Raoul Torero Ibad

Bilal Mazhar

Clémence Clémentine Vincent

Françoise Bernard

Julie Dégardin

Manuel Simonutti

Thomas Lamonerie

Ariel A Di Nardo

Alain Prochiantz

Kenneth L Moya
email: ken.moya@college-de-france.fr.

Clémence Bernard

Dr Mélanie Leboeuf

Anne-Cécile Boulay

OTX2 Non-Cell Autonomous Activity Regulates Inner Retinal Function

Keywords: homeoprotein non-cell autonomous, physiology retina, vision Significance

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

OTX2 is a homeoprotein transcription factor important for retinal development and maintenance. It is expressed early in the embryonic mouse optic vesicle and retinal pigmented epithelium (RPE) and is required for differentiation of photoreceptors by transactivation of Crx and Nrl and for differentiation of bipolar cells via regulation of PKCa [START_REF] Martinez-Morales | Otx genes are required for tissue specification in the developing eye[END_REF][START_REF] Nishida | Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development[END_REF][START_REF] Koike | Functional roles of Otx2 transcription factor in postnatal mouse retinal development[END_REF][START_REF] Muranishi | An essential role for RAX homeoprotein and NOTCH-HES signaling in Otx2 expression in embryonic retinal photoreceptor cell fate determination[END_REF]. In mice hypomorph for Otx2, visual deficits, retinal physiological dysfunction and age-and OTX2-dependent degeneration of the retina have been reported [START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF]. In adult mice, knockdown of Otx2 in RPE results in photoreceptor death, demonstrating that continued expression of OTX2 in RPE is necessary for photoreceptor survival [START_REF] Housset | Loss of Otx2 in the adult retina disrupts retinal pigment epithelium function, causing photoreceptor degeneration[END_REF]. Exogenous OTX2 protects adult retinal ganglion cells (RGCs) against NMDA-induced excitotoxicity and preserves visual acuity [START_REF] Ibad | Otx2 promotes the survival of damaged adult retinal ganglion cells and protects against excitotoxic loss of visual acuity in vivo[END_REF].

The capacity of homeoproteins (HPs) to transfer between cells allows different types of activities. Homeoproteins can act within the cells that produce them, thus in a cell autonomous fashion, but they can also exert their activity extracellularly or by transferring to cells that do or do not produce them, i.e., non-cell autonomously. Two separate sequences necessary and sufficient for HP cell exit and entry are in the DNA-binding homeodomain (for review, see Di [START_REF] Nardo | The physiology of homeoprotein transduction[END_REF]. A simple genetic approach cannot therefore be used to study their direct non-cell autonomous activity, as mutation of either sequence alters OTX2 DNA binding and thus also alters cell autonomous activities. An alternative genetic approach was used to specifically target extracellular OTX2 to only abolish non-cell autonomous activity. Conditional mice have been designed to express a neutralizing secreted anti-OTX2 single chain antibody (scFvOTX2 tg/o mice) in a Cre-dependent manner [START_REF] Bernard | A mouse model for conditional secretion of specific single-chain antibodies provides genetic evidence for regulation of cortical plasticity by a non-cell autonomous homeoprotein transcription factor[END_REF]. In the retina parvalbumin (PV) is only expressed by RGCs and amacrine cells that do not express Otx2. Thus it is anticipated that in PV Cre ::scFvOTX2 tg/o mice, OTX2-scFv expressed and secreted from RGCs and amacrine cells will sequester extracellular OTX2 in the vicinity of the producing cells, thus blocking its non-cell autonomous activities. This strategy based on anti-HP scFv in vivo secretion has been used with success in several animal models to neutralize extracellular PAX6, ENGRAILED, and OTX2 [START_REF] Lesaffre | Direct non-cell autonopous Pax6 activity regulates eye development in the zebrafish[END_REF][START_REF] Wizenmann | Extracellular Engrailed participates in the topographic guidance of retinal axons in vivo[END_REF][START_REF] Layalle | Engrailed homeoprotein acts as a signaling molecule in the developing fly[END_REF][START_REF] Bernard | A mouse model for conditional secretion of specific single-chain antibodies provides genetic evidence for regulation of cortical plasticity by a non-cell autonomous homeoprotein transcription factor[END_REF].

We show here that the sequestration of extracellular OTX2 by the OTX2-scFv secreted by RGCs and amacrine cells leads to a significant decrease in visual acuity. This decrease takes place in absence of any observable developmental defects, laminar abnormalities or changes in cell lineages. Electroretinogram (ERG) measurements show normal outer and inner nuclear function but show a twofold increase in amplitude in the response to 20 Hz flickers. Together, our results provide evidence for a direct non-cell autonomous activity of OTX2 for RGC function.

Materials and Methods

Production of transgenic mice. scFvOTX2 tg/o and scFvPAX6 tg/o mice were produced by the Institut Clinique de la Souris (Strasbourg, France) as described previously [START_REF] Bernard | A mouse model for conditional secretion of specific single-chain antibodies provides genetic evidence for regulation of cortical plasticity by a non-cell autonomous homeoprotein transcription factor[END_REF]. The mice were crossed with PV Cre mice obtained from The Jackson Laboratory (stock #8069). Mice were used without regard to sex, and males and females were used in all experiments. All animal experiments were conducted in accordance with European Directive number 86/609 (EEC Council for Animal Protection in Experimental Research and Other Scientific Utilization) and French authorization n°00702.01, "Viellissement, dégénération et régénération du système nerveux central adulte chez la souris," delivered by the French Ministère de l'Enseignement Supérieur et de la Recherche.

Immunoprecipitation and Western blot. Immunoprecipitation and Western blotting were conducted as described previously [START_REF] Bernard | A mouse model for conditional secretion of specific single-chain antibodies provides genetic evidence for regulation of cortical plasticity by a non-cell autonomous homeoprotein transcription factor[END_REF]. Retinas were dissected and suspended in immunoprecipitation lysis buffer (20 mM Tris pH 8, 120 mM NaCl, 1% NP-40, 1 mM MgCl 2 , 5% glycerol, Benzonase Nuclease, and protease inhibitors). Samples were centrifuged (10 min, 20,000 Â g) at 4°C, and the supernatants incubated overnight at 4°C, with rotation with anti-GFP-coupled magnetic beads (Chromotek). The beads were washed with lysis buffer and 1 M urea before Western blot analysis. Protein extracts were separated on an Invitrogen NuPAGE 4-12% Bis-Tris precast gel (Thermo Fisher Scientific) for 1 h at 200 V and transferred onto a methanol-activated PVDF membrane at 400 mA for 1 h. The membrane was blotted with an anti-MYC antibody (rabbit polyclonal, 1/4000; catalog #C3956, Sigma-Aldrich) and imaged with a LAS-4000 gel imager (Fujifilm).

In situ hybridization for OTX2scFV expression. In situ hybridization (ISH) was performed on retinal sections using RNAscope technology. Eyes were removed and fixed overnight at 4°C in buffered 4% paraformaldehyde (PFA) and cryoprotected in 20% sucrose overnight at 4°C. Forty micrometer cryostat sections were collected on Superfrost slides, dried, and stored at À20°C. On the day of processing, they were thawed to room temperature for 15 min before performing ISH according to the protocol of the manufacturer (Advanced Cell Diagnostics). Briefly, endogenous peroxidase was neutralized with H 2 O 2 , permeabilized with buffered Tween 20 using reagents provided by the manufacturer. Probes designed by Advanced Cell Diagnostics were hybridized for 2 h at 40°C, and the signal amplified in three steps was visualized either with Red (570 nm) or Far Red (690 nm). The probes PV (Mm-PV-C2) and Myc (Mm-MYC-C1) were used at 1:50 dilution.

Visual acuity. The optomotor test of visual acuity was used to screen for possible phenotype differences. Postnatal day 30 (P30) mice were used since it was thought that this age would allow sufficient time for scFv expression and secretion. The optomotor test was performed as described in the literature with an optotype of 0.375c/deg, which is known to elicit robust responses [START_REF] Ibad | Otx2 promotes the survival of damaged adult retinal ganglion cells and protects against excitotoxic loss of visual acuity in vivo[END_REF][START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF]. Briefly, mice were placed on an elevated platform centered in a rotating drum, and a square-wave 100% contrast optotype of 0.375c/deg was rotated at a speed of 2 rpm. Mice were filmed from above and scored in real time by two observers blind to genotype for head turns in the direction and speed of the moving grating. Any discrepancies between the two observers' real-time score was resolved by analyzing the video. A Mann-Whitney U test was used to compare the genotypes because of unequal variances.

