Marine Tessier

Marta Saez

Emmanuelle Goubert

Edith Blasco

Amandine Consumi

Benoit Dehapiot

Li Tian

Florence Molinari

Jerome Laurin

François Guillemot

Marta Saez Garcia

Christian A Hübner

Christophe Pellegrino

Claudio Rivera
email: claudio.rivera@helsinki.fi

Bumetanide induces post-traumatic microglia-interneuron contact to promote neurogenesis and recovery

Keywords: traumatic brain injury, microglia, chloride homeostasis, neuroinflammation, GABAergic transmission BDNF = Brain-derived neurotrophic factor, CCI = Controlled Cortical Injury

imaging, cognitive behavioral tests and flow cytometry to investigate the role of Nkcc1 inhibition by bumetanide in a mouse model of controlled cortical impact (CCI).

Here, we found that bumetanide rescues parvalbumin-positive interneurons by increasing interneuron-microglia contacts shortly after injury. The longitudinal phenotypic changes of microglia were significantly modified by bumetanide, including an increase in the expression of microglial-derived Bdnf. These effects were accompanied by the prevention of CCI-induced decrease in hippocampal neurogenesis. Treatment with bumetanide during the first week post-CCI resulted in significant recovery of working and episodic memory as well as changes in theta band oscillations one month later.

These results disclose a novel mechanism for the neuroprotective action of bumetanide mediated by an acceleration of microglial activation dynamics that leads to an increase of parvalbumin interneuron survival following CCI, possibly resulting from increased microglial Bdnf expression and contact with interneurons. Salvage of interneurons may normalize ambient gammaaminobutyric acid (GABA), resulting in the preservation of adult neurogenesis processes as well as contributing to bumetanide-mediated improvement of cognitive performance.

Introduction

Traumatic brain injury (TBI) is one of the most prevalent pathologies worldwide with more than 20 million people affected each year [START_REF] Gbd | Traumatic Brain Injury and Spinal Cord Injury Collaborators. Global, regional, and national burden of traumatic brain injury and spinal cord injury, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016[END_REF] . However, possibilities for intervention to prevent comorbidities and long-lasting sequelae that frequently follow TBI are limited. Understanding the changes and key players involved in the pathophysiology of the latent phase may help to prevent or reduce some of the comorbidities that accompany TBI.

TBI can be frequently associated with complications such as epileptic seizures, depression and memory impairment [START_REF] Nicholl | Neuropsychiatric sequelae of traumatic brain injury[END_REF] . This may partly result from the alteration of hippocampal information processing and neurogenesis [START_REF] Wang | Traumatic Brain Injury Severity Affects Neurogenesis in Adult Mouse Hippocampus[END_REF][START_REF] Zheng | Neurogenesis in adult human brain after traumatic brain injury[END_REF][START_REF] Graham | The nature, distribution and causes of traumatic brain injury[END_REF] . However, the mechanisms regulating neurogenesis following TBI remain elusive because of contradictory reports [START_REF] Wang | Traumatic Brain Injury Severity Affects Neurogenesis in Adult Mouse Hippocampus[END_REF][START_REF] Goubert | Bumetanide Prevents Brain Trauma-Induced Depressive-Like Behavior[END_REF] .

Adult neurogenesis is controlled by local GABAergic interneurons and GABAA receptors. In particular, the release of GABA by parvalbumin (PV)-expressing interneurons controls the quiescent state of radial glia-like cells (RGL) in the dentate gyrus (DG) [START_REF] Song | Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision[END_REF] . Studies in humans and mouse models of TBI have demonstrated a progressive loss of PV-expressing interneurons in both the ipsi-and the contralesional hippocampus that can persist over months [START_REF] Santhakumar | Long-term hyperexcitability in the hippocampus after experimental head trauma[END_REF][START_REF] Schiavone | The NADPH oxidase NOX2 mediates loss of parvalbumin interneurons in traumatic brain injury: human autoptic immunohistochemical evidence[END_REF][START_REF] Toth | Instantaneous perturbation of dentate interneuronal networks by a pressure wave-transient delivered to the neocortex[END_REF] . We have previously shown that the inhibition of chloride uptake by bumetanide, an inhibitor of Na-K-Cl (NKCC1) cotransport, significantly decreased PV interneuron loss with a positive effect on both depressive-like behavior and cognitive performance [START_REF] Goubert | Bumetanide Prevents Brain Trauma-Induced Depressive-Like Behavior[END_REF] . Despite the potential clinical relevance of these results, the underlying mechanisms remain unknown.

Previous studies on chloride uptake in the brain mainly focused on neuron-intrinsic mechanisms, although the expression of the principal chloride cellular uptake transporter Nkcc1 is mainly expressed in non-neuronal cells [START_REF] Glushakova | Delayed increases in microvascular pathology after experimental traumatic brain injury are associated with prolonged inflammation, blood-brain barrier disruption, and progressive white matter damage[END_REF] .

Glial cells are involved early on in the temporal sequence of the neuroinflammatory processes following TBI [START_REF] Lozano | Neuroinflammatory responses to traumatic brain injury: etiology, clinical consequences, and therapeutic opportunities[END_REF] . Indeed, quiescent glial cells are rapidly activated by a process called "reactive gliosis". The recruitment of peripheral neutrophils is also observed, which is followed by infiltration of lymphocytes and monocyte-derived macrophages. Simultaneously, the release of pro-inflammatory and anti-inflammatory cytokines promotes and/or inhibits the post-traumatic neuroinflammatory response [START_REF] Aguzzi | Microglia: scapegoat, saboteur, or something else?[END_REF] . Activated microglia trigger and maintain astrocytic activation through the release of cytokines, which in turn act on surrounding glial cells and neurons [START_REF] Morganti-Kossmann | Role of cerebral inflammation after traumatic brain injury: a revisited concept[END_REF] . In addition, recent reports showed an important pro-survival function of physical contact between activated microglia and interneurons, especially PV-positive interneurons [START_REF] Cserép | Microglia monitor and protect neuronal function through specialized somatic purinergic junctions[END_REF] .

In this study, we identified a novel mechanism involving cellular chloride uptake in microglia, interneuron survival and neurogenesis that is linked to TBI-induced cognitive decline.

Material and methods

For full description of experimental procedures please see supplementary material and methods.

Experimental Animals

Wild-type mice had a C57bl6-J background. Three transgenic lines were used : Nestin-GFP [START_REF] Mignone | Neural stem and progenitor cells in nestin-GFP transgenic mice[END_REF]

Controlled-cortical impact (CCI) model

Buprenorphine (0.03 mg/kg) was injected intraperitoneally (IP) into 10-week-old C57bl6-J males 30 minutes before surgery. Mice were then anesthetized using 4% isoflurane mixed with air and enriched with oxygen (0.3 %) and positioned in a stereotaxic frame (David Kopf Instruments®).

Body temperature was monitored throughout the procedure using a rectal probe and maintained at 37±2°C with a heating pad (Harvard Apparatus®). A unilateral craniotomy was performed at the level of the right posterior parietal cortex. CCI was performed using a Leica impactor with standardized parameters (tip diameter 3mm, 6 m/sec speed, 1.5 mm depth and 200 msec duration).

Animals were allowed to recover on the heating pad before being transferred to a post-surgical room. Before the experiment, animals were randomly assigned to one of the following groups:

Drug delivery

A bumetanide stock solution 20 mM (Sigma-Aldrich®, B3023) was prepared by dissolving 36.4 mg of powder into 1 ml of absolute ethanol. IP injections were performed twice daily 12h apart either during the first week post-CCI at 2 mg/kg or during one-week preceding CCI.

Minocycline (Sigma-Aldrich®, M9511) was injected twice daily, 12h apart (45 mg/kg) for one week. The vehicle solution consisted of the same preparation but lacked the bumetanide/minocycline powder to respect volume and diluent.

Immunohistochemistry

Immunohistochemistry was performed on 4% paraformaldehyde fixed free-floating sections.

Incubation with primary antibodies diluted in PBS with 5% NGS and 0.3% Triton X-100 was carried out at 4°C overnight using rabbit anti-doublecortin (ab18723, Abcam, 1:1000), mouse antiparvalbumin (p3088, Sigma, 1:500), rabbit anti-Iba1 (W1W019-19741, Wako, 1:500), mouse anti-GFAP (MAB360, Merck Millipore, 1:500), mouse anti-BDNF antibody (MAB#918), mouse anti-RFP antibody (MA5-15257, Thermofisher Scientific, 1:500), chicken anti-GFP antibody (AB_2307313, Avès Labs, 1:300) as well as corresponding Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific, 1:500).

