX. Subtypes of gammaaminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update[END_REF][START_REF] Farrant | Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors[END_REF]. These differences in stoichiometry and distribution support their different neurophysiological functions and pharmacological properties [START_REF] Olsen | GABA A receptors: subtypes provide diversity of function and pharmacology[END_REF][START_REF] Engin | An Emerging Circuit Pharmacology of GABAA Receptors[END_REF].

GABAARs are targeted by benzodiazepines (BZDs) and other drugs for the treatment of anxiety, epilepsy and sleep disorders [START_REF] Atkin | Drugs for Insomnia beyond Benzodiazepines: Pharmacology, Clinical Applications, and Discovery[END_REF][START_REF] Brodie | Epilepsy, Antiepileptic Drugs, and Aggression: An Evidence-Based Review[END_REF]. BZDs act as positive allosteric modulators (PAMs) of GABAARs by binding to a site at the interface between g2 subunit and α subunits [START_REF] Pritchett | Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology[END_REF]. Classical BZDs, such as diazepam (DZP, Fig. 1), bromazepam (BZP, Fig. 1) and lorazepam, exhibit similar pharmacological profile in behavioral tests [START_REF] Steru | Comparing benzodiazepines using the staircase test in mice[END_REF][START_REF] Bourin | Comparison of behavioral effects after single and repeated administrations of four benzodiazepines in three mice behavioral models[END_REF] and display poor selectivity over GABAARs which contain α1, or α2 or α3 or α5 (α1GABAAR, α2GABAAR, α3GABAAR, or α5GABAAR) [START_REF] Rudolph | Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes[END_REF], which explains their undesirable effects, including withdrawal symptoms, sedation, amnesia, cognitive impairments and aggressiveness. Indeed, α1GABAARs are associated with sedation, BZD addiction, anterograde amnesia, anticonvulsant activity and cortical plasticity [START_REF] Rudolph | Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes[END_REF][START_REF] Rudolph | Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes[END_REF][START_REF] Mckernan | Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype[END_REF]. α2GABAARs and α3GABAARs have been linked to anxiolysis, antihyperalgesia and myorelaxation [START_REF] Löwe | Molecular and neuronal substrate for the selective attenuation of anxiety[END_REF][START_REF] Dias | Evidence for a significant role of alpha 3-containing GABAA receptors in mediating the anxiolytic effects of benzodiazepines[END_REF][START_REF] Atack | GABAA receptor alpha2/alpha3 subtype-selective modulators as potential nonsedating anxiolytics[END_REF].

α5GABAARs are believed to be correlated to sedation, cognitive impairments and more recently, anxiolysis [START_REF] Dias | Evidence for a significant role of alpha 3-containing GABAA receptors in mediating the anxiolytic effects of benzodiazepines[END_REF][START_REF] Atack | GABAA receptor alpha2/alpha3 subtype-selective modulators as potential nonsedating anxiolytics[END_REF][START_REF] Savic | Are GABAA receptors containing alpha5 subunits contributing to the sedative properties of benzodiazepine site agonists?[END_REF][START_REF] Behlke | A Pharmacogenetic 'Restriction-of-Function' Approach Reveals Evidence for Anxiolytic-Like Actions Mediated by alpha5-Containing GABAA Receptors in Mice[END_REF].

Etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride, EFX, Fig. 1) is a non-BZD compound that exhibits anxiolytic and anticonvulsant effects in rodents [START_REF] Boissier | Experimental psychopharmacologic study of a new psychotropic drug, 2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine[END_REF] and is used for the treatment of anxiety-related disorders in humans [START_REF] Nguyen | Efficacy of etifoxine compared to lorazepam monotherapy in the treatment of patients with adjustment disorders with anxiety: a double-blind controlled study in general practice[END_REF][START_REF] Stein | Etifoxine versus alprazolam for the treatment of adjustment disorder with anxiety: a randomized controlled trial[END_REF]. EFX also displays anti-hyperalgesic and anti-inflammatory properties in different animal models [START_REF] Aouad | Etifoxine stimulates allopregnanolone synthesis in the spinal cord to produce analgesia in experimental mononeuropathy[END_REF][START_REF] Simon-O'brien | Etifoxine improves sensorimotor deficits and reduces glial activation, neuronal degeneration, and neuroinflammation in a rat model of traumatic brain injury[END_REF]. Both in vitro and in vivo studies in rats suggested that the anxiolytic effects of EFX involve a dual mechanism of action, by directly binding to central GABAARs and to the mitochondrial 18-kDa translocator protein (TSPO) with, as a result, potentiation of the GABAergic function [START_REF] Schlichter | Modulation of GABAergic synaptic transmission by the non-benzodiazepine anxiolytic etifoxine[END_REF][START_REF] Hamon | The modulatory effects of the anxiolytic etifoxine on GABA(A) receptors are mediated by the beta subunit[END_REF]. Indeed, it has been shown that EFX activates TSPO through a direct binding and consecutively stimulates the synthesis of neurosteroids, such as, allopregnanolone, which act as PAMs of GABAARs [START_REF] Verleye | The anxiolytic etifoxine activates the peripheral benzodiazepine receptor and increases the neurosteroid levels in rat brain[END_REF][START_REF] Costa | The Anxiolytic Etifoxine Binds to TSPO Ro5-4864 Binding Site with Long Residence Time Showing a High Neurosteroidogenic Activity[END_REF][START_REF] Liere | Differential effects of the 18-kDa translocator protein (TSPO) ligand etifoxine on steroidogenesis in rat brain, plasma and steroidogenic glands: Pharmacodynamic studies[END_REF]. Although the affinity of EFX for GABAARs was twice higher than the one for TSPO (Ki of 6.1 µM vs Ki of 12.7 µM), the predominance of one of the effect over the other, i.e. direct GABAARs binding or through TSPO activation, in mediating its anxiolytic effect, is still debated [START_REF] Verleye | The anxiolytic etifoxine activates the peripheral benzodiazepine receptor and increases the neurosteroid levels in rat brain[END_REF][START_REF] Verleye | Interactions of etifoxine with the chloride channel coupled to the GABA(A) receptor complex[END_REF][START_REF] Verleye | Investigation of the anticonvulsive effect of acute immobilization stress in anxious Balb/cByJ mice using GABA A-related mechanistic probes[END_REF].

The importance of the β subunit in the mode of action of EFX on GABAARs has been clearly evidenced [START_REF] Hamon | The modulatory effects of the anxiolytic etifoxine on GABA(A) receptors are mediated by the beta subunit[END_REF]. Constitutively-open homopentameric βGABAARs are inhibited by EFX. In addition, α1GABAARs and α2GABAARs embedding β2 or β3 are more sensitive to EFX than α1GABAARs and α2GABAARs with β1. These data underline the importance of the β subunit in the EFX-GABAAR interaction and for EFX-potentiation of GABA-induced currents of heteromeric GABAARs. However, homopentameric β2GABAARs are less sensitive to EFX than homopentameric β1GABAARs [START_REF] Hamon | The modulatory effects of the anxiolytic etifoxine on GABA(A) receptors are mediated by the beta subunit[END_REF],

suggesting that the nature of the α subunit might also play a role in EFX-GABAARs interaction. In addition, at anxiolytic doses, EFX has no sedative effects nor locomotion impairment in humans [START_REF] Nguyen | Efficacy of etifoxine compared to lorazepam monotherapy in the treatment of patients with adjustment disorders with anxiety: a double-blind controlled study in general practice[END_REF] or rodents [START_REF] Verleye | Effects of stress and etifoxine on pentobarbitalinduced loss of righting reflex in Balb/cByJ and C57BL/6J mice[END_REF] and this could hardly be explained by the equal potency of EFX on α2GABAAR and α1GABAAR since the latter is associated with sedation [START_REF] Rudolph | Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes[END_REF][START_REF] Mckernan | Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype[END_REF]. Thus, we hypothesized that the pharmacological profile of EFX reflects different sensitivities of all α subtypes containing-GABAARs.