Retinal histology. Eyes were removed and placed in 4% PFA, 2% ZnCl, and 20% isopropyl alcohol [START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF]. Five micrometer paraffin sections were cut in the vicinity of the optic nerve and stained for hematoxylin and eosin (Excalibur Pathology, Inc.). Three sections per retina were analyzed per animal. Total number of cells in the entire ganglion cell layer (GCL; i.e., RGCs and displaced amacrine cells) were counted for each of three sections that included the optic nerve head and averaged. The thickness of the inner nuclear layer (INL) and outer nuclear layer (ONL) proximal to the optic nerve head was measured on the three sections per animal and averaged.

Quantitative PCR for OTX2scFv expression and retinal cell types. RNA was extracted from frozen retinas of P30 mice with RNeasy Lipid Tissue Mini Kit (Qiagen). cDNA was generated with the Quantitect Reverse transcription kit (Qiagen). Using the 2 -DDCt method, sample expression levels were normalized to hprt and to PV Cre mice levels. Primers for the following cell types were used: Otx2-scFv (transgene expression), Brn3A (ganglion cells), Chx10 (bipolar cells), Syntaxin (amacrine and horizontal cells), Lim1 (horizontal cells), Rhodopsin (rods), and Red Opsin (L/M cone cells). Since the data are normalized to the value for PVCre mice, the nonparametric Mann-Whitney U test was used to evaluate the difference for Lim1 expression.

Electroretinogram. Mice were anesthetized with xylazine/ketamine (100 mg/kg, Imalgene 500, Virbac; 10 mg/ kg, Rompun 2%, Bayer). Tropicamide (Mydriaticum 0.5%, Théa) was used to dilate the pupil, and the cornea was locally anesthetized with oxybuprocaine hydrochloride. Gold electrodes were placed in contact with each eye, reference electrodes were placed subcutaneously in the submandibular area, and a ground electrode was placed subcutaneously on the back of the animal. ERG was performed with a mobile apparatus (SIEM Bio-Medical) with LED lamps in a Ganzfeld chamber controlled by the VisioSystem software. ERG recordings were obtained from seven PV Cre and nine PV-Cre::OTX2-scFv mice 30-31 d of age. The right and left eye of each mouse was considered to be independent. A Mann-Whitney U test was used to evaluate the difference between genotypes for 20 Hz flickers and oscillatory potential.

Results

In the retina, PV is expressed in RGCs and amacrine cells [START_REF] Haverkamp | Immunocytochemical analysis of the mouse retina[END_REF][START_REF] Haverkamp | Expression analysis of green fluorescent protein in retina neurons of four transgenic mouse lines[END_REF]. As shown in Figure 1A, quantitative real-time PCR (qRT-PCR) shows significant expression of the OTX2-scFv transgene in the retinas of PV Cre ::scFvOTX2 tg/o mice and no detectable expression in the retinas of PV Cre mice (raw ct PVCre = 38.2 6 1.7; PV::Cre;scFvOTX2 tg/o = 28.9 6 0.1). Because the antibody is fused with GFP [START_REF] Bernard | A mouse model for conditional secretion of specific single-chain antibodies provides genetic evidence for regulation of cortical plasticity by a non-cell autonomous homeoprotein transcription factor[END_REF], we immunoprecipitated the OTX2-scFv protein from PV Cre ::scFvOTX2 tg/o retina using an anti-GFP antibody followed by Western blot analysis. We detected a doublet at the expected 78 kDa molecular weight, that was not present in extracts from retina of PV Cre control mice (Fig. 1B, arrow). In situ hybridization (Fig. 1C) revealed PV-expressing cells in the inner retina in the reported location of PV-containing cells [START_REF] Haverkamp | Immunocytochemical analysis of the mouse retina[END_REF]. In PV Cre ::scFvOTX2 tg/o retinas, each PVexpressing cell also expressed mRNA for the scFv in PVexpressing cells, as expected (Fig. 1D). This demonstrates the expression of the transgene in PV cells and the presence of full-length OTX2-scFv protein in the retinas of PV Cre ::scFvOTX2 tg/o mice.

One-month-old mice were tested for visual acuity by examining their optokinetic response to a 100% contrast optotype square wave grating of 0.375c/deg spatial frequency. [START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF] reported that this spatial frequency was most robust in revealing differences in mice hypomorph for OTX2 activity, and the number of head movements in response to this optotype is sensitive to changes in visual acuity because of loss of RGCs (Torero [START_REF] Ibad | Otx2 promotes the survival of damaged adult retinal ganglion cells and protects against excitotoxic loss of visual acuity in vivo[END_REF]. A significant reduction in visual performance in the PV Cre ::scFvOTX2 tg/o mice was observed compared with PV Cre littermates (Fig. 2, Table 1). As an additional control, we also evaluated PV Cre ::scFvPAX6 tg/o mice. PAX6 homeoprotein is reported to also have noncell autonomous activities [START_REF] Lullo | Paracrine Pax6 activity regulates oligodendrocyte precursor cell migration in the chick embryonic neural tube[END_REF][START_REF] Kaddour | Extracellular Pax6 regulates tangential Cajal-Retzius ell pigration in the developing mouse neocortex[END_REF], and these mice showed no visual acuity loss. These results demonstrate that neutralizing extracellular OTX2 in the retina leads to decreased visual acuity at P30, but the present results do not allow us to determine whether this persists at later ages.

In search of a cellular correlate, we analyzed retinal structure by hematoxylin and eosin staining of sections from 1-month-old mice. There was no difference in thickness of the ONL (photoreceptors) or of the INL (horizontal, bipolar, amacrines, and Müller glial cells) between PV Cre :: scFvOTX2 tg/o and PV Cre littermates (Fig. 3). Nor could we find a difference in the number of cells in the GCL containing RGCs and displaced amacrine cells. These results thus show no gross structural abnormalities in the retinas of PV Cre ::scFvOTX2 tg/o mice or obvious differences in the number of cells in the ONL, INL, or GCL.

Quantitative RT-PCR was reported to faithfully reflect retinal cell number (Torero [START_REF] Ibad | Otx2 promotes the survival of damaged adult retinal ganglion cells and protects against excitotoxic loss of visual acuity in vivo[END_REF]. We observed no differences between P30 PV Cre ::scFvOTX2 tg/o and PV Cre littermates for the expression of mRNAs for Brn3A (RGCs), syntaxin (amacrine and horizontal cells), Chx10 (bipolar cells), rhodopsin (rod photoreceptors), and red/ green opsin (long/medium-wavelength cone photoreceptors; Fig. 4). A small increase in Lim1 expression by horizontal cells was observed that might reflect a non-cell autonomous repressor activity of OTX2 on Lim1 expression [START_REF] Nakano | OTX2 directly interacts with LIM1 and HNF-3b[END_REF][START_REF] Puelles | Otx2 controls identity and fate of glutamatergic progenitors of the thalamus by repressing GABAergic differentiation[END_REF]. Thus, based on these markers, histologic studies, and cell counting, it can be concluded that all cell types involved in retinal visual processing are present in normal numbers.