PV microglia contact quantification

Confocal stack with double immunofluorescent labeling (Parvalbumin and Iba1) were acquired using a LSM-800 Zeiss confocal microscope Plan Flour 40x/1,30 NA oil immersion objective.

Optical sections were collected from the ipsi-and contralesional hippocampi of mice treated and non-treated with bumetanide, at 3 and 7 days post-CCI. Images were then reconstructed in 3D with

Flow cytometry analysis

Fresh cortical tissues were dissected and gently chopped and minced in 1 mL ice-cold flow cytometry staining buffer (PBS + 1% fetal calf serum) through a 100 µm filter on ice. The homogenates were first blocked in PBS with 10% rat serum with gentle rotation in a cold room and then stained for 1 h protected from light at 4°C with CD206-FITC (cat no. 141704, Biolegend®) + MHCII-PE (cat no. 107608, Biolegend®) + CD11b-PerCP/Cy5.5 (cat no.101228, Biolegend®) + CD45-APC (cat no.103112, Biolegend®). After staining, cells were fixated with 8% PFA for 30 mins, centrifuged at 2000 rpm. Samples were acquired with a 2-laser, 6-color cytometer Gallios (Beckman Coulter®). Gating strategy was determined based on the specific staining of markers for microglial populations [START_REF] Li | Microglia are polarized to M1 type in high-anxiety inbred mice in response to lipopolysaccharide challenge[END_REF] .

Live Imaging

Imaging was performed on 5-month-old male mice (B6.129P-Cx3cr1tm1Litt/j, n=5) carrying a cranial window surrounded by a metallic head plate. One month after surgery, trained awake animals were head-fixed on a Mobile HomeCage® platform (MHC V5, Neurotar®) under a custom-built two-photon microscope (Femtonics®) equipped with Mai Tai® Ti:Sapphire Ultrafast Laser (Spectra-Physics) and imaged longitudinally using a Nikon Apo LWD 25X objective with 1.10 numerical aperture for 5 days. An image stack with a 5 mm step size covering 300 mm was acquired. After a 30 min baseline recording, injury was then achieved by focal excitation of 1 cell for 20s at 75 us/pixel at 100% laser intensity. After the injury, animals were treated (Vehicle/Bumetanide) twice per day and imaged, and z-stacks were collected with 5 min intervals for 1 h.

To compute the microglial cell dynamics, we designed a semi-automated procedure using a using Python. Sub-volumes were cropped around each cell before undergoing spatial registrations using pystackreg (G. Lichtner, pystackreg, (2022), https://github.com/glichtner/pystackreg). Cell

Behavioral tests

One month post-CCI, mice were tested on different tasks using the object recognition paradigm to test the individual components of episodic-like memory, namely the novel object recognition (NOR) and the object displacement task (ODT) [START_REF] Inostroza | Specific impairment of "what-where-when" episodiclike memory in experimental models of temporal lobe epilepsy[END_REF] . For NOR, mice were placed in the center of a box (Noldus apparatus®, 38.5 cm x 38.5 cm) and allowed to freely explore the space for 10 mins, then two identical objects were added. After a 3 min retention time, one of the objects was replaced and the time of exploration was measured for a 3 min period. The same experiment was also performed with 1 h retention time. A similar protocol was used for ODT with the difference that after the 3 min retention time, one of the objects was moved and the time of exploration of each object was recorded.

Mice were also tested in the Barnes maze (BM) test. One-month post-CCI, mice were trained twice daily for 4 days. On trial probe day (day 5), the target box was replaced with a false escape. The distance travelled, the number of errors, the time spent in the target zone and the latency to reach the target hole were measured. Finally, mice were tested for anxiety using the elevated plus maze (EPM) test (Ugo Basile company®). For each test, mice were placed in the center square, facing an open arm, and allowed to move freely for 5 min. Entries and time spent in one of the two open or closed arms and center were monitored. Recording and analyses were done using the Ethovision software (Noldus®).

In vivo electrophysiological recordings

Scalp electroencephalographic (EEG) recordings were performed in freely moving mice 3 weeks post-CCI. Telemetric recording electrodes were implanted at the level of the posterior parietal cortex (2 mm posterior to and 1.5 mm left of the bregma). A reference electrode was placed rostral For EEG recordings Animal temperature as well as movement were used to determine active and non-active time windows. Delta (1-4 Hz), Theta (4-12 Hz) and Gamma (30-80 Hz) bands [START_REF] Cohen | Analyzing Neural Time Series Data: Theory and Practice[END_REF] were normalized using the ratio of power in the first to the last minute within each recording session.

The mean normalized power of active time windows was subtracted from non-active for each recording.

From intrahippocampal recordings individual units were isolated from multisite LFP recordings using Spyking-Circus [START_REF] Yger | A spike sorting toolbox for up to thousands of electrodes validated with ground truth recordings in vitro and in vivo[END_REF] . The Single-unit activity were further classified into wide-spiking (WS) and narrow-spiking (NS) units [START_REF] Watson | Network homeostasis and state dynamics of neocortical sleep[END_REF] based on the spike waveform, autocorrelogram, and crosscorrelogram (CCG). NS and WS presumably correspond to putative inhibitory and excitatory units, respectively [START_REF] Csicsvari | Reliability and state dependence of pyramidal cellinterneuron synapses in the hippocampus: an ensemble approach in the behaving rat[END_REF][START_REF] Sirota | Entrainment of neocortical neurons and gamma oscillations by the hippocampal theta rhythm[END_REF] .

Statistical Analysis

All mean values are given with the standard error mean (SEM). Normality was tested using a D'Agostino-Pearson test. Two-tailed Student's t tests were used for testing statistical significance between two groups while Mann-Whitney tests were used when the normality test failed. Ordinary one-way ANOVA tests were used to compare three or more groups. In the event that the normality assumption was violated here, the Kruskal-Wallis test was used. When variances were unequal, the Brown-Forsythe ANOVA was used.

A C C E P T E D

M A N U S C R I P T
Statistics were done using Prism Version 8.3.0 (GraphPad software, Inc., La Jolla, CA, USA) and are represented as follows: * p < 0.05; ** p < 0.01 and *** p < 0.001; all results and statistical significance are reported in Appendix table S1. . These results show that CCI has a negative impact on learning performance and spatial memory, that it is not linked with anxiety.

Results

Bumetanide ameliorates CCI-induced memory impairment and pathological neuronal activity

Memory impairment following TBI are characterized by changes in theta rhythms [START_REF] Fedor | Hippocampal θ dysfunction after lateral fluid percussion injury[END_REF][START_REF] Winson | Loss of hippocampal theta rhythm results in spatial memory deficit in the rat[END_REF][START_REF] Bragin | Pathologic electrographic changes after experimental traumatic brain injury[END_REF] . Using telemetric EEG, we found that at 1-month post-CCI, the power of theta band oscillations in vehicle treated animals was significantly lower compared to sham (Sham 6.50 % ±0. 18

Bumetanide rescues CCI-induced changes in secondary neurogenesis and PV interneuron loss

Interneurons are important contributors to brain oscillations [START_REF] Moxon | From adagio to allegretto: The changing tempo of theta frequencies in epilepsy and its relation to interneuron function[END_REF] . PV-positive interneurons are a particularly vulnerable subpopulation following CCI [START_REF] Song | Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision[END_REF] . In addition, the impact of CCI on interneuron activity and reduction in numbers suggest changes in the interneuron population of the hippocampus following CCI. We quantified PV-positive interneurons in the granular layer of the Both sides showed a significant reduction in the number of PV interneurons at 3 dpCCI (normalized value on sham: sham contra 1.00 ±0.20 vs contra Veh 0.52 ±0.18 and sham ipsi 1.00 ±0.22 vs ipsi Veh 0.22 ±0.18, Figs 2A andB). This loss was significantly reduced by bumetanide treatment in the contra-(0.77 ±0.23) and ipsilesional side (0.51 ±0.14, Fig 2A andB). We also found a significant reduction in the PV-containing interneurons survival at 7 dpCCI in both sides (normalized value on sham: sham contra 1.0 ±0.27 vs contra Veh 0.59 ±0.18 and sham ipsi 1.00 ±0.27 vs ipsi Veh 0.14 ±0.12, Fig 2C andD). This loss was significantly reduced at the contra-(1.02 ±0.34) and ipsilesional sides (0.71 ±0.24, Fig 2C andD) by bumetanide treatment.

Bumetanide-induced interneuron survival was not restricted to the DG, as we found a similar effect on other hippocampal regions (in ipsilesional CA3: Sham 1.00 ±0.12 vs Ipsi Veh 0.52 ±0.09; and in ipsilesional CA1: Sham 1.00 ±0.25 vs Ipsi Veh 0.37 ±0.07).