In this study, we first compared EFX and BZP in anxiolysis, sedation and locomotor impairment behavioral tests in acute conditions, in mice. The pharmacological effects of both EFX and BZP already appear after a single administration [START_REF] Steru | Comparing benzodiazepines using the staircase test in mice[END_REF][START_REF] Bourin | Comparison of behavioral effects after single and repeated administrations of four benzodiazepines in three mice behavioral models[END_REF][START_REF] Verleye | Investigation of the anticonvulsive effect of acute immobilization stress in anxious Balb/cByJ mice using GABA A-related mechanistic probes[END_REF][START_REF] Verleye | Effects of stress and etifoxine on pentobarbitalinduced loss of righting reflex in Balb/cByJ and C57BL/6J mice[END_REF]. Here, we determined the anxiolytic doses of EFX and their possible influences on motor performance and arousal. We then assessed the impact of α subunit isoforms on the effects of EFX on GABA-evoked currents. We characterized the pharmacological profile of EFX on murine synaptic GABAARs (α1b3γ2S, α2b3γ2S, α3b3γ2S, α4b3γ2S and α6b3γ2S) and extrasynaptic GABAARs (α5β3γ2S, α4β3δ and α6β3δ) using electrophysiology. This pharmacological study was completed with a 3D model, showing the interaction between EFX and GABAARs. Our results demonstrate that the EFX mode of action involves both α and β, but not γ or δ, subunits.

Materials and methods

Ethical statements

All animal procedures were carried out in accordance with the European Community council directive 2010/63/EU for the care and use of laboratory animals and were approved by our respective local ethical committees (N°CEEA.45 and N°CEEA.72 for mice and N°CEEA.2012.68 for Xenopus, https://www.ceea-paysdelaloire.com/) in addition to the French Ministry of Agriculture (authorization N°B49071 and N° 02200.02). The NC3R's ARRIVE guidelines were followed in the conduct and reporting of all experiments using animals.

Animal care and conditioning

Experiments were carried out using 7-to 9-week-old Balb/cByJ mice (25-30 g) purchased from Charles River Laboratory (Les Oncins, France). Ten mice per translucent polypropylene cage (internal dimensions in mm: 375 x 375 x 180, L x W x H) were housed under standard laboratory conditions (22 ± 2° C, 12-h light/dark cycle, lights on at 7:00 AM) with food (AO4, SAFE, France) and tap water available ad libitum. No less than one week of rest followed their arrival. Mice were habituated to the testing room at least 60 min before performing any behavioral evaluation. All tests occurred between 9:00 AM and 3:00 PM. The behavioral tests were performed by two well-trained experimenters, who remained unaware of the administered treatment. In addition, all equipment was wiped with 70% ethanol between animals to erase the olfactory stimuli. All experiments were performed in a randomized manner. Single administrations of EFX (12.5-150 mg/kg, expressed as hydrochloride salt) or BZP (0.25-1 mg/kg) were given by the intraperitoneal (IP) route, 30 min before each test, except in the stress-induced hyperthermia test in which the compounds were administered 60 min before the test. Studies have shown that both compounds have a similar profile with plasma peak at 15-30 min [START_REF] Verleye | The anxiolytic etifoxine activates the peripheral benzodiazepine receptor and increases the neurosteroid levels in rat brain[END_REF][START_REF] Sansone | Benzodiazepines and amphetamine on avoidance behaviour in mice[END_REF][START_REF] Rastogi | Effect of a new benzodiazepine bromazepam on locomotor performance and brain monoamine metabolism[END_REF][START_REF] Verleye | Differential effects of etifoxine on anxiety-like behaviour and convulsions in BALB/cByJ and C57BL/6J mice: any relation to overexpression of central GABAA receptor beta2 subunits?[END_REF]. The control animals received an equivalent volume of vehicle (0.9% NaCl, 1% tween 80 (v/v)). One male C57Bl/6N mouse was used for the cloning experiments. This mouse had been included in a control group (not treated) from a previous protocol in which mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France). Euthanasia was performed using CO2 (3 ml/min, 4 min). Adult female Xenopus laevis were purchased from CRB (Rennes, France) and had been bred in the laboratory in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the European Community. Oocytes were harvested from mature female Xenopus laevis frogs under 0.15% tricaine anaesthesia. All animals recovered after 2-3 h. Every female is operated every three months, not less. A single female was used no more than 5 times.

3 Compounds

EFX hydrochloride (batch 653, Biocodex, France), and BZP (batch 5788, Francochim, France) (Fig. 1) were suspended in vehicle and administered (IP) in a volume of 10 ml/kg of body weight. For electrophysiology, EFX and DZP (batch 105F0451, Sigma, France) were dissolved in dimethylsulfoxide (DMSO) resulting in a maximal concentration of 0.1% DMSO, for oocyte perfusion (control experiments were performed to demonstrate no effect of DMSO). All other reagents and solvents were obtained from Sigma-Aldrich Merck (Saint-Louis, MO, USA) or Thermo Fisher Scientific (Waltham, Massachusetts, USA).

Etifoxine (EFX)

Diazepam (DZP) Bromazepam (BZP)

Anxiety-related behavioral assessment

Stress-induced hyperthermia (SIH) is defined as the increase in body temperature observed when a subject is exposed to an external stressor [START_REF] Olivier | Stress-induced hyperthermia and anxiety: pharmacological validation[END_REF]. The day preceding the experiment, the animals were isolated in smaller cages (dimensions: 265x160x140 mm). The body temperature (± 0.1°C) of one singly housed mouse was measured twice via a rectal probe (YSI n°423; 2 mm diameter) coupled to a thermometer (Letica-Temp812 model-Italia) at an interval of 10 min. The rectal temperature measurement procedure (handling, insertion of the probe) constitutes the stressor. Drugs were injected 60 min before the first measurement (T1), followed by a second temperature measurement 10 min later (T2). Methodological experiments have shown that the optimal conditions for drug testing are found with an injection-test interval of 60 min or longer to avoid residual effects of the injection procedure. Indeed, using a 60 min-injection-stress interval results in a hyperthermic response comparable to animals that are not injected [START_REF] Zethof | Stress-induced hyperthermia in mice: a methodological study[END_REF][START_REF] Van Der Heyden | Stress-induced hyperthermia in singly housed mice[END_REF]. The reduction of SIH (∆T = T2-T1) is considered to reflect an anxiolytic-like effect [START_REF] Olivier | Stress-induced hyperthermia and anxiety: pharmacological validation[END_REF][START_REF] Zethof | Stress-induced hyperthermia in mice: a methodological study[END_REF][START_REF] Van Der Heyden | Stress-induced hyperthermia in singly housed mice[END_REF]. Defensive burying after novel object exploration is a behavior that can be elicited in rodents in response to aversive or new stimuli [START_REF] De Boer | Defensive burying in rodents: ethology, neurobiology and psychopharmacology[END_REF]. Mice were singly housed in smaller cages (see above) with a sawdust depth of 2.5 cm the day before the test. Each mouse was confronted with an unfamiliar object (4x4x6 cm; aluminium) introduced in the centre of the cage. The time of contact or exploring the object (snout pointing toward the object at a distance < 1 cm with or without burying) was recorded for 10 min. The object was cleaned with alcohol (10%) between each trial. This behavioral approach reveals an anxiety-like or fear state and its suppression is associated with a reduction of the anxiety-like behavior [START_REF] De Boer | Defensive burying in rodents: ethology, neurobiology and psychopharmacology[END_REF].