To determine whether there was a functional defect in photoreceptors and/or bipolar cells, we turned to ERG. The negative a-wave reflects photoreceptor hyperpolarization in response to light. The amplitude is related to photoreceptor number and the implicit time to photoreceptor physiology [START_REF] Hood | A computational model of the amplitude and implicit time of the b-wave of the human ERG[END_REF][START_REF] Lamb | Dark adaptation and the retinoid cycle of vision[END_REF]; for review, see [START_REF] Weymouth | Rodent electroretinography: methods for extraction and interpretation of rod and cone responses[END_REF]. Figure 5, A and B, shows representative scotopic traces for retinas from PV Cre :: scFvOTX2 tg/o and PV Cre mice. The a-wave and b-wave implicit times and amplitudes were plotted against log intensity (Fig. 5C-F). Two-way ANOVA with repeated measures revealed no significant differences between the two genotypes (a-wave latency, F (1,14) = 0.011; a-wave amplitude, F (1,14) = 0.100; b-wave latency, F (1,14) = 3.826; b-wave amplitude F (1,14) = 0.77, all nonsignificant) or for genotype  stimulus intensity interaction (a-wave latency, F (3,42) = 0.660; awave amplitude, F (3,42) = 0.913; b-wave latency, F (3,41) = 1.915; b-wave amplitude, F (3,42) = 0.139; all nonsignificant). The absence of a difference in a-wave amplitude or implicit time in the PV Cre ::scFvOTX2 tg/o mice compared with PV Cre littermates indicates normal number and function of rod photoreceptors (Fig. 5; also see Fig. 7). The b-wave is mainly driven by bipolar activity, and the amplitude reflects the number and the implicit time bipolar function. Both were indistinguishable between PV Cre ::scFvOTX2 tg/o mice and PV Cre littermates, indicating the normal number and response of bipolar cells. The b-wave latency and amplitude under photopic conditions was similar in PV Cre ::scFvOTX2 tg/o mice and PV Cre littermates (Fig. 6). Interestingly, we observed a significant decrease in the amplitude in oscillatory potential (OP) 4 in PV Cre :: scFvOTX2 tg/o mice (Fig. 7A,B). There were no changes in earlier or later OPs. Additionally, for the flicker response, the PV Cre ::scFvOTX2 tg/o mice had an increase of ;33% in amplitude at 10 Hz, and at 20 Hz the response significantly increased by twofold (Fig. 7C,D). Both of these results suggest inner retinal dysfunction at the level of amacrine cells and/or RGCs.

Discussion

OTX2 homeoprotein is part of the HP family of transcription factors that transfer between cells and have neuroprotective effects in the brain [START_REF] Sugiyama | Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity[END_REF][START_REF] Ibad | Otx2 promotes the survival of damaged adult retinal ganglion cells and protects against excitotoxic loss of visual acuity in vivo[END_REF][START_REF] Rekaik | Engrailed homeoprotein protects mesencephalic dopaminergic neurons from oxidative stress[END_REF] for review, see Di [START_REF] Nardo | The physiology of homeoprotein transduction[END_REF]. We were interested in assessing the importance of the direct non-cell autonomous activity of OTX2 in the retina since the Otx2 locus is silent in cells of the GCL, yet they contain OTX2 protein [START_REF] Rath | Ontogenetic expression of the Otx2 and Crx homeobox genes in the retina of the rat[END_REF][START_REF] Sugiyama | Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity[END_REF]. Since the amino acid sequences for OTX2 to exit and enter cells lie in the DNA binding homeodomain, mutations in these sequences might alter DNA binding and thus cell autonomous activity. [START_REF] Bernard | A mouse model for conditional secretion of specific single-chain antibodies provides genetic evidence for regulation of cortical plasticity by a non-cell autonomous homeoprotein transcription factor[END_REF] used a genetic approach to neutralize extracellular OTX2 by expressing a secreted anti-OTX2 single-chain antibody. Here, we used this approach to drive the expression of the OTX2-scFv in cells that do not themselves produce OTX2, thus eliminating a putative cell autonomous effect of the antibody. PV is reported to be in AII amacrines in rats, cats, bats, and rabbits [START_REF] Lee | AII amacrine cells in the mammalian retina show disabled-1 immunoreactivity[END_REF]). In the mouse, direct immunofluorescence showed low PV expression in amacrines and displaced amacrines, and strong PV expression in RGCs [START_REF] Haverkamp | Immunocytochemical analysis of the mouse retina[END_REF]. A more sensitive transgenic approach using the PV promoter to drive expression of enhanced GFP [START_REF] Haverkamp | Expression analysis of green fluorescent protein in retina neurons of four transgenic mouse lines[END_REF] confirmed this expression pattern and showed that PV was expressed in some amacrines in the INL with notably strong expression in RGCs [START_REF] Haverkamp | Expression analysis of green fluorescent protein in retina neurons of four transgenic mouse lines[END_REF]. [START_REF] Kim | Morphological classification or parvalbumincontaining retinal ganglion cells in mouse: single cell injection after immunocytochemistry[END_REF] and later [START_REF] Yi | Types of parvalbumin-containing retinotectal ganglion cells in mouse[END_REF], using single-cell iontophoresis and retrograde tracing followed by PV localization, showed that at least eight different RGC types express PV. Thus, we anticipated widespread expression of the scFv throughout the GCL and inner INL. It is interesting to note that the ISH results showed scFv expression in fewer cells than expected, but it was sufficient to neutralize extracellular OTX2 and cause functional and vision changes.

Parvalbumin starts to be expressed in RGCs at approximately P11, after the birth of all retinal cell types [START_REF] Young | Cell proliferation during postnatal development of the retina in the mouse[END_REF][START_REF] Cepko | Cell fate determination in the vertebrate retina[END_REF]. Thus, it is expected that interference with OTX2 non-cell autonomous activity starting at the earliest at P11 would have no effect in establishing the normal layers and structure of the retina, as we observe. It was reported that in Otx2 hypomorph mice, in which both cell autonomous and non-cell autonomous phenotypes can be expected, there were fewer photoreceptors at P30, but those that were present have normal a-wave implicit time [START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF]. In PV Cre ::scFvOTX2 tg/o mice, the presence of normal numbers of OTX2-expressing photoreceptors and bipolar cells and their normal ERG a-wave and b-wave activity, respectively, confirms that OTX2 cell autonomous activity in these cells is not affected by expression of the secreted OTX2-scFv. It further suggests that OTX2 noncell autonomous activity on these cells does not contribute measurably to their survival and function, at least at 1 month of age.

It is notable that we observed no changes in retinal structure or organization in the PV Cre ::scFvOTX2 tg/o mouse retina and that both ONL and INL function, at least in terms of ERG a-and b-wave activity, was comparable to PV Cre littermates, and yet the PV Cre ::scFvOTX2 tg/o mice had a deficit in visual acuity. How might this occur? This was not simply because of the expression of an scFv since PV Cre ::scFvPAX6 tg/o mice did not show any loss of visual acuity compared with PV Cre mice. PV is expressed in other regions of the nervous system and could interfere with OTX2 non-cell autonomous activity in these regions, thus contributing to the reduction of visual acuity. The optokinetic response does not depend on visual cortex since large lesions of posterior cortex do not diminish visual acuity, indicating that the brain circuits for this response are entirely subcortical [START_REF] Douglas | Independent visual threshold measurements in the two eyes of freely moving rats and mice using a virtual-reality optokinetic system[END_REF]. The basic circuit for the optokinetic response is retinal projection to the nucleus of the optic tract that sends input to the reticular tegmental nucleus of the pons with output going to the vestibular nucleus driving the oculomotor nucleus [START_REF] Wada | Role of granule-cell transmission in memory trace of cerebellum-dependent optokinetic motor learning[END_REF]. None of these structures are known to express OTX2, and only the vestibular nucleus has PV cells. Thus, the only structure in the basic circuit that has both PV and OTX2 expression is the retina.

We propose that the loss of non-cell autonomous signaling OTX2 induces inner retinal physiological dysfunction, as suggested by the altered OP and flickers response. OPs are an indication of inner retinal function and appear to be driven mainly by rod activity [START_REF] Wachtmeister | Oscillatory potentials in the retina: what do they reveal[END_REF][START_REF] Lei | Study or Rodand Cone-driven oscillatory potentials in mice[END_REF]. OP ERG has been used to evaluate inner retina function in both human and experimental glaucoma [START_REF] Gur | Changes in the oscillatory potentials of the electroretinogram in glaucoma[END_REF][START_REF] Rangaswamy | Effect of experimental glaucoma in primates on oscillatory potentials of the slow-sequence mfERG[END_REF]. Early OPs have been attributed to GABAergic inhibitory activity in the ON pathway, while later OPs have been pharmacologically characterized as being generated by glycinergic inhibitory amacrine feedback synapses in the OFF pathway [START_REF] Wachtmeister | Oscillatory potentials in the retina: what do they reveal[END_REF]. Flickers reflect cone pathway activity [START_REF] Tanimoto | Electroretinographic assessment of rod-and conemediated bipolar cell pathways using flicker stimuli in mice[END_REF], and the cone photoreceptor function and cone bipolar function, as measured in the photopic a-wave and b-wave ERGs, is not compromised in the PV Cre ::scFvOTX2 tg/o retinas. Thus, the altered flicker responses that we observe represent a change in cone pathway activity at the level of the inner retina. Retinal ganglion cell dysfunction and degeneration in a model of glaucoma greatly alter the flicker response [START_REF] Grozdanic | Laser-induced mouse model of chronic ocular hypertension[END_REF], and the flicker ERG has been used to assess RGC function and hemodynamic changes in the mouse retina [START_REF] Chou | Adaptation of retinal ganglion cell function during flickering light in the mouse[END_REF]. The flicker response in macaques has been shown to be largely driven by spiking inner retinal neurons since it is increased at some frequencies by TTX and NMDA [START_REF] Viswanathan | Inner-retinal contributions to the photopic sinusoidal flicker electroretinogram of macaques[END_REF]. Here, we observed the same direction of change in PV Cre :: scFvOTX2 tg/o mice at 10 Hz, and significantly at 20 Hz. OPs and flickers are elicited by different stimuli. The OPs are part of the b-wave response elicited in our case by scotopic flash. The flickers are elicited by flicker stimuli in lightadapted conditions. Thus, two independent stimuli in different light adaptation conditions reveal inner retinal dysfunction when there is a decrease in OTX2 signaling in the retina.