Ambient GABA, provided by the ongoing activity of DG interneurons and particularly PV interneurons, plays a role in the proliferation and migration of granular cell progenitors [START_REF] Goubert | Bumetanide Prevents Brain Trauma-Induced Depressive-Like Behavior[END_REF][START_REF] Duan | Development of neural stem cell in the adult brain[END_REF] . A loss of PV interneurons may significantly contribute to the TBI-induced changes in stem cell proliferation by reducing GABA release. At 7 dpCCI, we observed a significant reduction in the number of DCX-positive neurons within both the ipsi-and contralesional DG (normalized value on sham: sham ipsi 1.00 ±0.31 vs. CCI Veh ipsi 0.19 ±0.09 and sham contra 1.00 ±0.31 vs. CCI Veh contra 0.78 ±0.24, Figs 2E, F, H andI). This coincided with an increase in the number of Nestin-positive cells within the DG (sham ipsi 1.00 ±0.17 vs. CCI Veh ipsi 2.08 ±0.29 and sham contra 1.00 ±0.18 vs. CCI Veh contra 1.37 ±0.34 Figs 2E, G, H andJ). Bumetanide treatment reduced the number of Nestin-positive cells (CCI Bum ipsi 1.4 ±0.24 and CCI Bum contra 1.0 ±0.15, Figs 3E, G, H andJ) and triggered an increase in the number of DCX-positive cells (CCI Bum ipsi 0.47 ±0.16 and CCI Bum contra 0.98 ±0.23, Figs 2E, F, H andI).

Bumetanide rescues CCI-induced changes in microglia morphology/phenotype and increases contacts with PV interneurons

Neuroinflammation is present in the primary (acute) and secondary (chronic) stages of TBI [START_REF] Lozano | Neuroinflammatory responses to traumatic brain injury: etiology, clinical consequences, and therapeutic opportunities[END_REF] where microglia and astrocyte activation are involved in the mechanisms leading to both adverse and beneficial effects. Under physiological conditions Nkcc1 is predominantly expressed in astrocytes and microglia compared to neurons [START_REF] Hammond | Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes[END_REF] . Therefore, we wondered whether these cell populations contribute to the effect of bumetanide. This was monitored in GFAP-Nkcc1-KO mice. Although we observed an abnormal morphology of astrocytes in this transgenic line, we did not find a significant increase in interneuron survival following CCI, suggesting that astrocytic chloride homeostasis is not relevant in CCI-induced apoptosis. (See full description in Supplementary Results and Fig. S1).

TBI is known to induce a strong inflammatory reaction [START_REF] Simon | The far-reaching scope of neuroinflammation after traumatic brain injury[END_REF] . To see if TBI-induced effects on PV survival and proliferation are sensitive to microglial activation, we used the anti-inflammatory drug minocycline. Interestingly, treatment with this agent resulted in similar effects on both PV survival (Contra Veh 0.59 ±0.18 vs Contra Mino 1.21 ±0.34 and ipsilesional side Ipsi Veh 0.14 ±0.12 vs Ipsi Mino 0.74 ±0.39, Fig 2A, B, C andD) and newborn neurons (CCI Mino ipsi 0.63 ±0.17 and CCI Mino contra 1.12 ±0.10, Fig 2E, F, G andH). These results suggest that microglia activation affect post-CCI interneuron survival and adult born neuron production.

To investigate whether the effect of bumetanide depends on Nkcc1 in microglia, we used a transgenic approach for Tamoxifen (Tam) inducible knock-down of Nkcc1 in CX3CR1-positive microglia. Here, we observed that bumetanide treatment induced a significant increase in the Microglia process motility and their interactions with interneurons play an important role in PV survival [START_REF] Cserép | Microglia monitor and protect neuronal function via specialized somatic purinergic junctions[END_REF] . We found significant differences in the number of contacts between microglial cells and PV interneurons between vehicle and bumetanide-treated animals at 3 dpCCI in both the contra- Using flow cytometry, we found that microglia were more likely to express the pro-inflammatory markers CD45, MHCII and CD172a (Veh MHCII + 13.55 % ±4.91 vs Bum MHCII + 21.59 % ±6.03; Fig 4E andF) after 3 days of bumetanide treatment. To confirm this, we measured interleukins in brain tissue and found a significant increase in IL-6 in the hippocampus of CCI animals treated with bumetanide (CCI Bum 21.94 pg/mL ±6.03 vs Sham 11.49 pg/mL ±0.91 and CCI Veh 12.85 pg/mL ±3.89, Fig 4G). Since TBI is characterized by inflammatory cell infiltration promoted by opening of the blood brain barrier (BBB), we also looked at blood cells. The CD45 high -CD11 low cells (e.g., possibly lymphocytes) were few in our samples and included not only neutrophils but also other innate immune cells (e.g., monocytes, macrophages and DCs) as reported in the second gating plot of Figure 4E. However, systemic inflammation is not influenced by IP bumetanide treatment, while intracortical inflammation is reduced by the same treatment [START_REF] Tóth | The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner[END_REF] . Thus, it is plausible that the effects of bumetanide may result from resident microglial cells and not infiltrating cells.

At 7 dpCCI, we found less prominent morphological changes in microglia cells with no effect of bumetanide in both the ipsi-and contralateral side: a decrease in the number of endpoints (Sham 900.7 ±167.9 vs CCI Veh 696.5 ±86. 72 5E andF). In line with these results, we also found a significant increase of interleukin 10 (IL-10) expression in CCI animals treated with bumetanide compared to control and vehicle-treated animals (CCI Bum 5.32 pg/mL ±0.86 vs Sham 1.69 pg/mL ±0.78 and CCI Veh 2.57 pg/mL ±0. 65, Fig 5G).

These results suggest that bumetanide protects PV interneurons by increasing microglia contacts during the first post-traumatic week as well as by regulation of microglia phenotypes. This allows us to postulate that bumetanide treatment protects PV interneurons through anti-inflammatory and pro-survival mechanisms mediated by microglia.

Bumetanide increases microglia Bdnf expression

Microglia are a central source of Bdnf, an important neurotrophin controlling neuronal survival and plasticity [START_REF] Ferrini | Microglia control neuronal network excitability via BDNF signalling[END_REF][START_REF] Lima Giacobbo | Brain-Derived Neurotrophic Factor in Brain Disorders: Focus on Neuroinflammation[END_REF][START_REF] Coull | BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain[END_REF] . Therefore, we measured the level of BDNF expression in microglial-like BV2 cells after 24 and 72h of treatment. 24 hours of bumetanide treatment led to a significant increase of Bdnf levels (Ctrl 0.18 pg/mL ±0.02 vs Bum 2.79 pg/mL ±0. 18

Bumetanide regulates post-CCI microglia process motility

To further investigate microglia in vivo, we performed longitudinal 2-photon imaging in Cx3CR1 +/GFP mice following laser-induced injury on the posterior parietal cortex (see supplementary information for a full description of the procedure). Quantification of microglial process length and motility during the first week post-injury revealed that bumetanide induced a E). These experiments are in line with the effect of bumetanide on process length in CCI animals, and a decreased motility ratio is consistent with the increased number of contacts between microglia and PV interneurons.

The effect of bumetanide is physiological state dependent

Brain injury is paradoxically aggravated in microglia NKCC1-deficient mice [START_REF] Tóth | The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner[END_REF] . As under these conditions chloride uptake is impaired prior to the trauma we asked if bumetanide treatment prior to CCI would have a different effect than when administered after CCI. Interestingly, administration of bumetanide during one week before CCI worsened CCI outcomes and, more particularly, PV survival. Indeed, bumetanide-treated animals showed a significant decrease of PV interneurons compared to Sham and vehicle treated animals on the ipsilesional side (normalized value on sham: Sham 1 ± 0.05 vs CCI Veh 0.54 ±0.09 vs CCI Bum 0.10 ±0.02) and compared to Sham on the contralesional side (normalized value on sham: Sham 1 ±0.05 vs CCI Veh 0.90 ±0.12 vs CCI Bum 0.57 ±0.12) (Fig 7A andB). These results indicate that the impact of bumetanide on PV-interneuron survival is dependent of the physiological state and the mechanism disclosed in this study is mainly relevant under pathological conditions.