Spontaneous locomotor activity

Testing was conducted in a quiet room under a light level of approximately 400 lux. The motor activity cages (dimensions: 265x160x140 mm) were made of clear plastic and were changed between each animal; these cages contained a minimum amount of sawdust. The locomotor activity was measured by infra-red beam interruptions that were counted by a control unit (OptaVarimex, Columbus, Ohio, USA). The sensitivity of this unit was set so that walking (horizontal activity) and rearing (vertical activity) were measured. The beam breaks corresponding to spontaneous locomotor activity were measured for 15 min.

Rotarod performance

The rotarod test assesses motor performance by measuring the capacity of mice to remain on a 3-cm-diameter rod revolving 16 rpm (model 7600; Ugo Basile, Comerio, Italy). The mice were trained to walk on the rotarod until they could complete three consecutive 120 s sessions without falling off the rod. Twenty-four hours later, selected animals were treated with drugs before being challenged. The rotarod performance time was measured three times, up to 120 s, and the mean was adopted as the performance time for each animal.

GABAAR subunit cDNA expression vectors

pRK7 plasmids containing cDNAs encoding rat α2 subunits were a kind gift from Professor

Harmut Lüddens (University of Mainz, Mainz, Germany). The GABAAR α2 subunits from rat and mouse are identical in amino acid sequence. pGW1 (=pRK5) plasmids containing cDNAs encoding mouse β3 and γ2S subunits were kindly provided by Professor Steven J. Moss (Tufts University, Boston, USA). The cDNAs encoding the α3, α4, α5, α6 and d subunits used in this work were cloned in mouse brain as described below.

RNA extraction, RT-PCR and cloning of full-length cDNAs encoding α3-6 and d subunits

The brain was dissected from a male C57Bl/6N mouse for RNA extraction and purification.

Total RNA was then extracted using TRIzol® Reagent (Ozyme/Biogentex, France). First strand cDNAs were synthesised from 5 µg of total RNA using SuperScriptTM III First-Strand Synthesis System Super Mix (Invitrogen, USA) in the presence of oligo (dT)20, according to the manufacturer's instructions. cDNAs encoding α3-6 and d subunits were amplified using gene-specific primer pairs encompassing each ORF (Supplemental Table 1) and high-fidelity thermostable DNA polymerase (Advantage 2 Proofreading Polymerase kit, Clontech, Saint-Germain-en-Laye, France). cDNAs fragments were purified with the Nucleospin PCR Cleanup Kit (Macherey-Nagel, Hoerdt, Germany)

and were subsequently cloned into PCR® 4 TOPO® (Invitrogen). Each clone was sequenced twice on both strands using universal sense and reverse primers by GATC Biotech (Konstanz, Germany).

Sequence analyses were performed using BioEdit sequence analysis software. To transfer α3-6 and d subunits ORFs into the pRK5 expression vector, we adapted the ligase-free method for directional cloning [START_REF] Walker | A method for generating sticky-end PCR products which facilitates unidirectional cloning and the one-step assembly of complex DNA constructs[END_REF]. Plasmids and cDNA inserts were separately prepared by PCR using the proof reading polymerase, KOD DNA polymerase (Merck Millipore, Fontenay sous Bois, France). To generate sticky-end cDNAs, two individual PCR reactions were performed, PCR1 and PCR2, with genespecific primers containing short overhangs that allow annealing with the complementary overhangs of the plasmid. pRK5 plasmid was modified to clone the GABAAR subunit ORFs flanked at their 5' end by alfalfa mosaic virus (AMV) coat protein (RNA 4) and at their 3'end by 3ʹ -untranslated regions (UTRs) from the Xenopus β-globin gene (3UTRXBG). The combination of both UTRs has been shown to improve expression in both oocytes and mammalian cells [START_REF] Venkatachalan | Optimized expression vector for ion channel studies in Xenopus oocytes and mammalian cells using alfalfa mosaic virus[END_REF]. First, a fusion of AMV and 3'UTRXBG was constructed and cloned into pRK5 between EcoRI and XbaI. The resulting modified vector (pRK5-5AMV-3UTRXBG) was used as a template for two individual PCRs with the following pair primers: 5'-TAAACCAGCCTCAAGAACACCCGA-3' with 5'

GGTGGAAGTATTTGAAAGAAAATTAAAAATA-3' (PCR1), and 5'-AAGCTTGATCTGGTTACCACTAAACC-3' with 5'-AAAATTAAAAATAAAAACGAATTCAATCGATA-3' (PCR2). PCR1 and PCR2 products were purified and mixed in T4 ligase buffer. To generate cDNA with sticky ends, the amplicons were subjected to melting and reannealing, as previously described [START_REF] Walker | A method for generating sticky-end PCR products which facilitates unidirectional cloning and the one-step assembly of complex DNA constructs[END_REF]. Inserts containing GABAAR subunit ORFs were also prepared in two individual PCRs (LIC-PCR1 and 2) with gene-specific primer pairs (see Table S1 in supplementary data). Sticky-end inserts were obtained as described for the plasmid preparations. For each construct, sticky-ends plasmid and GABAA cDNA preparation were assembled in T4 ligase buffer and incubated for 2 h at 22°C. One to two microliters of this assemblage were used to transform chemically competent E. coli cells (DG1, Eurogentec, Seraing, Belgium). The resulting clones were sequenced as described above.

Expression of GABAARs in Xenopus oocytes

Adult female Xenopus laevis (CRB, Rennes, France) were anaesthetized in ice-cold water with 0.15% Tricaine (3-aminobenzoic acid ethyl ester, Sigma). Ovarian lobes were collected and washed in standard oocyte saline (SOS containing 100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4). Stage V-VI oocytes were partially defolliculated by enzymatic treatment with 2 mg/ml collagenase (type IA, Sigma) in Ca 2+ -free SOS for 60 min. To express functional GABAAR, cDNA mixtures were directly injected into the nucleus (animal pole) of individual defolliculated oocytes in different volumes of DNA solution at a concentration of 50 ng/µl using a nanoinjector (Drummond Nanoject) (see Table S2 for receptor stoichiometry and DNA quantity injected). Following injection, the oocytes were kept at 18°C in SOS supplemented with gentamycin (50 μg/ml), penicillin (100 UI/ml), streptomycin (100 μg/ml), and sodium pyruvate (2.5 mM). The incubation medium was replaced every two days. Oocytes were incubated 1 to 2 days after DNA injection, depending on the GABAAR subtype.