We propose that non-cell autonomous OTX2 signaling in the inner retina is affected without loss of RGCs or amacrine cells in the GCL in the PV Cre ::scFvOTX2 tg/o mouse retina. What could be the nature of this signaling? Homeoproteins including OTX2 are capable of transferring between cells and, in addition to their transcriptional activity, can stimulate protein translation, alter chromatin remodeling, and repress transposable element expression (for review, see Blaudin de [START_REF] De Thé | Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression[END_REF][START_REF] Nardo | The physiology of homeoprotein transduction[END_REF]. OTX2 from the retina can reach inhibitory PV cells in V1 cortex and interference with its non-cell autonomous activity perturbs the normal opening and closing of the critical period for ocular dominance plasticity by altering the balance of excitation and inhibition [START_REF] Sugiyama | Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity[END_REF][START_REF] Bernard | A mouse model for conditional secretion of specific single-chain antibodies provides genetic evidence for regulation of cortical plasticity by a non-cell autonomous homeoprotein transcription factor[END_REF]. The change in flicker amplitude we recorded in the PV Cre ::scFvOTX2 tg/o mouse retina and OP may be related to a change in the excitation/inhibition of the inner retinal circuitry. It was reported that extracellular OTX2 neutralization in the retina can retard the opening of critical period plasticity in V1 cortex [START_REF] Sugiyama | Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity[END_REF]. Since visual acuity measured by the optomotor test is independent of the visual cortex, this means that OTX2 signaling in the retina has separate physiological consequences both on subcortical and cortical visual circuits.

Non-cell autonomous signaling by the homeoproteins ENGRAILED 1/2 is important for RGC axon guidance in Xenopus and chick tectum, and this signaling involves stimulating ATP production and release from the RGC growth cone and subsequent activation of adenosine A1 receptor [START_REF] Brunet | The transcription factor Engrailed-2 guides retinal axons[END_REF][START_REF] Wizenmann | Extracellular Engrailed participates in the topographic guidance of retinal axons in vivo[END_REF][START_REF] Stettler | Engrailed homeoprotein recruits the adenosine A1 receptor to potentiate ephrin A5 function in retinal growth cones[END_REF]. Retinal ganglion cells have particularly high energy demands, and energy homeostasis is critical for their function; and energy deficits are hypothesized to underlie RGC dysfunction and degeneration in diabetes, glaucoma, and optic neuropathies [START_REF] Osborne | Maintenance of retinal ganglion cell mitochondrial functions as a neuroprotective strategy in glaucoma[END_REF][START_REF] Yu | Retinal ganglion cells: energetics, compartmentation, axonl transport, cytoskeletons and vulnerability[END_REF][START_REF] Ito | Mitochondrial dynamics, transport, and quality control: a bottleneck for retinal ganglion cell viability and opti neuropathies[END_REF]. Bipolar cell degeneration in Otx2 hypomorph mice has been attributed to mitochondrial dysfunction that can be reversed by exogenous OTX2 [START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF][START_REF] Kim | Mitochondrial Protection by Exogenous Otx2 in Mouse Retinal Neurons[END_REF]. While our finding could possibly be explained by a metabolic defect, it remains to be determined whether RGC mitochondrial energetics are altered in the PV Cre ::scFvOTX2 tg/o retina and whether this affects inner retina activity. Because displaced amacrine cells and RGCs in the GCL, major components of the inner retinal circuit, do not themselves express the OTX2 gene but are capable of taking up exogenous OTX2 [START_REF] Sugiyama | Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity[END_REF][START_REF] Ibad | Otx2 promotes the survival of damaged adult retinal ganglion cells and protects against excitotoxic loss of visual acuity in vivo[END_REF], it can be proposed that interfering in vivo with OTX2 non-cell autonomous activity at the level of the mature retina leads to an alteration in inner retinal cell functions in early life and causes the observed deficit in visual acuity. Future studies will be required to establish whether these functional changes are longer lasting.

Figure 1 .

 1 Figure 1. Expression of OTX2-scFV in P30 mouse retinal tissue. A, qRT-PCR for OTX2scFV mRNA was conducted on extracts from PV Cre and PV Cre ::scFvOTX2 tg/o mice and raw ct values normalized and converted to arbitrary units. B, Retinal lysates from PV Cre and PV Cre ::scFvOTX2 tg/o mice were immunoprecipitated for GFP followed by Western blotting with an anti-MYC antibody. Molecular weight markers are in the left lane in kDa. The middle lane contains the lysate from PV Cre mice. Right lane contains lysate from PV Cre ::scFvOTX2 tg/o mice. The arrow indicates the presence of the bands at the expected migration position of the full-length OTX2scFv-GFP protein. C, In situ hybridization using RNAscope technology of PV Cre retina shows PV expression (red) in the inner retina. DAPI is in blue. Inset arrows show details of PV-expressing cells in the GCL. D, In situ hybridization using RNAscope technology of PV Cre ::scFvOTX2 tg/o retina. mRNA for PV is red, and mRNA for the myc tag of the OTX2scfv is green. Arrows indicate scFvexpressing PV cells in the GCL (i.e., displaced amacrines and/or RGCs); arrowheads indicate scFv-expressing PV cells in the innermost part of the INL in the place of amacrine cells. Scale bars: low-magnification images, 100 mm; insets, 50 mm. Values: mean 6 sem.

Figure 2 .

 2 Figure2. Optomotor test monitoring visual acuity. Thirty-day-old mice were subjected to the optomotor test using an 100% contrast optotype of 0.375c/deg. PV Cre and PV Cre ::scFvPax6 tg/o mice made an average of about five head turns during the 2 min test period. PV Cre ::scFvOTX2 tg/o mice made significantly fewer head turns revealing reduced visual acuity. ppp , 0.01 or pppp , 0.001, Mann-Whitney U test, two tailed. N = 25 for PV Cre , N = 12 for PV Cre ::scFvPax6 tg/o , and N = 13 for PV Cre ::scFvOTX2 tg/o . Values: mean 6 sem.

Figure 3 .

 3 Figure 3. Expression of OTX2scFv does not alter retinal organization in the P30 mouse. Top, All retinal layers organization and approximate sizes are similar in retina from mice expressing OTX2scFv and their PV Cre littermates. Bottom, The number of cells in the GCL, the INL thickness, and the ONL thickness are similar between the two genotypes. Scale bar, 50 mm. N = 8 for PV Cre mice and N = 6 for PV Cre ::scFvOTX2 tg/o mice. Values: mean 6 sem.

Figure 4 .

 4 Figure 4. Expression of retinal cell type-specific genes in P30 mice of the two genotypes. Genes specific of RGCs, bipolar cells, amacrine cells, rods, and cones show similar expression levels in the two genotypes. Lim1 mRNA expression in horizontal cells is significantly increased in the OTX2-scFv-expressing mice. *p , 0.05, Mann-Whitney U test, two tailed. N = 4 for each genotype. Values: mean 6 sem.

Figure 5 .

 5 Figure 5. ERG of P30 mice expressing OTX2scFV and their PV Cre littermates. A, B, Representative traces under scotopic conditions of the right eye at 3.19 cd/m 2 . C-F, Scotopic ERG parameters plotted against log intensity. Two-way ANOVA for repeated measures showed no significant differences based on genotype or genotype by intensity interaction (see text). N = 7 for PV Cre mice and N = 9 for PV Cre ::scFvOTX2 tg/o mice. Values: mean 6 sd.

Figure 6 .