Discussion

How cognitive deficits observed in experimental models and clinical TBI develop following an initial concussion is not well understood [START_REF] Graham | The nature, distribution and causes of traumatic brain injury[END_REF] . In this study, we were interested how chloride homeostasis might be involved in post-traumatic changes in working memory [START_REF] Kent | Working Memory: A Selective Review[END_REF] . Theta rhythm is a potential electrophysiological biomarker of altered neuronal activity [START_REF] Buzsáki | Theta rhythm of navigation: link between path integration and landmark navigation, episodic and semantic memory[END_REF][START_REF] Vertes | Hippocampal theta rhythm: a tag for short-term memory[END_REF] and is believed to synchronize activity both within local networks and between distal cortical regions involved in cognitive processing [START_REF] O'neill | Theta Oscillations in the Medial Prefrontal Cortex Are Modulated by Spatial Working Memory and Synchronize with the Hippocampus through Its Ventral Subregion[END_REF][START_REF] Ward | Synchronous neural oscillations and cognitive processes[END_REF] . In both humans and rats, the power of theta rhythm increases during the acquisition phase of spatial learning and object recognition. Treatment-or injury-induced inhibition

A C C E P T E D M A N U S C R I P T
of theta oscillations correlates with cognitive dysfunction [START_REF] Winson | Loss of hippocampal theta rhythm results in spatial memory deficit in the rat[END_REF][START_REF] Paterno | Mild Traumatic Brain Injury Decreases Broadband Power in Area CA1[END_REF][START_REF] Pevzner | Making Waves in the Brain: What Are Oscillations, and Why Modulating Them Makes Sense for Brain Injury[END_REF] . In agreement, we found a positive correlation between decreased theta-band power and poor performance in the spatial cognitive test after one-month post-CCI. Interestingly, we found that an early transient treatment with the chloride cellular uptake inhibitor bumetanide was effective to counteract the long-term changes in both behavior and theta oscillations.

Several studies link cognitive processes with normal adult neurogenesis in the hippocampus. This corresponds with the generation of functional neurons from adult neural stem cells. TBI can induce significant changes in the proliferation and maturation of granular cells [START_REF] Tunc-Ozcan | Activating newborn neurons suppresses depression and anxiety-like behaviors[END_REF][START_REF] Gao | Moderate traumatic brain injury promotes proliferation of quiescent neural progenitors in the adult hippocampus[END_REF] . In the present study we found a significant increase in the number of Nestin-positive cells in both ipsi-and contralesional DG of mice 7 dpCCI. Nestin is a radial glia (RGL) cell marker, and represents a pool of quiescent cells that divide only occasionally [START_REF] Mignone | Neural stem and progenitor cells in nestin-GFP transgenic mice[END_REF] . The division of these RGLs leads to the generation of intermediate progenitor cells (IPCs) that will undergo a limited number of rapid divisions before entering the neuronal differentiation pathway [START_REF] Ming | Adult neurogenesis in the mammalian brain: significant answers and significant questions[END_REF] . IPCs are divided into two subtypes: i) type 2a cells that are positive for Nestin and negative for double-cortin (DCX), an immature neuronal marker, and ii) type 2b cells, positive for Nestin and DCX [START_REF] Kronenberg | Subpopulations of proliferating cells of the adult hippocampus respond differently to physiologic neurogenic stimuli[END_REF] . IPCs then give rise to neuroblasts (type 3 cells), which express DCX [START_REF] Brandt | Transient calretinin expression defines early postmitotic step of neuronal differentiation in adult hippocampal neurogenesis of mice[END_REF] . The increased number of Nestin-positive cells after TBI might intuitively suggest that there will be an increase in the number of newborn neurons. This is, however, not the case since we found a significant decrease of DCX positive cells in both the contra-and ipsilesional side of the DG. These results indicate that TBI leads to an increase in the quiescent cell pool and a resulting decrease in the number of immature granule cells consistent with previous findings [START_REF] Goubert | Bumetanide Prevents Brain Trauma-Induced Depressive-Like Behavior[END_REF] . Furthermore, treatment with bumetanide during the first week post-TBI significantly reduced the CCI-induced decrease in Nestin-positive cells, as well as increased the number of DCX-positive cells. These results suggest that the effects of bumetanide on cognitive performance could be related to positive effects on post-traumatic granule cell production. In addition, they may suggest a common upstream mechanism for the action of bumetanide.

GABA, especially released by PV interneurons [START_REF] Song | Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision[END_REF][START_REF] Giachino | GABA suppresses neurogenesis in the adult hippocampus through GABAB receptors[END_REF] , appears to play a central role in regulating the activity of RGLs by inhibiting their proliferation. Thus, post-traumatic changes in granule cells could be related to abnormal interneuron function. Indeed, we found loss of PV interneurons in the granular layer of the hippocampus, as early as 3 dpCCI in both the ipsi-and contralesional side that

A C C E P T E D M A N U S C R I P T
was sustained at 7 dpCCI and one month after trauma [START_REF] Goubert | Bumetanide Prevents Brain Trauma-Induced Depressive-Like Behavior[END_REF] . This loss of PV interneurons may also contribute to abnormal brain activity in TBI [START_REF] Almeida-Suhett | GABAergic interneuronal loss and reduced inhibitory synaptic transmission in the hippocampal CA1 region after mild traumatic brain injury[END_REF] .

The increase of Nestin-positive cells and decrease in DCX cells that we detected after TBI can thus be related to the loss of PV interneurons, which results in less ambient GABA [START_REF] Anacker | Adult hippocampal neurogenesis and cognitive flexibility -linking memory and mood[END_REF][START_REF] Ge | GABA sets the tempo for activity-dependent adult neurogenesis[END_REF] . In accordance with this assumption, treatment with bumetanide significantly counteracted the loss of PV interneurons and normalized the CCI induced change in Nestin and DCX positive cells. As these cell populations were not significantly affected in sham animals by bumetanide a direct effect on progenitors may have less prominent importance.

Neuroinflammation is observed in both the acute and chronic stages of TBI [START_REF] Lozano | Neuroinflammatory responses to traumatic brain injury: etiology, clinical consequences, and therapeutic opportunities[END_REF] . It appears to be responsible for both detrimental and beneficial effects by contributing to primary injury and secondary damage, but also facilitating tissue repair [START_REF] Woodcock | The role of markers of inflammation in traumatic brain injury[END_REF] . Both astrocytes and microglia are important players in the mechanisms of brain inflammation and, in addition, display one of the highest expression levels of Nkcc1 in the brain. Thus, it is plausible that the post-traumatic effects produced of bumetanide could be related to an influence on the dynamics of inflammatory processes [START_REF] Woodcock | The role of markers of inflammation in traumatic brain injury[END_REF][START_REF] Steenerson | Pathophysiology of Sports-Related Concussion[END_REF] .

Following this idea, we first studied the role of Nkcc1-mediated chloride uptake in astrocytes using conditional transgenic animals. Despite bumetanide having substantial effects on the morphology of GFAP-positive cells in both the contra-and ipsilateral sides in sham animals, the ablation of Nkcc1 in these cells did not rescue the loss of PV-positive interneurons. This indicates that astrocytes are not significantly involved in the bumetanide-induced increase in interneuron survival. Nevertheless, in this study we only monitored the first week post-CCI and we cannot exclude effects at more delayed stages of injury.

To define if CCI induced neuroinflammation through microglia could influence PV interneuron survival we first treated animals with minocycline, a second-generation tetracycline derivative that has an anti-inflammatory and neuroprotective action. Minocycline is known to have antiinflammatory effect via the inhibition of microglial cells activation [START_REF] Garrido-Mesa | Minocycline: far beyond an antibiotic[END_REF] and induce microglial Bdnf expression [START_REF] Miao | Minocycline promotes posthemorrhagic neurogenesis via M2 microglia polarization via upregulation of the TrkB/BDNF pathway in rats[END_REF] . Minocycline increased the survival of interneurons and the number of newborn neurons, suggesting that microglia inflammation is involved.

Interestingly, bumetanide treatment induced phenotypic changes in microglia and lead to a faster activation of microglial cells into a pro-inflammatory phenotype at early stages, which is required for cell debris removal and phagocytosis of apoptotic cells. At 7 dpCCI, microglial cells of

A C C E P T E D M A N U S C R I P T
bumetanide-treated animals acquired pro-phagocytotic characteristics. Microglia in control animals acquired pro-inflammatory characteristics slightly later, but importantly it persisted more than 7 dpCCI, resulting in a deleterious inflammatory state. Intriguingly, bumetanide increased the concentration of interleukin 6 (IL-6) 3 dpCCI in line with a recent study using the same trauma model showing that hippocampal granule cells derived release of IL-6 is triggered by renewed microglial cells. This increase in neuronal survival, allows adult neurogenesis to proceed normally and prevents behavioral deficits [START_REF] Willis | Repopulating Microglia Promote Brain Repair in an IL-6-Dependent Manner[END_REF] . One-week post-CCI, we detected higher levels of IL-10 in bumetanide-treated animals, along with a phenotype switch. Expression of IL-10 can promote neuronal and glial cell survival and dampen inflammatory responses via a number of signaling pathways [START_REF] Chen | Interleukin-10 mediates the neuroprotection of hyperbaric oxygen therapy against traumatic brain injury in mice[END_REF] . To further investigate if chloride uptake is important for microglia function, we knocked down the expression of Nkcc1 in primary microglia. We observed identical morphological changes and the same higher phagocytosis activity. This is interesting as microglia mediated phagocytosis is important for recovery as well as clearance of degenerated cells [START_REF] Fu | Phagocytosis of microglia in the central nervous system diseases[END_REF] . Inhibition of phagocytosis can lead to increase neuronal damage and decrease neuronal cell viability [START_REF] Neumann | Microglia Cells Protect Neurons by Direct Engulfment of Invading Neutrophil Granulocytes: A New Mechanism of CNS Immune Privilege[END_REF] .