Electrophysiological recordings

Injected oocytes were tested for GABAAR expression, at a holding potential of -60 mV using a two-electrode voltage clamp amplifier (TEV-200, Dagan Corporation, Minneapolis, USA). Digidata 1440A interface (Axon CNS Molecular Devices, California, USA) and pCLAMP 10 software (Axon CNS Molecular Devices) were used for acquisition. Cells were continuously superfused with standard oocyte saline (SOS) at room temperature and were challenged with drugs in SOS. Electrodes were filled with 1 M KCl / 2 M K acetate and had typical resistances of 0.5-2 MΩ in SOS. Drugs were perfused at a flow rate close to ~4 ml/min. EFX and DZP were applied for 2 min before co-application of GABA at EC10, (determined for each GABAAR subtype, see Table S2 in supplementary data) until the current response peaked. EFX was tested at concentrations ranging from 2 to 100 μM, corresponding to its clinical use. Between the two applications, the oocytes were washed in SOS for 10-15 min to ensure full recovery from receptor desensitization (see Fig. 4A inbox). To control whether GABA-evoked currents were mediated by ternary α1-6β3γ2SGABAARs, control experiments were performed using SOS containing 10 µM Zn 2+ to inhibit binary GABAARs [START_REF] Draguhn | Functional and molecular distinction between recombinant rat GABAA receptor subtypes by Zn 2+[END_REF]. Data were analysed using pCLAMP 10 software. Data are expressed as the mean ± SEM of 6-10 oocytes generated from at least two collections. Concentration-effect relationships were analysed using the following equation: Y=Ymin + (Ymax-Ymin)/(1+10 ((LogEC50-X).nH) ), where X is the concentration of EFX, Ymin and Ymax are the minimum and highest responses, and nH is the Hill coefficient.

Molecular model design

The template chosen for homology modelling was the recently solved structure of a GABAAR (pdb code 4COF). The sequences of the human α2, β3 and γ2S GABAAR subunits were aligned with those of the template (β3) using T-Coffee software [START_REF] Notredame | T-Coffee: A novel method for fast and accurate multiple sequence alignment[END_REF]. The model was then prepared by homology modeling using Modeler version 9.5 software [START_REF] Sali | Comparative protein modelling by satisfaction of spatial restraints[END_REF] with default settings. One hundred models were prepared, and the best model, according to the Discrete Optimized Protein Energy function (DOPE), was selected. Side chains in the models were improved with Scwrl4 [START_REF] Krivov | Improved prediction of protein side-chain conformations with SCWRL4[END_REF]. The whole model was then improved with CHARMM [START_REF] Brooks | CHARMM: the biomolecular simulation program[END_REF][START_REF] Henin | A predicted binding site for cholesterol on the GABAA receptor[END_REF]. Disulfide bridges formed between neighbouring cysteines both in the 'Cys-loop' and between the M1 and M3 transmembrane helices in α and γ subunits, as recently proposed [START_REF] Henin | A predicted binding site for cholesterol on the GABAA receptor[END_REF]. The model was then subjected to minimization with decreasing harmonic potential.

Docking

The docking had been performed with AutoDock Vina [START_REF] Trott | AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[END_REF]. The ligands and proteins were prepared with prepare_ligand4.py and prepare_receptor4.py scripts, respectively. The side chains of amino-acids in the binding site were made flexible (a3: Ser257 Ser258 and β3 Gln66, Tyr87, Gln89, Tyr91). Each ligand was docked 100 times in a large cube of 30 Å in each dimension. Fig. 7 was prepared with PyMOL (DeLano W.L. (2010) The PyMOL Molecular Graphics System, version 1.6, Schrodinger, LLC, New York).

Data analysis and statistics

Data are presented as the mean ± SEM. Behavioral data were analysed by one-way ANOVA followed by Dunnett's post-hoc test for comparison with the vehicle group. In cases in which the two conditions (normality of the data and equality of variances) were not fulfilled, the non-parametric Kruskal-Wallis procedure was used, followed by the post-hoc Dunn's test to evaluate the statistical significance between the vehicle and treated groups. All test analyses were carried out by observers who were blinded to the experimental procedures. Sample sizes (number of animals in the behavioral studies) were not predetermined by a statistical method. Each behavioral experiment group included at least 10 animals and this sample size needed to detect significant effects was based on experience from previous studies. Significance tests between groups in the electrophysiological studies were performed using variance analysis (one-way ANOVA) followed by Tukey's post-hoc test for comparison of all groups or the non-parametric Mann and Whitney test when appropriate. Concerning the electrophysiological experiments, we compiled data from different batches of oocytes and we excluded data, in case of potential drift (> 0.6 mV) after pulling out the electrodes from the oocytes and when current amplitudes were <10 nA or > 2 µA. GraphPad Prism 7.02 (GraphPad Software, San Diego, USA) was used for all graphs and statistical analyses. Differences with p<0.05 were considered significant (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001).

Results

Anxiolytic effects of EFX

Previous studies have shown that EFX exhibits anxiolytic effects using conventional behavioral tests such as elevated plus maze and dark-light box tests [START_REF] Verleye | Differential effects of etifoxine on anxiety-like behaviour and convulsions in BALB/cByJ and C57BL/6J mice: any relation to overexpression of central GABAA receptor beta2 subunits?[END_REF][START_REF] Ugale | Neurosteroid allopregnanolone mediates anxiolytic effect of etifoxine in rats[END_REF]. Here, we evaluated the effect of EFX on stress and anxiety-related behaviors (stress-induced hyperthermia and novel object exploration) to determine and confirm its anxiolytic doses in comparison with BZP. In non-treated animals, handling stress resulted in a rise in body temperature close to 1°C (Fig. 2A to D). BZP significantly lowered body temperature in animals at 1 mg/kg dose, before handling (T0) revealing hypothermia (H(3)=11.343, p=0.010, then p<0.05, Dunn's test) (Fig. 2A). BZP dose-dependently prevented stress-induced hyperthermia (F 3,41 =18.290, p<0.001) (Fig. 2B). Compared to the vehicletreated animals, BZP was effective at doses of 0.5 and 1 mg/kg (p<0.05, Dunnett's test). EFX also induced changes in body temperature but without hypothermia at the highest dose (50 mg/kg) compared with control animals (F 3,41 =2.269, p=0.095) (Fig. 2C). As observed for BZP, the temperature increase was dose-dependently prevented by EFX (H(3)=20.072, p<0.001) with a significant effect at 25 and 50 mg/kg dose (p<0.05, Dunn's test) (Fig. 2D). The anxiolytic effects of BZP and EFX were also assessed by evaluating the behavioral approach in the presence of an unfamiliar object. The duration of contact with a novel object was significantly decreased in animals treated with BZP (H(3)=15.304, p=0.002) from the dose of 1 mg/kg (p<0.05, Dunn's test) (Fig. 2E).

The same was observed with EFX (H(3)=17.536, p<0.001), with a significant effect observed at doses of 25 and 50 mg/kg compared with the control animals (p<0.05, Dunn's test) (Fig. 2F). In conclusion, these two different behavioral tests led to similar anxiolytic doses of EFX (25-50 mg/kg, IP).

Motor performance assessment

We then compared the impact of EFX and BZP on locomotion performance and motor coordination (Fig. 3). As illustrated in Fig. 3, BZP (IP route) decreased spontaneous locomotor activity (H(3)=8.229, p=0.042). These effects were statistically significant for the 1 mg/kg dose (p<0.05; Dunn's test) (Fig. 3A). EFX (IP route) decreased spontaneous locomotor activity with a significant difference at 100 mg/kg (H(4)=9.633, p=0.047) compared to control animals (p<0.05, Dunn's test) (Fig. 3B). BZP reduced the time on the rotarod (H(3)=9.167, p=0.027), with a significant effect at the 1 mg/kg dose (p<0.05, Dunn's test) (Fig. 3C). EFX was devoid of any effect up to the 100 mg/kg dose and affected motor coordination at the 150 mg/kg dose compared with control animals (H(5)=19.006, p=0.002 then p<0.05, Dunn's test) (Fig. 3D). In conclusion, BZP triggers motor impairments at anxiolytic doses (1 mg/kg, IP), while EFX exhibits no locomotor effects at efficient anxiolytic doses (25 to 50 mg/kg, IP). with the respective vehicle groups (see "data analysis and statistics" in the Material and Methods section). 387

EFX effects on GABA currents depends on α subunit isoforms 388

Because the distribution of GABAAR α subunits within the CNS is heterogeneous and 389 contributes to their receptor functions, we next investigated the involvement of α subunits in the EFX 390 mode of action using electrophysiology. To achieve this goal, we compared the effects of EFX (2 to 391 100 µM) on GABA-induced currents elicited by α1GABAARs, α2GABAARs, α3GABAARs, 392 α4GABAARs, α5GABAARs or α6GABAARs, containing β3 together with γ2S or d, when appropriate.