 6 Figure 6. The b-wave amplitude in photopic lighting in the two genotypes of 1-month-old mice. N = 7 for PV Cre mice and N = 9 for PV Cre ::scFvOTX2 tg/o mice. Values: mean 6 sem.

Figure 7 .

 7 Figure 7. Inner retinal function. A, Extracted oscillatory potential traces of a PV Cre mouse (left) and PV Cre ::scFvOTX2 tg/o mouse (right). B, The amplitude of an early OP (OP2) is not altered in the PV Cre ::scFvOTX2 tg/o mice, while the amplitude of a late OP (OP4) is significantly reduced by ;50%. C, Representative traces of 20 Hz flickers of the left eye of mice of the two genotypes. D, The amplitude of the flicker response to 10 Hz stimulation after light adaptation is slightly increased, and at 20 Hz the response is significantly doubled in amplitude. Bottom, There was no difference in the implicit time of the flicker responses between mice expressing OTX2scFv and their PV Cre littermates. ***p , 0.005, Mann-Whitney U test, two tailed. N = 7 for PV Cre and N = 9 for PV Cre :: scFvOTX2 tg/o mice. Values mean 6 sem.

Table 1 :

 1 Statistical table

	Data structure	Type of test	Power
	Figure 2	Mann-Whitney U	0.92
	PV cre vs PVCre::scFvOTX2 tg/o
	Not considered normal due to unequal N
	Figure 4	Mann-Whitney U	0.78
	PV cre vs PVCre::scFvOTX2 tg/o
	Data normalized
	Figure 5	Two-way ANOVA with repeated measures	n.s.
	PV cre vs PVCre::scFvOTX2 tg/o		n.s.
	a-wave latency, F (1,14) = 0.011		n.s.
	a-wave amplitude, F (1,14) = 0.100		n.s.
	b-wave latency, F (1,14) = 3.826		n.s.
	b-wave amplitude, F (1,14) = 0.77		n.s.
	Genotype  stimulus intensity interaction		n.s.
	a-wave latency, F (3,42) = 0.660		n.s.
	a-wave amplitude, F (3,42) = 0.913
	b-wave latency, F (3,41) = 1.915
	b-wave amplitude, F (3,42) = 0.139,
	Figure 7 OPs	Mann-Whitney U	0.97
	PV cre vs PVCre::scFvOTX2 tg/o
	Dissimilar variance
	Figure 7D Flickers	Mann-Whitney U	0.96
	PV cre vs PVCre::scFvOTX2 tg/o
	Dissimilar variance

September/October 2020, 7(5) ENEURO.0012-19.2020 eNeuro.org

This research was supported by grants HOMEOSIGN (Grant ERC-2013-AdG 339379) and NeuroprOtx (Grant ANR-16-CE16-0003-02). *R.T.I., B.M., and C.V. contributed equally to this work. [fn_type;


Killian Denoue

François Cheviré

Christophe Calers

Louisiane Verger
email: louisiane.verger@univ-rennes1.fr

David Le Coq

Laurent Calvez
email: laurent.calvez@univ-rennes1.fr

Killian Dénoue

Mechanochemical synthesis and structural characterization of gallium sulfide Ga2S3

Keywords: Gallium sulfide, Mechanochemical synthesis, X-ray diffraction, Chalcogenide

Introduction

Gallium (III) sulfide (Ga 2 S 3 ) is a semiconducting material with attractive properties for a large number of applications. Ga 2 S 3 combined with other compounds is used as anode materials as well as for solid-state electrolytes in Li-ion and Na-ion batteries to improve the cyclability and the stability of the electrodes thanks to its self-healing properties [START_REF] Wang | A novel carbon-coated Ga 2 S 3 anode material derived from post-synthesis modified MOF for high performance lithium ion and sodium ion batteries[END_REF][START_REF] Cozic | GeS 2 -Ga 2 S 3 -LiCl Glass System: Electrical Conductivity and Structural Considerations[END_REF][START_REF] Paraskiva | Na + ion conducting glasses in the NaCl-Ga 2 S 3 -GeS 2 system: A critical percolation regime[END_REF][START_REF] Senoh | Gallium (III) sulfide as an active material in lithium secondary batteries[END_REF][START_REF] Wang | Exploring the sodium ion storage mechanism of gallium sulfide (Ga 2 S 3 ): a combined experimental and theoretical approach[END_REF]. Moreover, luminescence properties can be achieved either by using nanocrystalline Ga 2 S 3 quantum dots [START_REF] Hu | Synthesis and tunable emission of Ga 2 S 3 quantum dots[END_REF] or by doping Ga 2 S 3 -based chalcogenide glasses with rare-earth [7,[START_REF] Lin | Enhanced Up-Conversion Luminescence in Er 3+ -Doped 25GeS 2 -35Ga 2 S 3 -40CsCl Chalcogenide Glass-Ceramics[END_REF] for light emission or fluorescence applications. When associated with lanthanum, the synthesized glasses possess excellent manufacturing properties to design optical lenses, optical amplifiers, laser optical elements used in telecommunications and sensors [START_REF] Morgan | Lanthanum-fluoride addition to gallium-lanthanumsulphide[END_REF][START_REF] Kityk | NIR and visible luminescence features of erbium doped Ga 2 S 3 -La 2 S 3 glasses[END_REF]. Ga 2 S 3 can also be associated with germanium sulfide and halides or iodides, to obtain chalcogenide glasses and glass-ceramics presenting non-linear optical properties such as second and third harmonic generation [START_REF] Tsisar | Tl 2 S-Ga 2 S 3 -GeS 2 glasses for optically operated laser third harmonic generation[END_REF][START_REF] Ebothe | GeSe 2 -Ga 2 S 3 -PbI 2 glass alloys like second-order non-linear optical materials[END_REF][START_REF] Masselin | CsCl effect on the optical properties of the 80GeS 2 -20Ga 2 S 3 base glass[END_REF][START_REF] Lin | Second-order optical nonlinearity and ionic conductivity of nanocrystalline GeS 2 -Ga 2 S 3 -LiI glass-ceramics with improved thermo-mechanical properties[END_REF]. Finally, Ga 2 S 3 is used for its semiconductor properties in the fields of microwave frequencies [START_REF] Qasrawia | Characterization of the Al/Ge/In 2 Se 3 /Ga 2 S 3 /Al hybrid tunneling barriers designed for Gigahertz/Terahertz applications[END_REF][START_REF] Al Garni | Tunable Au/Ga 2 S 3 /Yb Varactor Diodes Designed for High Frequency Applications[END_REF], photo-detection [START_REF] Zhou | Nonlayered Two-Dimensional Defective Semiconductor γ-Ga 2 S 3 toward Broadband Photodetection[END_REF][START_REF] Alsaif | Atomically Thin Ga 2 S 3 from Skin of Liquid Metals for Electrical, Optical, and Sensing Applications[END_REF], and as a precursor for solar panels [START_REF] Wei | Fabrication of in-situ Ti-doped CuGaS2 thin films for intermediate band solar cell applications by sputtering with CuGaS 2 :Ti targets[END_REF] and for photo-catalysis to generate hydrogen from water splitting [START_REF] Hu | Janus Group-III Chalcogenide Monolayers and Derivative Type-II Heterojunctions as Water-Splitting Photocatalysts with Strong Visible-Light Absorbance[END_REF].

Four different crystallographic forms of Ga 2 S 3 have been reported in the literature [START_REF] Guymont | Electron Microscope Study of γ-Ga 2 S 3[END_REF][START_REF] Tomas | X-Ray Diffraction and Electron Microscopy Studies of α-and β-Ga 2 S 3[END_REF][START_REF] Neuhaus | Studies on the binary system zinc sulfide-gallium sesquisulfide at pressures up to 150 kbars and temperature T = 800°[END_REF][START_REF] Tomas | Détermination des structures des formes α et β de Ga 2 S 3 , structural determination of α and β Ga 2 S 3[END_REF][START_REF] Collin | Préparation et Structure de Ga 2 S 3 α Type Wurtzite Lacunaire[END_REF][START_REF] Pardo | Diagramme de phases gallium-soufre et études structurales des phases solides[END_REF].