In addition, we found that bumetanide treatment leads to more contact sites between microglia and PV interneurons in vivo. This is consistent with previous results showing pro-survival signaling mediated by microglia contacts on interneurons [START_REF] Cserép | Microglia monitor and protect neuronal function through specialized somatic purinergic junctions[END_REF] . These results imply that the mechanism by which bumetanide rescues PV interneurons can be related to additional effects on microglia morphology.

In accordance, bumetanide treatment leads to a significant increase of microglia processes and ramifications 3 dpCCI. Moreover, it also significantly changes microglia process motility in vivo.

The neurotrophic factor Bdnf is crucial for post-traumatic neuronal survival and plays a central role in microglia sensitive learning paradigms [START_REF] Shulga | The loop diuretic bumetanide blocks posttraumatic p75NTR upregulation and rescues injured neurons[END_REF][START_REF] Parkhurst | Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor[END_REF] as well as microglia modulation of post-traumatic network function [START_REF] Coull | BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain[END_REF] . Interestingly, we found here a significant increase of Bdnf in microglia induced by bumetanide following CCI. There is a complex interplay between neurotrophic signaling and chloride homeostasis in neurons e.g Bdnf can normalize post-traumatic GABAA mediated responses from depolarizing and hyper excitable to hyperpolarizing but have the opposite effect under physiological conditions. This could contribute to neuronal survival under pathological conditions [START_REF] Rivera | Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2[END_REF][START_REF] Shulga | Posttraumatic GABA(A)-mediated [Ca2+]i increase is essential for the induction of brain-derived neurotrophic factor-dependent survival of mature central neurons[END_REF] . Thus, changes in GABAergic transmission in combination with increased Bdnf release from microglia and increased contact between microglia and interneurons could contribute to bumetanide induced increased survival of PV interneurons. The effects observed here suggest that bumetanide directly acts on microglia. Indeed, recent work by Tόth et al [START_REF] Tóth | The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner[END_REF] using microglia Nkcc1 deficient mice are in agreement with our results. In addition, they showed that systemic application of bumetanide had positive post-traumatic effects. In contrast, deletion of Nkcc1 in microglia resulted in more severe tissue damage after ischemic stroke. Although these effects are compelling and indicate complicated actions of bumetanide, they do not provide a mechanistic view on the post-traumatic benefits of bumetanide. Moreover, Tόth et al disrupted chloride uptake before induction of ischemia. We obtained similar results, when we administered bumetanide already before induction of TBI. Thus, these results show a complex and context dependent effect of bumetanide and indicate that the mechanism disclosed in this study may apply to post-traumatic conditions. Some limitations of this study could be addressed in future research. For example, we only used males [START_REF] Caplan | Spatiotemporal Distribution of Microglia After Traumatic Brain Injury in Male Mice[END_REF][START_REF] Siebold | Cosyntropin Attenuates Neuroinflammation in a Mouse Model of Traumatic Brain Injury[END_REF][START_REF] Witcher | Traumatic Brain Injury Causes Chronic Cortical Inflammation and Neuronal Dysfunction Mediated by Microglia[END_REF] and, as previously described [START_REF] Berry | The effect of gender on patients with moderate to severe head injuries[END_REF][START_REF] Coimbra | Does sexual dimorphism influence outcome of traumatic brain injury patients? The answer is no![END_REF] , there are likely sex differences in clinical outcomes after TBI. On the other hand, no significant differences between males and females in lesion volume, neurodegeneration, blood-brain barrier (BBB) alteration and microglia activation have been found in animal models [START_REF] Jullienne | Male and Female Mice Exhibit Divergent Responses of the Cortical Vasculature to Traumatic Brain Injury[END_REF][START_REF] Mollayeva | Systematic Review of Sex and Gender Effects in Traumatic Brain Injury: Equity in Clinical and Functional Outcomes[END_REF] . A second potential limitation is that we did not fully study the infiltrating immune cells due to the limited sample size. However, it would be interesting to address this, either as in Tóth et al [START_REF] Tóth | The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner[END_REF] by using specific markers e.g. CD3, CD20 and Ly-6G, or by using transgenic mice like CX3CR1creERT2 x iDTR as in Willis et al [START_REF] Willis | Repopulating Microglia Promote Brain Repair in an IL-6-Dependent Manner[END_REF] .

In conclusion, this study shows that a treatment targeting inhibition of the chloride co-transporter Nkcc1 can regulate the activation kinetics of microglia and provides a mechanistic link for the positive effect of bumetanide on post-traumatic cognitive decline. These results also open an additional avenue to better understand chloride uptake dependent pathophysiological mechanisms in the post-traumatic brain as well as to identify the elements involved in the development of longterm sequelae following TBI.

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  , hGFAP-Cre 17 (line 77.6 mice Stock No. 024098; The Jackson Laboratory) x Nkcc1 flox 18 and B6.129P-Cx3cr1tm1Litt/j 19 and were maintained on a mixed genetic background. All animal experiments complied with the French and Finnish ethical committee approved all experimental procedures (N°: APAFIS#2797 and ESAVI/3183/2022), the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines and EU Directive 2010/63/EU for animal experiments.

  sham-vehicle (Sham Veh), sham-bumetanide (Sham Bum), CCI-vehicle (CCI Veh), and CCIbumetanide (CCI Bum).

  Imaris Software. Then contacts between microglia Parvalbumin interneurons were quantified with imageJ® plugin SynapCountJ.

  dynamics were evaluated from the segmented image by calculating the percentage of binary pixels changing in value from one time point to another[START_REF] Thévenaz | A pyramid approach to subpixel registration based on intensity[END_REF] .

  Downloaded from https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awad132/7134130 by guest on 04 May 2023 in the cerebellum. After a 72-hour recovery, EEG (amplified 31,000, filtered at 0.1-120Hz pass, acquired at 1000Hz) was monitored using a telemetric system (Emka Technologies S.A.S) for 3 days, 24 hours per day.Multisite intrahippocampal recordings were performed by implanting a one shank 16 channels linear probe (A1x16-3mm-100-177, Neuronexus) through a 1mm diameter craniotomy at the posterior parietal level (2 mm posterior, 1.5 mm left of the bregma and 1mm ventral) on head restrained animals trained on Mobile HomeCage® (MHC V5, Neurotar®). Animals were recorded 3 times 10 minutes. Recordings were acquired using the Allego software (SmartBox Pro™, Neuronexus) and a sampling rate of 30K Hz per channel.

  To investigate the effects of bumetanide on post-TBI cognitive behavior, we performed four cognitive performance tests: the Novel Object Recognition (NOR) and the Object Displacement Task (ODT). CCI induced a significant decrease in the recognition ratio of the tasks examined after normalization to Sham conditions unless otherwise stated (ODT: CCI Veh 0.38 ±0.06; NOR 5 mins: CCI Veh 0.44 ±0.20; NOR 1 h Sham 1.17 ±0.58 vs CCI Veh 0.507 ±0.31, Fig1B, C and D).Interestingly, the strongest effect of bumetanide was found in NOR with 1 h retention (CCI Bum 0.86 ±0.40, Fig 1D). We then used a spatial memory test, the Barnes Maze (BM), which showed that CCI induced a significant increase of the latency in reaching the target hole (Sham 8.00 ±0.58 vs CCI Veh 44.00 ±7.61, Fig 1E). Similarly, bumetanide treatment resulted in a better performance compared to vehicle (CCI Bum 13.00 ±1.95, Fig 1E). Finally, as NOR and ODT are strongly influenced by anxiety, we used the Elevated Plus Maze (EPM) to test this parameter and did not find significant differences in the number of entries that animals made into each zone (Center: Sham 40.00 ±0.58 vs CCI Veh 44.00 ±4.02 vs CCI Bum 41.40 ±3.11; Open arms: Sham 35.33 ±2.186 vs CCI Veh 35.00 ±6.18 vs CCI Bum 46.00 ±1.86; Closed arms: Sham 65.33 ±10.11 vs CCI Veh 79.75 ±10.82 vs CCI Bum 58.00 ±12.03) (Fig 1F). There were also no differences in the time that animals spent in the center (Sham 51.18 ±1.657 vs CCI Veh 35.00 ±5.343 vs CCI Bum 43.048 ±5.306), nor in the open arms (Sham 82.44 ±18.79 vs CCI Veh 100.70 ±23.10 vs CCI Bum ±12.39), nor in the closed arms (Sham 145.72 ±9.97 vs CCI Veh 151.96 ±18.27 vs CCI Bum 176.264 ±8.27) (Fig 1G)