393

We first challenged EFX effects on GABA-currents elicited by synaptic α1β3γ2S, α2β3γ2S 394 and α3β3γ2S GABAARs expressed in Xenopus oocytes (Fig. 4). We observed that EFX displays both agonist and potentiating effects as previously described [START_REF] Hamon | The modulatory effects of the anxiolytic etifoxine on GABA(A) receptors are mediated by the beta subunit[END_REF]. The agonist effects of EFX were revealed by its perfusion before co-application with GABA at EC10 (Table S2). EFX exhibits almost no agonist effects on α1β3γ2S GABAARs (12.4 ± 5.8 and 23.7 ± 10.4 % of GABA EC10 at 50 and 100 µM EFX, respectively) (Fig. 4A). In contrast, comparable agonist effects were observed with α2β3γ2S GABAARs (171 ± 24.9 % of GABA EC10, at 100 µM EFX, Fig. 4B) and α3β3γ2S GABAARs (153.6 ± 27.3% of GABA EC10, at 100 µM EFX, Fig. 4C). In comparison with GABA, EFX exerted weaker agonist effects (~100 fold less efficient). For these three receptors, EFX potentiation of GABA EC10-evoked currents was dose-dependent, reaching a plateau at 50 µM (Fig. 4). At this concentration, EFX induced a potentiation of GABA EC10-evoked currents by 68.9 ± 11.6%, 160.3 ± 40.2% and 410.7 ± 20.2% of α1β3γ2S, α2β3γ2S and α3β3γ2S, respectively. The potentiating effects induced by EFX from 2 to 100 µM, was ~2.4-6.0-fold stronger (p<0.05) with α3β3γ2S than with α1β3γ2S and α2β3γ2S GABAARs. Taking in account both agonist and potentiating effects, α1β3γ2S GABAARs are found much less sensitive to EFX than α2β3γ2S and α3β3γ2S GABAARs. Based on pharmacokinetic data [START_REF] Verleye | Effects of stress and etifoxine on pentobarbitalinduced loss of righting reflex in Balb/cByJ and C57BL/6J mice[END_REF], we estimated that 20 µM matches the concentration of free EFX in the mouse brain after injection of anxiolytic doses (25-50 mg/kg, Fig. 2). Thus, we compared agonist and potentiating effects of 20 µM EFX on α(1-3) β3γ2S GABAARs (Fig. 5A andB). EFX (20 µM) acts as a partial agonist on α2GABAARs and α3GABAARs, while these agonist effects are not significant on α1GABAARs (Fig. 5A). As for the potentiating effects, α3GABAARs

were much more sensitive to EFX (245.6 ± 41.8%) than α1 (50.1 ± 12.5%) and α2 (101.7 ± 12.2% increase) -containing GABAARs (Fig. 5B). In the synaptic cleft the GABA concentration rapidly rises up to the millimolar range [START_REF] Barberis | Impact of synaptic neurotransmitter concentration time course on the kinetics and pharmacological modulation of inhibitory synaptic currents[END_REF], we thus compared the GABA concentration-response relationships in the presence and absence of 20 µM EFX at α1GABAARs, α2GABAARs and α3GABAARs (Fig. 5C, Table 1). EFX induced a decrease of GABA EC50 with α1GABAARs and α2GABAARs in a similar extent (~4-fold). In contrast, the GABA potency on α3GABAARs was increased by 20.6 (Fig. 5C).

Taken together, our electrophysiological data show that, EFX behaves as a selective PAM of α3GABAARs at concentration equivalent to anxiolytic doses. The concentration-response relationships were analyzed using the Hill-Langmuir equation with variable slope. nH : Hill slope. The data are mean ± SEM of at least two independent experiments.

C

Next, we tested EFX on synaptic α4β3γ2S GABAARs and extrasynaptic α4β3d GABAARs. In both cases, EFX did not exhibit any agonist effects nor significantly potentiate GABA-induced currents, even at high concentrations (Fig. 6A,B). Conversely, DZP at 2 µM enhanced GABA currents elicited by α4β3γ2S GABAARs, as previously reported [START_REF] You | Benzodiazepine modulation of the rat GABAA receptor alpha4beta3gamma2L subtype expressed in Xenopus oocytes[END_REF], but it did not affect α4β3d GABAARs (Fig. 6A,B). The extrasynaptic α5β3γ2S GABAARs appeared to be also insensitive to EFX at low concentrations (2 and 20 µM), and weakly sensitive to EFX at 50 µM (54.26 ± 29.19%) (Fig. 6C andD). At 100 µM, EFX effects were significantly increased but did not reach a plateau (212.0 ± 31.97%).

The α6 subunit was expressed with γ or d in accordance with the native GABAAR composition in the cerebellum [START_REF] Nusser | Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells[END_REF]. Similar agonist effects were observed with α6β3γ2S GABAARs (76.9 ± 20.3% of GABA EC10, at 100 µM EFX,) and α6β3δ GABAARs (46.8 % of GABA EC10, at 100 µM EFX) (p=0.23, Mann and Whitney test) (Fig. 6E,F) as seen with α2GABAARs and α3GABAARs (Fig. 4B,C).

Moreover, the PAM effects of EFX revealed equal sensitivities of synaptic α6β3γ2SGABAARs and extrasynaptic α6β3δ GABAARs. EFX-potentiation of GABA-evoked currents reached a plateau at 50 µM EFX (170.5 ± 48.4% increase for α6β3γ2S GABAARs and 143.6 ± 22.3% for α6β3δ GABAARs, p>0.99, Mann and Whitney test) in accordance with a specific concentration-dependent mode of action (Fig. 6E andF). In conclusion, taking into account both agonist and potentiating EFX effects, GABAARs can be ranked in three categories: i) resistant (α4GABAARs and α5GABAARs), ii) moderately sensitive (α1GABAARs, α2GABAARs and α6GABAARs) and iii) highly sensitive (α3GABAARs) to EFX.

Modelling of EFX-α3β3γ2s GABAAR interaction

To gain further insight into the mechanism of action of EFX, we generated a homology model of α3β3γ2s GABAAR (Fig. 7A) to predict how EFX binds to its receptor site. The resulting computed docking model was consistent with an EFX binding site located between α and β subunits in the extracellular domain (Fig. 7B andC). The pocket found at the interface between α3 and β3 subunits, homologous to the GABA binding sites, was large enough to accommodate EFX. Among the putative binding modes, one was found in which EFX bound in the proximity of five amino acid residues of the β3 subunit: N66, Y87, Q89, Y91 and R194 (Fig. 7D). In α3 subunit, we identified two amino acid residues (S257, S258) that may be involved in the EFX-GABAAR interaction. Two residues of β3, N66 and R194, are conserved in β2 and β3 and are different in β1 subunits (R66 and N194). This pair of amino acids might therefore control the binding mode of EFX as variations at these residues might explain the subunit selectivity.