Hexagonal β-Ga 2 S 3 crystallizes in the P6 3 mc space group, in a wurtzite-type structure (Figure 1 a). Ga atoms are statistically distributed with an occupation of 2/3 on the 2b site of the lattice and are located on tetrahedral sites formed by S, with Ga-S bonds almost equivalent (3 at 2.254 Å and 1 at 2.268 Å). Hexagonal α-Ga 2 S 3 (space group P6 1 ) is a superstructure of the β wurtzitetype phase. They differ by a partial ordering of the Ga atoms. In the α structure, Ga atoms can occupy 3 different sites, but only partially because the anionic network is the same as the wurtzite structure. Pardo et al. [START_REF] Pardo | Polymorphism of Ga 2 S 3 and Phase Diagram of Ga-S[END_REF] showed that while one site is fully occupied, the other 2 are randomly occupied. This phase was not identified in our work by X-ray diffraction (XRD) characterizations and is not further discussed. Monoclinic α'-Ga 2 S 3 (space group Cc) is also a superstructure of the wurtzite, but in this case a full ordering between vacancies and Ga atoms in the cationic network is observed (Figure 1 b). Ga is still in a tetrahedral environment characterized by 3 Ga-S bonds almost equivalent (around 2.31 Å) and a shorter one (around 2.20 Å). S atoms exhibit 2 distinct environments as shown in Figure 1 b: while S1 and S3 are surrounded by 3 Ga atoms with a Ga-S distances around 2.31 Å, S2 is linked to only 2 Ga with much shorter Ga-S bonds at 2.20 Å. Finally, a cubic sphalerite-type structure is also reported for γ-Ga 2 S 3 , (space group F-43m) with a random distribution of vacancies and Ga atoms on the cationic sites with an occupation of 2/3 and at the center of tetrahedra formed by S with Ga-S at 2.258 Å (Figure 1 c).

Synthesis of Ga 2 S 3 with a control of polymorphism and stoichiometry remains a challenge.

While monoclinic α'-Ga 2 S 3 is exactly stochiometric, the other polymorphs exhibit a sulfur deficiency [START_REF] Pardo | Diagramme de phases gallium-soufre et études structurales des phases solides[END_REF]. Various techniques have been reported to synthesize crystalline bulk Ga 2 S 3 . It was initially obtained by reacting Ga and S in sealed silica tube at temperature above 1000°C.

The monoclinic α' form can be synthesized this way and is stable from room temperature to melting. By controlling the Ga/S ratio introduced and the quenching temperature, the other polymorphs can also be obtained [START_REF] Pardo | Diagramme de phases gallium-soufre et études structurales des phases solides[END_REF][START_REF] Pardo | Polymorphism of Ga 2 S 3 and Phase Diagram of Ga-S[END_REF][START_REF] Hahn | Zur Struktur des Ga 2 S 3[END_REF][START_REF] Goodyear | The unit cell of α-Ga 2 S 3[END_REF][START_REF] Zavrazhnov | The phase diagram of the Ga-S system in the concentration range of 48[END_REF][START_REF] Hahn | Über die Kristallstrukturen von Ga 2 S 3 , Ga 2 Se 3 und Ga 2 Te 3[END_REF]. A reaction involving sulfur vapors on metallic gallium at 1250 °C has been demonstrated by Brukl et al. [START_REF] Brukl | Die Sulfide des Galliums[END_REF]. The most common reported synthesis is the treatment at 750 °C of gallium (oxo)hydroxides, GaO(OH) or Ga(OH) 3 , under flowing H 2 S [START_REF] Collin | Préparation et Structure de Ga 2 S 3 α Type Wurtzite Lacunaire[END_REF][START_REF] Pardo | Diagramme de phases gallium-soufre et études structurales des phases solides[END_REF][START_REF] Pardo | Polymorphism of Ga 2 S 3 and Phase Diagram of Ga-S[END_REF]. Only the α'-Ga 2 S 3 polymorph is formed by this technique. The flammability of H 2 S gas brings safety concerns. Furthermore, all the bulk syntheses mentioned above are unsafe due to possible explosion hazard at the temperatures used caused by the high vapor pressures of sulfur involved. Recently, three safer processes have been described in the literature: (i) a green synthesis involving the reaction of GaCl 3 and Na 2 SO 4 in aqueous solution leading to the formation of nanoparticles of α-Ga 2 S 3 [START_REF] Ahamad | Green Synthesis and Characterization of Gallium (III) Sulphide (α-Ga 2 S 3 ) Nanoparicles at Room Temperature[END_REF], (ii) thermal decomposition of gallium xanthate precursors to obtain γ-Ga 2 S 3 [START_REF] Alderhami | Accessing γ-Ga 2 S 3 by solventless thermolysis of gallium xanthates: a low-temperature limit for crystalline products[END_REF] and (iii) a mechanical milling approach starting from metallic gallium and sulfur [START_REF] Kristl | Mechanochemical Route for the Preparation of Nanosized Aluminum and Gallium Sulfide and Selenide[END_REF].

Mechanochemical synthesis is a technique used more and more recently to produce unique materials. It combines several advantages. From a processing point of view, it is a simple, rapid, no-waste and energy-efficient pathway compared to other methods. It also offers the possibilities to synthesize materials without solvent or gas, and to obtain metastable phases or materials that usually require high temperatures. It is also a top-down approach to produce nanoparticles [START_REF] Baláž | Hallmarks of mechanochemistry: from nanoparticles to technology[END_REF].

Mechanochemical processes find multiple applications from the synthesis of semiconductor materials [START_REF] Baláž | Bio-inspired mechanochemical synthesis of semiconductor nanomaterial using eggshell membrane[END_REF], to the treatment of wastes such as metallurgical residues [START_REF] Wang | Improvement in the floatability of CuO by dry grinding with sulphur[END_REF], leaded glasses [START_REF] Yuan | Mechanochemical sulfidization of lead oxides by grinding with sulfur[END_REF], through catalysis, cosmetics and the medical field [START_REF] Boldyreva | Mechanochemistry of inorganic and organic systems: What is similar, what is different[END_REF]. Finally, a large number of chalcogenides have already been produced using this synthesis method, ranging from binary to quaternary [START_REF] Baláž | Chalcogenide mechanochemistry in materials science: insight into synthesis and applications (a review)[END_REF][START_REF] Baláž | Ultrafast mechanochemical synthesis of copper sulfides[END_REF]. This technique was chosen to carry out this work.

In the present study, we investigate the synthesis of crystalline Ga 2 S 3 by mechanochemical technique from pure Ga and S as raw materials. The as-prepared crystalline powder is characterized by a laser diffraction particle sizer and a scanning electron microscope (SEM) and its crystalline forms are determined by the means of XRD analyses and Rietveld refinements. [START_REF] Tomas | Détermination des structures des formes α et β de Ga 2 S 3 , structural determination of α and β Ga 2 S 3[END_REF], (b) monoclinic α' phase (space group Cc) [START_REF] Collin | Préparation et Structure de Ga 2 S 3 α Type Wurtzite Lacunaire[END_REF], and (c) cubic γ phase (space group F-43m) [START_REF] Guymont | Electron Microscope Study of γ-Ga 2 S 3[END_REF]. Gallium atoms are represented in blue, sulfur atoms are represented in yellow, orange or red.

Experimental section

Synthesis

Ga 2 S 3 was prepared by mechanochemical synthesis using a planetary ball mill (Fritsch, Pulverisette 7). 8 g of stoichiometric proportions of gallium (Neyco, 99.99%) and sulfur (Strem Chemical Inc., 99.999%) was placed into a tungsten carbide (WC) vessel (internal volume of 45 mL) with 10 WC balls (10 mm in diameter) resulting in balls to powder mass ratio of 10:1. The mechanochemical reaction was performed from 1 to 10 hours at a fix rotation speed of 400 rpm.

The vessel was opened every hour to scrub and hand grind the powder stuck to the walls in order to obtain a uniform powder. All processes were performed in a dry N 2 atmosphere.