  vs CCI Veh 3.42 % ±0.41, Fig 1I). Bumetanide treatment normalized the theta band power and was significantly different from vehicle treated animals (CCI Bum 6.14 % ±0.29, Fig 1I). The power of the delta band was lower in CCI animals and was not affected by bumetanide treatment (Sham 5.44 % ±0.18 vs CCI Veh 1,14 % ±0,17 vs CCI Bum 1.82 % ±0.53, Fig 1H). Similarly, the power of the gamma band was significantly higher in vehicle-treated animals compared to sham but not changed in bumetanide animals (Sham 0.39 % ±0,03 vs CCI Veh 0.89 % ±0,13 vs CCI Bum 0,70% ±0.08, Fig 1J). To assess the impact on single-unit activity we isolated putative interneuron and principal neuron single-units from intrahippocampal multisite recordings. Here we found that the spike rate of putative interneurons was significantly decreased in vehicle compared to Sham animals (Sham 23,38 ±2,42 Hz vs CCI Veh 3,26 ±0,6824 Hz) but not in bumetanide animals (Bum 15,08 ±2,274 Hz; Fig 1K). Interestingly, putative principal cell unit spike rate was not affected in all groups (Fig 1L). The number of total recorded putative interneurons was much lower in Vehicle (5) animals than Sham (10) and Bumetanide (12) treated whereas principal cell number was constant in all conditions (Sham: 47 cells; Veh:48 cells; Bum:48 cells; Fig 1M). These results suggest that bumetanide ameliorates CCI-induced changes in interneuron function.

  Downloaded from https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awad132/7134130 by guest on 04 May 2023 ipsi-and contralesional DG during the first post-traumatic week at 3 and 7 days post-CCI (dpCCI).

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  average cell size in the control condition (Tam + Bum 105732 µm² ±18324 vs ShNKCC1 + RFP 27650 µm² ±4322 vs Tam + RFP 17482 µm² ±2164). Interestingly, knockdown of Nkcc1 also induced an increase in size (Tam + ShNKCC1 + RFP 127090 µm² ±29570 vs ShNkcc1 + RFP 27650 µm² ±4322 vs Tam + RFP 17482 µm² ±2164) (Fig 3A and C). Similar results were found in BV2 cells (Fig S2). Then, we investigated the phagocytosis capacity of the cells in the different conditions. Bumetanide treatment induced a significant increase in the number of engulfed beads (Tam + Bum 28.00 ±6.66 vs ShNkcc1 + RFP 9.34 ±3.45 vs Tam + RFP 6.08 ±3.64). Knockdown of Nkcc1 had similar effect (Tam + ShNkcc1 + RFP 25.46 ±3.58 vs ShNkcc1+ RFP 9.34 ±3.45 vs Tam + RFP 6.08 ±3.64) (Fig 3B and C). These results suggest that bumetanide can regulate microglia morphology and activation by modulating Nkcc1 activity. Furthermore, bumetanide treatment in vivo induced a significant increase of microglia endpoint numbers (Sham 715.9 ±81.03 vs CCI Veh 698.8 ±331.2 vs CCI Bum 1076.0 ±85.13), number of attachment points (Sham 41.86 ±12.40 vs CCI Veh 101.9 ±43.70 vs CCI Bum 140.8 ±54.96) and Downloaded from https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awad132/7134130 by guest on 04 May 2023 soma area (Sham 1458 µm² ±652.7 vs CCI Veh 3494 µm² ±757.6 vs CCI Bum 4985 µm² ±850,6) (Fig 4A and B) in the ipsilesional DG 3 dpCCI.

(

  CCI Veh 40.00 ±24.92 vs CCI Bum 103.50 ±23.55) and ipsilesional side (CCI Veh 2.75 ±2.48 vs CCI Bum 63.20 ±9.04) (Fig 4C and D). In addition, there was no significant difference between Sham and CCI Bum in the contralesional side (Sham contra 115.9 ±13.50 vs CCI Bum contra 93 ±12.48) but a slight difference in the ipsilesional side (Sham ipsi 99.57 ±13.01 vs CCI Bum ipsi 63.20 ±9.04) (Fig 4C and D).

  Fig S2I). In addition, we quantified the immuno-like intensity of Bdnf in sham vehicle, sham bumetanide, CCI vehicle and CCI bumetanide treated animals and found a consistent increase in Bdnf expression within Iba1-positive microglia in bumetanide-treated animals either on the contralesional (normalized value on sham vehicle : Sham Veh 1.00 ±0.12 vs Sham Bum 3.55 ±0.30 vs CCI Veh 2.27 ±0,41 vs CCI Bum 5.60 ±0.75) or the ipsilesional side (normalized value on sham vehicle : Sham Veh 1.00 ±0.06 vs Sham Bum 4.28 ±0.20 vs CCI Veh 3.28 ±0.18 vs CCI Bum 12,86 ±1.06; Fig 5G, H and I). These results indicate that bumetanide induces Bdnf expression, which may play an important role in the survival of PV interneurons.

  Downloaded from https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awad132/7134130 by guest on 04 May 2023 significant decrease in process length one day post-laser injury. This difference was not significant 5 dpCCI (Veh Day 1 33.58 ±3.66 vs Bum Day 1 24.43 ±1.37; Veh Day 5 ±3.69 vs Bum Day 5 42.51 ±2.23; Fig 6B, D and C). Interestingly, bumetanide induced a significant decrease in the motility ratio at both 1-and 5-dpCCI (Veh Day 1 0.59 ±0.1 vs Bum Day 1 0.52 ±0.01; Veh Day 5 0.70 ±0.03 vs Bum Day 5 0.61 ±0.12, Fig 6B, D and

AA

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Figure 1

 1 Figure 1 Effect of bumetanide on CCI-induced behavioral changes. (A) Schematic representation of the experimental timeline. (B) The Object Displacement Task after a 5 min retention time. (C) The Novel Object Recognition task after a 1-hour retention time. (D) The Novel Object Recognition task after a 5-min retention time. For all experiments, the results are presented as a ratio of time of new versus familiar. n = 10, 11 and 10 respectively. (E) Time spent finding the target box in the Barnes Maze. (F) Number of entries in different zones of the elevated plus maze. (G) Time spent in different zones of the elevated plus maze. n = 3, 5 and 5 respectively. (H) Normalized spectral density of delta band in mice 1-month post-CCI. (I) Normalized spectral density of theta band. (J) Normalized spectral density of gamma band. n=6, 9 and 9 respectively. (K) Spike rate of narrow-spiking (NS) units. (L) Spiking rate of wide-spiking (WS) units. (M) Total number of isolated cells. Data from B were analyzed using the Kruskal-Wallis test. Data from C to G and L to M were analyzed using One-way ANOVA with Dunnett's post hoc test. Data from H to J were analyzed using One-way ANOVA with a Tukey post hoc test *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

 2 Figure 2 Effect of bumetanide and minocycline on CCI-induced changes in adult neurogenesis and PV interneuronal loss in the DG. (A) Parvalbumin (PV) labeling (white arrows) at 3 dpCCI in the contralesional (upper pictures) and ipsilesional (lower pictures) DG of sham, CCI vehicle and CCI bumetanide-treated animals (scale bar = 100 µm). (B) Quantification of PV-positive cells 3 dpCCI in the contralesional and ipsilesional DG of sham, CCI vehicle and CCI bumetanide-treated animals. (C) Same as (A) at 7 dpCCI with minocycline-treated animals added. (D) Same as (B) at 7 dpCCI. (E) Doublecortin (DCX, upper pictures) and Nestin (lower pictures) labeling at 7 dpCCI in the ipsilesional DG of sham, CCI vehicle, bumetanide-treated and minocycline-treated animals (scale bar = 100 µm). (F) Quantification of DCX-positive cells 7 dpCCI in the ipsilesional DG of sham, CCI vehicle, bumetanide-treated and minocycline-treated animals. (G) Quantification of Nestin-positive cells 7 dpCCI in the ipsilesional DG of sham, CCI vehicle, bumetanide-treated and minocycline-treated animals. (H) Same as in (E) in the contralesional DG. (I) Same as in (F) in the contralesional DG. (J) Same as in (G) in the contralesional DG. n = 7 animals per condition, 3 slices per animal. Sets of data from B, D, F, G