Discussion

The current study shows that a single administration EFX (25-50 mg/kg) induced a robust anxiolytic behavior in mice subjected to stress-induced hyperthermia and novel object exploration tests. In the same range of doses, and unlike classical BZDs, EFX did not evoke any secondary effects in the spontaneous locomotor activity and rotarod performance. Pharmacokinetic data in Balb/cByJ mice treated with anxiolytic doses indicated that EFX brain content reaches concentration range from 16 to 31 µM [START_REF] Verleye | Effects of stress and etifoxine on pentobarbitalinduced loss of righting reflex in Balb/cByJ and C57BL/6J mice[END_REF]. In addition, EFX exhibits lipophilic properties with an estimated partition coefficient (log P) of 2 and a brain/ plasma ratio range of 2.2-2.9 [START_REF] Bouillot | A microPET comparison of the effects of etifoxine and diazepam on [(11)C]flumazenil uptake in rat brains[END_REF]. Based on different reports in the literature, it is reasonable to assume that an equilibrium between the total and free fractions which depends on physicochemical properties of the compound occurs in the brain tissue [START_REF] Toutain | Free drug fraction vs free drug concentration: a matter of frequent confusion[END_REF][START_REF] Maurer | Relationship between exposure and nonspecific binding of thirty-three central nervous system drugs in mice[END_REF][START_REF] Summerfield | Central nervous system drug disposition: the relationship between in situ brain permeability and brain free fraction[END_REF] and, as a result this could support the relevance of the effective concentrations in the present electrophysiological studies. In this context, we demonstrated that the a subunit plays an important role in EFX-induced positive effects on GABAARs. EFX favours GABA potency over GABAARs with the following rank order: α3b3g2S > α2b3g2S > α6b3g2S and no or weak effects on α1b3g2S, α4b3g2S and α5b3g2S.

EFX displays anxiolytic properties with weak side effects

Our findings confirm the anxiolytic properties of EFX at similar doses to those previously used in other anxiety mouse models [START_REF] Verleye | Effects of stress and etifoxine on pentobarbitalinduced loss of righting reflex in Balb/cByJ and C57BL/6J mice[END_REF]. In comparison, EFX displays approximately 50-fold less potent anxiolytic effects than BZP and DZP [START_REF] Steru | Comparing benzodiazepines using the staircase test in mice[END_REF][START_REF] Bourin | Comparison of behavioral effects after single and repeated administrations of four benzodiazepines in three mice behavioral models[END_REF]. However, both BZP and DZP strongly alter locomotor performance and awakening at anxiolytic doses, while EFX does not. It is noteworthy that pharmacokinetic factors involving, for example, active metabolites or differences in the extent of metabolism could explain the differences in the effective doses of EFX and BZP. We reasoned that BZDs, which have a high potency (submicromolar) for enhancing GABA-evoked currents, will produce effects at a lower concentration than EFX which has a lower potency (micromolar) for GABAARs. Interestingly, EFX exhibits higher efficacy for α2GABAARs and α3GABAARs, known to mediate anxiolytic effects (up to 171% and 410% for α2GABAARs and α3GABAARs, respectively) than DZP (108% and 160% for α2GABAARs and α3GABAARs, respectively) [START_REF] Lippa | Selective anxiolysis produced by ocinaplon, a GABA(A) receptor modulator[END_REF].

Compelling evidence indicates that the anxiolytic effects of BZP cannot be dissociated from its sedative and myorelaxant effects, while the therapeutic margin is wider with EFX. In addition, previous results have shown that EFX is devoid of amnesic effects at anxiolytic doses (50 mg/kg, IP route) in the rat [START_REF] Verleye | Effects of etifoxine on stress-induced hyperthermia, freezing behavior and colonic motor activation in rats[END_REF]. On the other hand, BZP and DZP display amnesic activity at doses producing anxiolytic effects (from 0.25mg/kg, IP) in the mouse [START_REF] Porsolt | Antagonism by exifone, a new cognitive enhancing agent, of the amnesias induced by four benzodiazepines in mice[END_REF]. As observed in rodents, patients treated with EFX for adjustment disorders with anxiety do not exhibit adverse effects, such as the memory and vigilance disturbances [START_REF] Nguyen | Efficacy of etifoxine compared to lorazepam monotherapy in the treatment of patients with adjustment disorders with anxiety: a double-blind controlled study in general practice[END_REF][START_REF] Micallef | A double blind parallel group placebo controlled comparison of sedative and mnesic effects of etifoxine and lorazepam in healthy subjects [corrected[END_REF][START_REF] Deplanque | Etifoxine impairs neither alertness nor cognitive functions of the elderly: A randomized, double-blind, placebo-controlled crossover study[END_REF]. This is perfectly in line with our electrophysiological data showing the absence of effects of EFX on a4GABAARs and a5GABAARs, known to be involved in cognitive functions [START_REF] Engin | An Emerging Circuit Pharmacology of GABAA Receptors[END_REF].

We cannot rule out the possibility that EFX anxiolytic properties rely on both direct and indirect GABAAR stimulation mechanisms. Since EFX has been shown to stimulate the synthesis of neurosteroids, such as allopregnenolone, which directly boosts GABAAR activity [START_REF] Liere | Differential effects of the 18-kDa translocator protein (TSPO) ligand etifoxine on steroidogenesis in rat brain, plasma and steroidogenic glands: Pharmacodynamic studies[END_REF][START_REF] Krivov | Improved prediction of protein side-chain conformations with SCWRL4[END_REF][START_REF] Wolf | Enhancing neurosteroid synthesis--relationship to the pharmacology of translocator protein (18 kDa) (TSPO) ligands and benzodiazepines[END_REF], this may account for its anxiolytic effects. Neurosteroids equally enhance GABA-evoked currents mediated by a1GABAARs, a3GABAARs and a6GABAARs, while they have little effects on α2GABAARs, α4GABAARs and α5GABAARs [START_REF] Porsolt | Antagonism by exifone, a new cognitive enhancing agent, of the amnesias induced by four benzodiazepines in mice[END_REF][START_REF] Belelli | The influence of subunit composition on the interaction of neurosteroids with GABA(A) receptors[END_REF], suggesting that EFX should induce both sedation and anxiolysis. However, because EFX did not induce sedation at anxiolytic doses, this minors the involvement of neurosteroids in the EFX mode of action. We hypothesize that EFX may exert its anxiolytic effect through a direct enhancement of the activity of GABAARs. To date, there is no experimental data on EFX modulation of GABAARs to conclude a plausible mode of action. However, using recombinant murine GABAARs expressed in Xenopus oocytes, it has been shown that both efficacy and potency of GABA are enhanced by EFX [START_REF] Hamon | The modulatory effects of the anxiolytic etifoxine on GABA(A) receptors are mediated by the beta subunit[END_REF]. The effect of EFX might be explained by either mechanism, i.e. an increased frequency of the open state of GABAARs and/or an increase of the duration of burst openings.

Further studies using a chronic treatment are warranted to support the specificity of EFX compared to BZDs in the development of tolerance complex phenomenom involving in part selective alterations in GABAAR receptor subunit expression [START_REF] Gravielle | Activation-induced regulation of GABAA receptors: Is there a link with the molecular basis of benzodiazepine tolerance?[END_REF].