Characterization techniques

XRD measurements were performed at room temperature on samples protected from air by a Kapton (polyimide) window to avoid moisture corrosion of fine submicron particles. They were recorded in the 10-90° 2θ range with a 0.0261° step size with each data point having an effective total counting time of 40 s/step using a PANalytical X'Pert Pro diffractometer (Cu K α1 , K α2 radiations, λ Kα1 = 1.54056 Å, λ Kα2 = 1.54439 Å, 40 kV, 40 mA, PIXcel 1D detector). Data collector and HighScore Plus software were used for recording and analysis of the patterns, respectively. The powder XRD patterns for Rietveld refinements were collected in the 2θ 10-120° range with a 0.026° step size with each data point having an effective total counting time of 400 s/step. All calculations were carried out using Fullprof and WinPlotr programs [START_REF] Rodríguez-Carvajal | Recent advances in magnetic structure determination by neutron powder diffraction[END_REF][START_REF] Roisnel | WinPLOTR: A Windows tool for powder diffraction pattern analysis[END_REF]. High temperature XRD patterns were acquired from room temperature up to 800°C with a step of 50°C (rate: 5°C/min) under a flowing nitrogen atmosphere in the 10-90° 2θ range with a 0.026° step size with each data point having an effective total counting time of 40 s/step using a PANalytical Empyrean diffractometer (Cu K α1 , K α2 radiations, λ Kα1 = 1.54056 Å, λ Kα2 = 1.54439 Å, 40 kV,40 mA, PIXcel 3D detector) equipped with an Anton Paar HTK1200 temperature chamber.

Laser grain size measurements were performed on Fritsch, Laser particle sizer Analysette 22 NanoTec, using a wet dispersion unit, after different milling times. SEM images were obtained on JEOL JSM-IT 300 using an acceleration voltage of 15 kV and a magnification x5000. Rietveld refinements were carried out to confirm and quantify the relative amount of these two polymorphic species of Ga 2 S 3 after 4 h of milling. As illustrated in Figure 3, the diffraction peaks show a substantial half-height width, which can influence the refinement parameters. This observation occurs for crystalline compounds obtained by mechanochemistry since their particle size is definitively smaller comparatively to the particle size of compounds obtained by other techniques [START_REF] Baláž | Hallmarks of mechanochemistry: from nanoparticles to technology[END_REF]. According to the refinements, a quantitative estimation shows that the hexagonal β phase represents 68 wt% (±5%) of the crystallized mixture and the remaining 32 wt% (±5%) are the cubic γ phase. After 4 h of milling, it is clearly shown that the experimental data and the refinements are much better when both hexagonal and cubic phases are included (Figure 3 b), comparatively to the refinement with only the hexagonal phase (Figure 3 a).

Results and discussion

As shown in

Preferential orientation and morphology anisotropy of nanocrystals can typically result in a modification of the relative intensity and width of diffraction peaks. However, this effect in our case can be excluded as mechanical milling results in the formation of isotropic particles with no preferential orientation as shown latter by SEM. Kristl et al. also used mechanochemistry to synthesis Ga 2 S 3 [START_REF] Kristl | Mechanochemical Route for the Preparation of Nanosized Aluminum and Gallium Sulfide and Selenide[END_REF]. Milling conditions are different than this work, they used stainless steel vessel with balls to powder mass ratio of 20:1. After 2 h of milling time, they obtained a material with a similar XRD pattern than the powder obtained in this work after 4 h. The condition used here are then milder than Kristl et al. Although they identify their material to be β single-phase [START_REF] Kristl | Mechanochemical Route for the Preparation of Nanosized Aluminum and Gallium Sulfide and Selenide[END_REF], they rather probably have a mixture of β and γ polymorphs like evidenced in this paper. As mentioned in the introduction, only α'-Ga 2 S 3 is stoichiometric and stable, the other polymorphs showing an S deficiency. This is especially true for γ-Ga 2 S 3 which is the furthest apart from the ideal composition Ga 2 S 3 in the binary Ga-S: its theoretical composition is reported to be close to Ga 2 S 2.76 [START_REF] Guymont | Electron Microscope Study of γ-Ga 2 S 3[END_REF] (see binary diagram in [START_REF] Hahn | Über die Kristallstrukturen von Ga 2 S 3 , Ga 2 Se 3 und Ga 2 Te 3[END_REF]). Pardo et al. have reported that an annealing treatment on β-Ga 2 S 3 results in the decomposition of the compound into α'-Ga 2 S 3 and GaS [START_REF] Hahn | Über die Kristallstrukturen von Ga 2 S 3 , Ga 2 Se 3 und Ga 2 Te 3[END_REF]. Besides, by heating a mixture of different amount of GaS and α'-Ga 2 S 3 , they also showed that γ-Ga 2 S 3 can be obtained together with one of the initial reactants. These results are consistent with the reactivity put forward in our study: after the formation of the two stable compounds α'-Ga 2 S 3 and GaS, mechanical milling enables the formation of substoichiometric polymorphs, β and γ. The presence of both the hexagonal and cubic phases of Ga 2 S 3 is not surprising since their structure is based on the occupancy of almost identical tetrahedral sites by Ga, with different atomic stacking (ABAB for β and ABCABC for γ) (Figure 1). Furthermore, γ-Ga 2 S 3 is always found in the presence of other polymorphs, and has only been isolated as a powder very recently by thermolysis of gallium xanthate precursors [START_REF] Alderhami | Accessing γ-Ga 2 S 3 by solventless thermolysis of gallium xanthates: a low-temperature limit for crystalline products[END_REF]. The conditions involved in the high-energy mechanical milling seem to promote the synthesis of metastable polymorphs of Ga 2 S 3 (β and γ) which can only be obtained by specific heating and quenching temperature by traditional high temperature synthesis.

Interestingly, the formation of GaS during the first hour of mechanical synthesis instead of pure Ga 2 S 3 means that probably a certain amount of S becomes poorly crystallized and not detected by XRD. To investigate further the influence of the initial S amount on the resulting phases obtained, two synthesis were tested with a 5 % molar excess of S, and 9 % molar default of S. The precursors were mechanically milled during 4 h, as described in the experimental section. 220) and (311) peaks indexed in the space group of γ-Ga 2 S 3 remains constant while the intensity of the other peaks increases with an excess of S. The sample synthesized with a 5 % molar excess of S is composed of 24.6 wt% of γ-Ga 2 S 3 and 75.4 wt% β-Ga 2 S 3 (±5%), while the sample obtained with a 9 % molar default of S is composed of 41.9 wt% of γ-Ga 2 S 3 and 58.1 wt% β-Ga 2 S 3 (±5%). The formation of the cubic γ-phase is then promoted by a S deficiency, which is consistent with the fact that this polymorph is the furthest apart from the ideal composition. When Ga and S are introduced in stoichiometric proportion, the milling time was extended up to 10 h to investigate a possible evolution of the phases. There was no obvious difference between the XRD patterns of Ga 2 S 3 powders after 4h and 10h of grinding (not shown). In addition, as underlined previously, the evolution of the powder color is very marked from 1 h to 4 h, but it remains similar after 10 h. These two observations are consistent with the formation of the final mixture of the 2 Ga 2 S 3 polymorphs after 4 h of mechanical milling.

Nevertheless, some evolutions can still be pointed out as depicted in Figure 5 a in which the morphology of the Ga 2 S 3 powder after 4 h and 10 h of mechanosynthesis is presented. This powder is composed of aggregates of different sizes (micron scale), themselves composed of spherical particles of submicron sizes. A diminution of the average grain size is visible when comparing the SEM photographs after 4 h and 10 h. To quantify this observation, the median grain size (Q50) of the powder was measured by laser particle sizer after different grinding time.

As shown both in Figure 5 b, the Q50 exponentially decreases as a function of grinding time from 420 nm down to 42 nm for 1 h and 10 h, respectively. Kristl et al. [START_REF] Kristl | Mechanochemical Route for the Preparation of Nanosized Aluminum and Gallium Sulfide and Selenide[END_REF] also obtained aggregates of spherical particles. However, using their conditions 2 h is enough to decrease the diameter of the particles down to 40 nm. As previously observed when comparing the XRD pattern, the condition used in this work are then milder. Although no further modification of the structure of Ga 2 S 3 is observed after 4 h based on the XRD patterns, the previous analyze clearly evidences that it is possible to control and reduce the particle size of the compound by extending the grinding time. Controlling the grain size of Ga 2 S 3 as a precursor is crucial depending on the subsequent reaction or application. In situ high temperature XRD measurements were carried out to investigate the stability in temperature of Ga 2 S 3 powders obtained after 4 h of milling time. Figure 6 shows the evolution of the XRD patterns acquired at room temperature, and then every 50°C from 50°C up to 800°C.