Figure 3

 3 Figure 3 Effect of impaired chloride uptake in microglia morphology and phagocytosis capacity. (A) Average size of primary microglia cells from CX3CR1-Cre ERT2 mice under different conditions: co-transfected with pSico-sh Nkcc1 and pCAG-RFP, treated with bumetanide and tamoxifen, transfected with pCAG-RFP and treated with tamoxifen, co-transfected with pSicosh Nkcc1 and pCAG-RFP and treated with tamoxifen. n = 4 wells per conditions. 5 cells chosen randomly per well. (B) Number of latex beads engulfed by cells in the different conditions. n = 4 wells per conditions. 12 cells chosen randomly per wells. (C) Immunostainings of the primary microglia cell culture from CX3CR1-Cre ERT2 mice in the different conditions. Anti-Iba1 was used to label microglial cells (purple), anti-RFP to label the pCAG-RFP plasmid (red), GFP to enhance the pSico-sh Nkcc1 fluorescence (green). YFP was expressed when cells were treated with tamoxifen (green) and the beads were labelled with a Rabbit IgG-DyLight 633 Complex (blue). All data set were analyzed using a one-way ANOVA test with Dunnett's post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

 4 Figure 4 Effect of bumetanide on microglia phenotypes and morphological changes after CCI in the DG 3 dpCCI. (A) Total area of Iba1+ cells soma, quantifications of Iba1+ process endpoints, attachment points and length in the ipsilesional DG. n = 5 animals, 2 slices per animal. (B) Iba1 immunostaining from sham, CCI-vehicle and CCI-bumetanide treated animals. (C) Quantification of the number of contacts between microglia (Iba1 staining) and PV interneurons at 3 dpCCI. n = 5 animals per condition, 3 slices per animal. (D) Example of PV and Iba1 immunostaining from sham, CCI-vehicle and CCI-bumetanide treated animals in the ipsilesional DG, 3D representations (upper pictures), and stack (lower pictures). (E) Flow cytometry gating plots at 3 dpCCI on the ipsilesional side. (F) Analysis of microglia phenotypes by detection of MHCII + (pro-inflammatory) and CD206 + (pro-phagocytosis) markers by flow cytometry on the ipsilesional side at 3 dpCCI. n = 5-6 animals per condition. (G) Quantity of IL-4, IL-10 and IL-6 produced in the ipsilesional hippocampus. Phenotype changes were analyzed by t-test. Number of contacts and morphological analysis (quantified using the ImageJ plugins Neurphology and SynapCountJ) were analyzed using

Figure 1 147x247

 1 Figure 1 147x247 mm ( x DPI)

  Figure 2 160x198 mm ( x DPI)

  Figure 3 145x247 mm ( x DPI) A C C E P T E D M A N U S C R I P T

  Figure 4 160x198 mm ( x DPI)

  Figure 5 160x198 mm ( x DPI)

  Figure 6 160x226 mm ( x DPI)

  Downloaded from https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awad132/7134130 by guest on 04 May 2023 microglia (Veh CD206 + 16.79 % ±2.957 vs Bum CD206 + 27.44 % ±8.635, Fig

	S C R I P T
	A N U
	M
	vs CCI Bum 659 ± 100.4) and of process length (Sham 256.7 µm ±46.75 vs CCI Veh 197.8 µm ±17.17 vs CCI Bum 192.7 µm ±42.21) (Fig 5A and B). A C C E P T E D
	Despite this, the number of contacts between microglial cells and PV interneurons still differed for
	the ipsilesional side (CCI Veh 25.20 ±12.30 vs CCI Bum 39.56 ±5.85), without difference between
	Sham and CCI Bum (Sham ipsi 57 ±6.382 vs CCI Bum ipsi 41.23 ±2.472) (Fig 5C and D).
	Surprisingly, bumetanide promoted the expression of the pro-phagocytosis marker CD206 by

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Acknowledgements

We thank Prof Eero Castren and Yves-Alain Barde for providing the BDNF antibody. We also thank dr. Tristen Hewitt for comment on the manuscript.

Funding

This work was supported by public Aix-Marseille Université (AMU); Eranet Neuron III program through the Acrobat, ANR GABGANG project and Academy of Finland project nr 341361 through CR; and by the "fondation des gueules cassées" 83-2020 through MT CAH was funded by grants of the BMBF (01EW1706) and the DFG (HU 800/10-1).

Data availability

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Competing interests

Authors report no conflict of interest.

Supplementary material

Supplementary material is available at Brain online.


Kevin Dedecker

Martin Drobek

Vincent Rouessac

Anne Julbe

A Palladium-based MOF for the Preferential Sorption of Benzene

Keywords: Metal Organic Framework, Palladium, sorption, benzene, aromatics, volatile organic compounds, Quartz Crystal Microbalance

The selective sorption of volatile aromatic compounds is a challenging issue for their total abatement. Despite the well-known affinity of palladium toward rich π systems, the studies dedicated to VOC capture with Pd(II) based-MOFs are still very scarce. Intending to shed more light on this complex topic, this work compares the adsorption properties of two isostructural MOFs [Cu(2-pymo)2]n and [Pd(2pymo)2]n, and their selectivity for the sorption of linear, cyclic or aromatic VOCs. The combination of both experimental and computational investigations highlights an increasing aromatic affinity over saturated hydrocarbons when palladium is chosen as metal center (nBenzene/nn-hexane= 1.8 at 0.5 p/p0) in the MOF framework instead of copper (nBenzene/nn-hexane= 0.7 at 0.5 p/p0). Furthermore, [Pd(2-pymo)2]n clearly exhibits a preferential adsorption of benzene over toluene (nBenzene/nToluene= 1.7 at 0.5 p/p0), due to the steric hindrance effects of the latter. The present results clearly underline the attractiveness of Pdbased MOFs for the design of selective aromatic adsorbents. Moreover, they also highlight the [Pd(2pymo)2]n MOF as a relevant candidate for the selective capture of benzene, by a synergistic combination of both charge interactions and steric hindrance effects.

Introduction

Unprecedented environmental threats resulting from human activities are altering the planet with climate change, ozone depletion, deforestation, water scarcity, and increasing pollution of water and air.

The growing emission of volatile organic compounds (VOCs) strongly contributes to the deterioration of the environment [START_REF] Carslaw | OH and HO2 Radical Chemistry in a Forested Region of North-Western Greece[END_REF][START_REF] Wang | Impacts of Biogenic Emissions of VOC and NOx on Tropospheric Ozone during Summertime in Eastern China[END_REF][START_REF] Carter | Development of Ozone Reactivity Scales for Volatile Organic Compounds[END_REF] and jeopardizes the integrity of living beings [START_REF] Li | A Review of Environmental Occurrence, Toxicity, Biotransformation and Biomonitoring of Volatile Organic Compounds[END_REF][START_REF] Sharma | Air Pollution and Control[END_REF][START_REF] Cheng | Global Research Trends in Health Effects of Volatile Organic Compounds during the Last 16 Years: A Bibliometric Analysis[END_REF] . Amongst the organic pollutants classified as VOCs, a particular attention is given to aromatic compounds such as benzene and its derivatives [START_REF] Montero-Montoya | Volatile Organic Compounds in Air: Sources, Distribution, Exposure and Associated Illnesses in Children[END_REF] , due to their deleterious effects on human health. These compounds are mainly generated by industrial emissions (e.g. petrochemical processes and derived products such as crude oil, petrol, glues, paints, furniture wax, and detergents) but also by tobacco smoke, gas stations, and motor vehicle exhausts. Benzene is notoriously responsible for a wide range of diseases including leukemia and anemia [START_REF] Belingheri | Benzene and Leukemia: From Scientific Evidence to Regulations. A Historical Example[END_REF][START_REF] Ifeanyi | A Review on Bezene and Haematological System[END_REF] . It is considered officially as a human carcinogen and the tolerated personal exposure limit is continuously decreasing (100 ppm in 1927, 1 ppm in 2000) [START_REF] Teixeira | Evaluation of Genotoxicity in a Group of Workers from a Petroleum Refinery Aromatics Plant[END_REF] . Despite a significant effort to reduce their emission, the presence of volatile aromatic compounds is hardly avoidable in urban areas. Moreover, it turns out that indoor exposure to benzene pollution may reach 1.5 times the outdoor street level due to its continuous accumulation in confined spaces [START_REF] Cocheo | Urban Benzene and Population Exposure[END_REF] . One of the considered options to preserve individuals from the noxious effects of these pollutants bears on an effective reduction/monitoring of the daily exposure by employing specifically designed materials for their capture and/or detection [START_REF] Dedecker | 5 -Metal-Organic Frameworks for the Capture of Volatile Organic Compounds and Toxic Chemicals[END_REF] . In this area, the application of activated carbons, zeolites and silica is largely discussed in the literature. However, these materials suffer from either a lack of selectivity (adsorption of a wide range of VOCs) and/or a low adsorption capacity [START_REF] Shen | Record-High Capture of Volatile Benzene and Toluene Enabled by Activator Implant-Optimized Banana Peel-Derived Engineering Carbonaceous Adsorbents[END_REF] . Despite their low cost, it may be difficult to overcome these aforementioned limitations. Hence, it makes sense to explore the potential of other relevant materials with greater chemical versatility.