The EFX mode of action depends on the GABAAR α subunits

Our electrophysiological data demonstrate that EFX behaves both as a partial agonist and a PAM of GABAARs. In fact, EFX strongly potentiates GABA-evoked currents mediated by α2GABAARs, α3GABAARs and/or α6GABAARs, with major effects on α3GABAARs in comparison to any other GABAARs. This was also highlighted by a larger enhancement of the GABA potency on α3GABAARs, than on α1GABAARs and α2GABAARs. However, the involvement of α3GABAARs in anxiolysis is still a matter of debate. As mentioned above, there is still a controversy concerning the implication of α2GABAARs, α3GABAARs and α5GABAARs in the control of anxiety-related behaviors [START_REF] Dias | Evidence for a significant role of alpha 3-containing GABAA receptors in mediating the anxiolytic effects of benzodiazepines[END_REF][START_REF] Atack | GABAA receptor alpha2/alpha3 subtype-selective modulators as potential nonsedating anxiolytics[END_REF][START_REF] Behlke | A Pharmacogenetic 'Restriction-of-Function' Approach Reveals Evidence for Anxiolytic-Like Actions Mediated by alpha5-Containing GABAA Receptors in Mice[END_REF][START_REF] Atack | (1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3ylmethoxy)-3-(2-fluorophenyl )-1,2,4-triazolo[4,3-b]pyridazine], an agonist selective for alpha2and alpha3-containing GABAA receptors, is a nonsedating anxiolytic in rodents and primates[END_REF]. Other non-BZD compounds such as TPA023, AZD6280 and AZD7325, have been shown to exert anxiolysis without sedative side effects in rodents and/or humans by preferentially enhancing α2GABAARs and a3GABAARs over the other GABAAR subtypes [START_REF] Atack | (1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3ylmethoxy)-3-(2-fluorophenyl )-1,2,4-triazolo[4,3-b]pyridazine], an agonist selective for alpha2and alpha3-containing GABAA receptors, is a nonsedating anxiolytic in rodents and primates[END_REF][START_REF] Christian | EEG-beta/gamma spectral power elevation in rat: a translatable biomarker elicited by GABA(Aalpha2/3)-positive allosteric modulators at nonsedating anxiolytic doses[END_REF].

However, these three compounds bind to the BZD site, while EFX does not [START_REF] Schlichter | Modulation of GABAergic synaptic transmission by the non-benzodiazepine anxiolytic etifoxine[END_REF]. In addition, another non-BZD compound, TP003 was first reported as a selective PAM of α3GABAAR, and initially considered to exhibit anxiolytic properties through this receptor [START_REF] Dias | Evidence for a significant role of alpha 3-containing GABAA receptors in mediating the anxiolytic effects of benzodiazepines[END_REF][START_REF] Fischer | Contribution of GABA(A) receptors containing alpha3 subunits to the therapeutic-related and side effects of benzodiazepine-type drugs in monkeys[END_REF]. However, two recent studies have revealed that this drug is not selective to α3GABAAR, but equally modulates GABA-evoked currents mediated by α5GABAAR and also moderately potentiates α1GABAARs and α2GABAAR [START_REF] Christian | EEG-beta/gamma spectral power elevation in rat: a translatable biomarker elicited by GABA(Aalpha2/3)-positive allosteric modulators at nonsedating anxiolytic doses[END_REF][START_REF] De Lucas | GABAA alpha5 subunit-containing receptors do not contribute to reversal of inflammatory-induced spinal sensitization as indicated by the unique selectivity profile of the GABAA receptor allosteric modulator NS16085[END_REF]. TP003 was indeed shown to counteract anxiety behaviors in both rodents and squirrel monkeys and thus highlights the medical use of α3GABAAR-selective molecules as efficient anxiolytics with no sedative secondary effects [START_REF] Dias | Evidence for a significant role of alpha 3-containing GABAA receptors in mediating the anxiolytic effects of benzodiazepines[END_REF][START_REF] Fischer | Contribution of GABA(A) receptors containing alpha3 subunits to the therapeutic-related and side effects of benzodiazepine-type drugs in monkeys[END_REF]. Therefore, we believe that the anxiolytic-like effects of EFX in mice are related to the modulation of α2GABAARs and α3GABAARs.

The EFX binding site

Our objective was to challenge the possible influence of the α subunit for at least three reasons.

First, β2 homopentamers are less sensitive to EFX than β3 homopentamers. However, when they are combined with α1 or α2 subunits, the resulting binary GABAARs display a different pharmacological profile: α1β2-3 and α2β2-3 GABAARs are equally potentiated, indicating that α1 and α2 subunits modulate EFX potentiation [START_REF] Hamon | The modulatory effects of the anxiolytic etifoxine on GABA(A) receptors are mediated by the beta subunit[END_REF]. Second, α1 and α2 subunits share a high amino acid sequence identity, while α4-6 are structurally more distant [START_REF] Simon | Analysis of the set of GABA(A) receptor genes in the human genome[END_REF] and thus could potentially have distinct pharmacological influences. Here, we bring compelling evidences demonstrating that stimulating effects of EFX are much stronger on α3β3γ2S than on α2β3γ2S, α6β3γ2S and α1β3γ2S GABAARs, while α4β3γ2S and α5β3γ2S are almost insensitive. Altogether, our findings indicate a strong regulatory effect of the α subunit on EFX mode of action.

We also examined the involvement of γ2S and d subunits in the mode of action of EFX and we observed that α4β3γ2S and α4β3d on one hand, α6β3γ2S and α6β3d GABAARs on the other hand, are equally sensitive to EFX. This reinforces the idea that, unlike BZDs [START_REF] Pritchett | Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology[END_REF], the third subunit is not involved in the EFX-GABAAR interaction.

Consecutively, we hypothesized that EFX site is likely located between the α and β subunits.

Our 3D docking simulation suggests that EFX binds in a pocket at the α/β subunit interface homologous to the GABA binding pocket recently described [START_REF] Zhu | Structure of a human synaptic GABAA receptor[END_REF], highlighting putative amino acid residues involved in EFX binding. Interestingly, among them, two residues of β3, N66 and R194, are conserved in β2 and β3 and differ in the β1 subunits (R66 and N194). This pair of amino acids may belong to the binding site of EFX and summarise its subunit selectivity. Site-directed mutagenesis experiments are required to validate this hypothesis and to define the residues underlying the EFX selectivity towards α3GABAARs. These experiments will allow us to construct genetic models in which specific α(1-6)GABAARs subtypes will be rendered insensitive to EFX to directly correlate specific α2GABAARs or α3GABAARs to its anxiolytic effects or test whether α3GABAAR functions are involved in the regulation of anxiety.

Conclusions

In conclusion, this study provides new information about the mode of action of EFX, a non-BZD anxiolytic compound, showing that it potentiates GABA transmission, mainly through the interaction with α2GABAARs and α3GABAARs and likely their associated functions. Modelling simulation indicates that EFX could interact with a pocket localized at the α/β subunits interface,homologous to the GABA binding site. To the best of our knowledge, EFX belongs to the group of non-BZD molecules which act at a site distinct from the classical BZDs site and exert positive effects on anxiety without secondary effects. EFX may therefore serve as a molecular template for the design of novel anxiolytics with similar mechanisms of action and higher potency.

Fig. 1 .

 1 Fig. 1. Structure of the positive allosteric modulators of GABAARs used in this study. The 2D structure of EFX (PubChem CID: 171544), DZP (PubChem CID: 3016) and BZP (PubChem CID: 2441) are illustrated.