A phase transition is observed starting at 450°C, from the mixture of the two polymorphs β and γ-Ga 2 S 3 to monoclinic α'-Ga 2 S 3 (Cc, JCPDS no. 01-076-0752). The latter is stable and continuously crystallizes up to 800 °C. Differential scanning calorimetry (DSC) was conducted on Ga 2 S 3 powders after 4 h of milling time. An exotherm was detected around 410°C, that coincide with the formation of α'-Ga 2 S 3 evidenced in the high temperature XRD data. No predominance of one of the two preliminary phases over the other is observed before the transition to the α'-phase. The XRD diagram acquired on the powder at room temperature after the in situ experiment shows that only the monoclinic phase is still present (blue pattern in Figure 7) meaning that the phase transition observed upon heating is then irreversible. This is consistent with the fact that β and γ are metastable polymorphs of Ga 2 S 3 . α'-Ga 2 S 3 is stoichiometric, meaning that a loss of Ga should be observed together with the decomposition of substoichiometric β and γ phases. Indeed, the presence of supplementary peaks attributed to monoclinic Ga 2 O 3 phase (JCPDS no. 00-041-1103), show that the excess of Ga reacts with some oxygen contamination, probably coming from handling the sample shortly in air during the setup of the experiment or from the nitrogen atmosphere.

Conclusion

Ga 2 S 3 samples have been prepared by mechanical alloying of pure raw Ga and S and the mechanism of reactivity has been investigated. Our work shows that after only 1 h of milling, the main phases identified are monoclinic α'-Ga 2 S 3 and GaS, that further react to form Ga 2 S 3 in 4 h.

It is therefore a fast and energy-efficient synthesis technique compared to melt quenching process performed at high temperature (above 1180 °C). Moreover, the evolution of the color as the synthesis progresses allows a simple and direct follow-up of the reaction progress. The XRD studies and the Rietveld refinements also show that the material mechano-synthesized is a mixture of phases: a majority hexagonal β phase (68% ± 5%) and a cubic γ phase (32% ± 5%).

The high temperature XRD analysis reveals that no evolution of the two phases occurs before a phase transition to monoclinic α' starting at 450°C. Furthermore, the relative amount of the two phases can be modify by the quantity of precursors added. This work provides new insight in the reactivity of Ga and S mixtures, and shows that structural modifications accessible by mechanical synthesis can be different than by traditional melting-quenching method in silica tube. Finally, the main advantage of this synthesis technique is that a large quantity of Ga 2 S 3 can be produced in a short time at a very low cost. This efficient process is interesting for all applications that do not need a specific polymorph of Ga 2 S 3 as a starting material such as gigahertz and terahertz applications, or glasses for optics and solar panels.

Figure 1 .

 1 Figure 1. Crystal structures of the different polymorphs of Ga 2 S 3 : (a) hexagonal β phase (space group P6 3 mc) [25], (b) monoclinic α' phase (space group Cc) [26], and (c) cubic γ phase (space

Figure 2 ,

 2 the color of the powders evolves from brown to yellow as a function of milling time. These color changes indicate the gradual disappearance of raw elements due to their progressive reaction to form Ga 2 S 3 . Figure2also shows the XRD patterns acquired on the powder after different milling times between 1 h and 4 h. The presence of both a diffraction halo, located between 15 and 25 ° 2θ, and the background drift are due to the Kapton window. After only 1 h of mechanical milling (black diagram in Figure2), monoclinic α'-Ga 2 S 3 (Cc, JCPDS no. 01-076-0752) is detected. The two diffraction peaks at 16.654 and 18.613 ° 2θ is an evidence for the presence of this polymorph, as they are not present in the α, β or γ form. However, their low relative intensity compared to the main characteristic peak of Ga 2 S 3 can be explained by the presence of β (P6 3 mc, JCPDS no. 01-084-1441) and γ (F-43m, JCPDS no. 00-043-0916) polymorphs as well, since their diffraction peaks are overlaid with the α' polymorph (see the Bragg positions in Figure2). GaS (P6 3 /mmc, JCPDS no. 01-074-0227) is also detected as a minority phase. The presence of GaS can explain the color of the powder. After 2 h of milling (red diagram in Figure2), GaS diffraction peaks intensity decreases and they disappear after 3 h of milling (blue diagram in Figure2). At the same time, some broadening and an evolution of the Ga 2 S 3 diffraction peaks are observed. The former point indicates a reduction of the crystallites size during prolongated ball milling. The latter suggests a structural modification of the Ga 2 S 3 phase. Firstly, the two low intensity peaks around 20 ° 2θ characteristic of α'-Ga 2 S 3 are not detected after 3 h and 4 h of milling time. An XRD acquisition of the powder after 4 h of milling time without the Kapton window was done for the high temperature study and also show the absence of these two peaks (Figure6bottom pattern). Secondly, the relative intensity of the peaks at 29.6, 49.4 and 58.7° 2θ noticeably increases compared to the other peaks. These two modifications can be explained by a reaction of the monoclinic α'-Ga 2 S 3 phase with GaS to form a mixture of hexagonal β-and cubic γ-Ga 2 S 3 . The intensity of the peaks aforementioned is the result of the contribution of the (002), (110) and (112) lines indexed in the P6 3 mc space group of the β phase, together with the (111), (220) and (311) lines indexed in the F-43m group of the γ phase, as illustrated on Figure2with the Bragg position of the β-and γ-Ga 2 S 3 polymorphs. To summarize at this point, reaction between Ga and S takes place within 1 h of milling, leading to a mixture of different polymorphs of Ga 2 S 3 and GaS, which further react to form a mixture of β and γ-Ga 2 S 3 after 4 h of milling.

Figure 2 .

 2 Figure 2. XRD diagrams of Ga 2 S 3 samples prepared by ball milling for different periods (from bottom to top: 1 h, 2 h, 3 h and 4 h) with associated photographs. The three lowermost diagrams represent the peaks position of monoclinic α'-Ga 2 S 3 (JCPDS no. 01-076-0752), hexagonal β-Ga 2 S 3 (JCPDS no. 01-084-1441) and cubic γ-Ga 2 S 3 (JCPDS no. 00-043-0916).

Figure 3 .

 3 Figure 3. Rietveld refinement (black line) of Ga 2 S 3 synthesized by mechanical milling for 4 h (red dots) considering (a) only the hexagonal β-Ga 2 S 3 phase (JCPDS no. 01-084-1441) and (b) the hexagonal β-Ga 2 S 3 phase (JCPDS no. 01-084-1441) and cubic γ-Ga 2 S 3 phase (JCPDS no. 00-043-0916). The blue lines plot the difference between observed and calculated intensities. Green and blue bars show the position of the Bragg reflections for γ-Ga 2 S 3 and β-Ga 2 S 3 , respectively.

Figure 4 a

 4 and Figure 4 b show Rietveld refinements of XRD patterns acquired on powders obtained with 9 % molar default of S and 5 % molar excess of S, respectively. The intensity of the (111), (

Figure 4 .

 4 Figure 4. Rietveld refinement (black line) of Ga 2 S 3 samples prepared by ball milling with 9 % molar default (a) and 5% molar excess of S (b). The blue lines plot the difference between observed and calculated intensities. Green and blue bars show the position of the Bragg reflections for γ-Ga 2 S 3 and β-Ga 2 S 3 , respectively.

Figure 5 .

 5 Figure 5. (a) SEM images of Ga 2 S 3 powder after 4 h (left) and 10 h (right) of mechanosynthesis (accelerating voltage: 15 kV, magnification: x 5000). (b) Evolution of the median size Q50 from 1 hours up to 10 hours determined by laser particle sizer.

Figure 6 .

 6 Figure 6. In situ high temperature XRD patterns of the Ga 2 S 3 sample obtained after 4 h of milling time, from 50°C to 800°C every 50°C.

Figure 7 .

 7 Figure 7. Comparison between XRD patterns at 800 ° C and 25 ° C post in-situ, and the monoclinic α'-Ga 2 S 3 phase (JCPDS no. 01-076-0752).

ACKNOWLEDGMENTS

Authors would like to acknowledge the IUF (Institut Universitaire de France) for their financial support.

AUTHOR INFORMATION

Corresponding Authors

Louisiane Verger -Univ Rennes CNRS, ISCR (Institut des Sciences Chimiques de Rennes) -UMR 6226, F-35000 Rennes, France ; Email : louisiane.verger@univ-rennes1.fr Laurent Calvez -Univ Rennes CNRS, ISCR (Institut des Sciences Chimiques de Rennes) -UMR 6226, F-35000 Rennes, France ; Email : laurent.calvez@univ-rennes1.fr

Notes

The authors declare no competing financial interest.