Fig. 2 .**T*

 2 Fig. 2. Comparison of the effects of EFX and BZP on anxiety-related behaviors in mice. (A-D) The graphs show the evolution of the mean rectal temperature (± SEM) at T0 and T0+10 min after treatment with vehicle (dose 0) or BZP (A) or EFX (C) through IP route, 60 min before the first temperature measurement at T0. Histograms represent the mean (± SEM) of the difference of the rectal temperature measured at T0 and T+10 min in the same mouse after treatment with vehicle or BZP (B) or EFX (D) at the indicated doses. *p<0.05 compared with the vehicle group (Dunn test). (E,F) Histograms illustrate the mean time (± SEM) of contact with an unfamiliar object after IP administration of BZP (E) or EFX (F). *p<0.05 compared with the respective vehicle groups (dose 0). (see "data analysis and statistics" in the Material and Methods section). Animal numbers are indicated inside the bars.

382Fig. 3 .

 3 Comparison of BZP and EFX on locomotor activity and motor coordination. (A, B) Spontaneous locomotor 383 activities were assayed in the actimeter test in mice after IP injections of BZP (A) or EFX (B). The bars represent the 384 means ± SEM of the number of infrared beam interruptions over15 min. (C,D) Histograms illustrate the time on the rod 385 (mean ± SEM) after injection of BZP (C) or EFX (D). Animal numbers are indicated inside the bars. *p <0.05 compared 386

10 L o c o m o to r a c t iv it y ( c o u n t s / 1

 101 o m o to r a c t iv it y ( c o u n t s / 1

Fig. 4 .

 4 Fig.4. Effects of EFX on GABA-activated currents mediated by three synaptic GABAARs (a1β3γ2S, a2β3γ2S and a3β3γ2S). EFX effects were investigated by TEVC in Xenopus oocytes expressing a1β3γ2S (A), a2β3γ2S (B) and a3β3γ2S GABAARs (C). (A-C) Increasing concentrations of EFX (2, 20, 50 and 100 µM) were applied 2 min before coapplication of GABA at EC10 (inbox). The amplitudes of EFX-evoked currents (• ) were normalized to the amplitude of control currents (n) obtained with GABA alone at EC10. The potentiation effects of EFX was determined as the percentage increase of EC10-GABA current amplitudes (• ). Left panel, GABA EC10-induced representative currents are illustrated, showing the partial agonist and positive modulatory effects of EFX. Right panel, data points (mean ± SEM of 6-11 oocytes from at least two different animals) were fitted by non-linear regression to the Hill equation with variable slope using GraphPad Prism 7. Statistical analyses were performed using one-way ANOVA tests followed by Tukey's post-hoc correction (comparison with data obtained at 2 µM, *p<0.05, **p <0.01; ***p <0.001; ****p <0.0001, ns: not significant).

  n t ia t io n ( % , ) E F Xe v o k e d c u r r e n t s ( n t ia t io n ( % , ) E F Xe v o k e d c u r r e n t s ( % , ) n t ia t io n ( % , ) E F Xe v o k e d c u r r e n t s (

Fig. 5 .

 5 Fig. 5. Pharmacological profile of EFX over a(1-3)b3g2S GABAARs. Comparison of EFX effects at 20 µM corresponding to anxiolytic doses. Detailed analysis of agonist (A) and potentiating (B) effects of 20 µM EFX on a1b3g2S, a2b3g2S and a3b3g2S GABAARs. One-way ANOVA followed by Tukey post-hoc test was used for the analysis ((*p<0.05, **p <0.01; ***p <0.001; ****p <0.0001). The number of recorded oocytes is indicated above or inside the bars. (C) Concentration-response curves of GABA-evoked currents in the absence (open circles) and presence (close circles) of 20 µM EFX. Statistical analyses were performed using a non-parametric Mann & Whitney unpaired t-test. All data are expressed as the mean ± SEM (n ≥ 6).

  n is t e ff e c t ( 2 0 µ M E F X , % )

Fig. 6 .

 6 Fig. 6. Effects of EFX on GABA EC10-activated currents mediated by synaptic (a4β3γ2S and a6 β3γ2S) and extrasynaptic (a4β3δ, a5β3γ2S and a6β3δ) GABAARs. (A) Superimposed current traces evoked by GABA EC10 in a representative cell expressing a4β3γ2S or a4β3δ GABAARs in the absence (black traces) or presence of DZP (2 µM) or EFX. (B) Graphs illustrating the mean (± SEM) EFX potentiation of GABA EC10-activated currents mediated by synaptic and extrasynaptic a4GABAARs. (C) Current traces evoked by GABA EC10 in a representative oocyte expressing extrasynaptic a5β3γ2S GABAARs in the absence (black trace) or presence of EFX (2 to 100 µM). (D) Graphs showing the mean (± SEM) EFX potentiation of GABA EC10-evoked currents. Statistical analyses were performed by one-way ANOVA followed by Tukey post-hoc test (*p<0.05, **p <0.01; ***p <0.001; ****p <0.0001, ns: not significant). (E) Current traces evoked by GABA EC10 with synaptic a6β3γ2S and extrasynaptic a6β3δ GABAARs in the absence (black traces) or presence of increasing concentrations of EFX (2 to 100 µM). (F) Graphs showing the mean (± SEM) EFX potentiation of GABA EC10-activated currents mediated by a6β3γ2S and a6β3δ GABAARs. Statistical analyses were performed using a non-parametric Mann & Whitney unpaired t-test (* p<0.05; ns: not significant).

  e v o k e d c u r r e n t s ( % , _ ) [E F X ] ( µ M ) P o t e n tia t io n ( % , e n t ia t io n ( % , ) E F Xe v o k e d c u r r e n t s (

Fig. 7 .

 7 Fig. 7. Binding modes of EFX obtained by docking on the mouse α 3 β 3 γ 2S GABA A R. (A) Model of the receptor viewed from the membrane plane. The protein is shown in cartoon representation with a different color code for each polypeptide. The position of the membrane is represented by a sphere positioned at the level of lipid head groups as determined by the Orientations of Proteins in Membranes database [51]. (B-D) Binding mode of EFX by docking on the mouse α 3 β 3 γ 2S GABA A R model. EFX (CPK representation) interacts with a pocket localized at the α 3 (cyan) β 3 (magenta) interface, homologous to GABA binding sites. The binding sites of BZD, GABA and EFX are indicated by arrows (B). Lateral view of the extracellular domain (C). Close-up showing the EFX-binding pocket (EFX appears in sticks) (D).

Table 1

 1 Parameters of the GABA concentration-response relationship at a1b3g2S, a2b3g2S and a3b3g2SGABAARs modulated by 20 µM EFX.

		control		+ 20 µM EFX
	GABAAR subtype	EC50 (µM)	nH	EC50 (µM)	nH
	a1b3g2S	9.99 ± 0.96	1.06 ± 0.09	2.37 ± 0.11	1.41 ± 0.08
	a2b3g2S	1.31 ± 0.12	1.69 ± 0.28	0.32 ± 0.03	1.14 ± 0.01
	a3b3g2S	10.89 ± 0.61	1.34 ± 0.09	0.53 ± 0.08	1.05 ± 0.15

Acknowledgements

We are indebted to Dr. Alain Hamon and Bastien Faucard for helpful discussions about the work. We are grateful to Sophie Quinchard for technical assistance. We also thank Dr. Ulrich Jarry, Charlène Labège, Céline Alamichel and Vincent Juif for their contribution to preparing the cDNAs encoding α3-6 subunits in the pRK5 vector and electrophysiology experiments.

Conflict of interest

The authors declare no conflicts of interest.