Metal-Organic Frameworks (MOFs) are hybrid materials which are well known for their rich structural diversity, the versatility of their properties, and their attractive sorption performance. They are composed of inorganic bricks connected through organic ligands to form highly porous structures [START_REF] Lu | Tuning the Structure and Function of Metal-Organic Frameworks via Linker Design[END_REF] which can be advantageously tuned at the nanoscale to fit properly the requirements of each selected application. For instance, the spacer length adjustment allows to tailor the unit cell dimensions which control both the molecular sieving effect [START_REF] Liang | An Ultramicroporous Metal-Organic Framework for High Sieving Separation of Propylene from Propane[END_REF] and host-guest interactions by confining guest molecules [START_REF] Dedecker | Metal-Organic Frameworks for Cultural Heritage Preservation: The Case of Acetic Acid Removal[END_REF] .

Furthermore, the affinity to specific adsorbates can be enhanced by a judicious functionalization of ligands [START_REF] Pires | Enhancement of Ethane Selectivity in Ethane-Ethylene Mixtures by Perfluoro Groups in Zr-Based Metal-Organic Frameworks[END_REF] . It must be underlined that metal centers may also significantly affect host-guest interactions either by the presence of open metal sites or by a specific affinity of the metal for the target adsorbate(s).

As an example, the investigation of the CPO-27 series (Ni, Co, Mg, Cu and Zn) for the desulfurization of fuel [START_REF] Van De Voorde | Adsorptive Desulfurization with CPO-27/MOF-74: An Experimental and Computational Investigation[END_REF] evidenced that CPO-27(Ni) offers the highest affinity toward thiophene due to strong nickelsulfur interactions.

Amongst different transition metals, palladium holds a special place due to its ability to interact strongly with hydrogen [START_REF] Griessen | Thermodynamics of the Hybrid Interaction of Hydrogen with Palladium Nanoparticles[END_REF] and with conjugated molecules such as alkenes [START_REF] Prentice | Infrared Spectroscopic Evidence for π-Complex Formation from Ethylene Chemisorbed on Palladium and Platinum[END_REF] , alkynes [START_REF] Greaves | Metal-Acetylene Complexes. II. Acetylene Complexes of Nickel, Palladium, and Platinum[END_REF] , and aromatics [START_REF] Takahashi | Aromatics/Aliphatics Separation by Adsorption: New Sorbents for Selective Aromatics Adsorption by π-Complexation[END_REF] via π-complexation mechanism. Hence, palladium is a significant asset to boost the sorption selectivity of porous materials for this series of molecules. It has been proven that the incorporation of palladium species (e.g. Pd atoms or nanoparticles, salts, oxides or complexes) within materials is able to catalyze reactions [START_REF] Luo | Metal Organic Frameworks as Robust Host of Palladium Nanoparticles in Heterogeneous Catalysis: Synthesis, Application, and Prospect[END_REF] , contribute to H2 sorption, dissociation, and spillover [START_REF] Szilágyi | Probing Hydrogen Spillover in Pd@MIL -101(Cr) with a Focus on Hydrogen Chemisorption[END_REF][START_REF] Liu | Synergy between Palladium Single Atoms and Nanoparticles via Hydrogen Spillover for Enhancing CO2 Photoreduction to CH4[END_REF][START_REF] Contescu | Detection of Hydrogen Spillover in Palladium-Modified Activated Carbon Fibers during Hydrogen Adsorption[END_REF] , enhance sensor sensitivity [START_REF] Weber | High-Performance Nanowire Hydrogen Sensors by Exploiting the Synergistic Effect of Pd Nanoparticles and Metal-Organic Framework Membranes[END_REF] and promote VOCs capture [START_REF] Qin | Metal-Organic Framework MIL-101 Doped with Palladium for Toluene Adsorption and Hydrogen Storage[END_REF] .

Despite the appealing potentialities of palladium-based materials, only few MOFs with Pd as a metal node were reported in the literature. This is mainly due to their tedious synthesis which often requires post-synthetic modification strategies to circumvent the formation of undesired by-products obtained in one pot synthesis (e.g. isolated PdO or amorphous materials) [START_REF] Parshamoni | Synthesis of a Palladium Based MOF: Via an Effective Post-Synthetic Modification Approach and Its Catalytic Activity towards Heck Type Coupling Reactions[END_REF][START_REF] Navarro | Borderline Microporous-Ultramicroporous Palladium(Ii) Coordination Polymer Networks. Effect of Pore Functionalisation on Gas Adsorption Properties[END_REF][START_REF] He | A Practice of Reticular Chemistry: Construction of a Robust Mesoporous Palladium Metal-Organic Framework via Metal Metathesis[END_REF][START_REF] Navarro | CO2 Sorption Properties of a Series of Robust Sodalite-Type Microporous Coordination Polymers[END_REF] . In order to clearly assess the potential of Pd-MOFs for the abatement of aromatic vapors, especially the benzene removal, the present work reports a comparative study of two isostructural MOFs referred as [Cu(2-pymo)2]n 33 and [Pd(2pymo)2]n [START_REF] Navarro | CO2 Sorption Properties of a Series of Robust Sodalite-Type Microporous Coordination Polymers[END_REF] (2-pymo = 2-pyrimidinolate) exposed to four model VOC compounds: n-hexane (linear alkane), cyclohexane (cyclic alkane), benzene and toluene (aromatics). Experimental isotherms obtained by QCM (Quartz Crystal Microbalance) measurements were combined with computational adsorption results in a comprehensive study in order to highlight the role of palladium nodes for the selective sorption/capture of aromatics.

Experimental section

Materials and methods

All reagents were purchased from commercial suppliers and used without any further purification.

The structure and purity of the synthesized materials were verified by Powder X-Ray Diffraction (PXRD), N2 adsorption-desorption measurements, thermogravimetric analysis (TGA), and Fourier-Transformed infrared (FTIR) spectroscopy. The powder diffraction patterns of crystalline materials were recorded by using PANanalytical X'Pert Pro diffractometer (Ni-filtered Cu-Kα radiation, 40 kV, 20 mA) from 5 to 50° (2θ). N2 adsorption-desorption isotherms were measured at 77 K (liquid nitrogen bath) using Micromeritics ASAP 2020 equipment. Samples were outgassed for 12 h at 130°C under vacuum. Thermogravimetric analyses were carried out with TA instruments SDT 2960 under dry air at a constant heating rate of 5°C/min from 25 to 600°C. FTIR spectra were recorded from 500 to 4000 cm -1 (resolution of 4 cm -1 ) with Nicolet Nexus FT-IR apparatus in reflection mode. Vapor adsorption isotherms were recorded with a Quartz Crystal Microbalance (QCM). The QCM setup is composed of a quartz resonator (AT-cut quartz, 14 mm, 6 MHz) working with a Maxtek TM-400 controller and 0.5 L chamber with injected VOC vapors [START_REF] Rouessac | Three Characterization Techniques Coupled with Adsorption for Studying the Nanoporosity of Supported Films and Membranes[END_REF] . The pumping system (Alcatel Drytel 1025), with membrane and turbomolecular pumps, reached pressured below 10 -1 Pa for both purge and complete desorption.

Synthesis of [Pd(2-pymo)2]n and [Cu(2-pymo)2]n

The MOF synthesis protocols reported in literature were followed with only slight modifications and the final materials were analyzed to identify the products and check their purity. [Pd(2-pymo)2]n was obtained in two steps: synthesis of [PdCl2(2-Hpymo)2] followed by its base-promoted polymerization to [Pd(2-pymo)2]n [START_REF] Navarro | Borderline Microporous-Ultramicroporous Palladium(Ii) Coordination Polymer Networks. Effect of Pore Functionalisation on Gas Adsorption Properties[END_REF][START_REF] Navarro | CO2 Sorption Properties of a Series of Robust Sodalite-Type Microporous Coordination Polymers[END_REF] . In more detail, a fresh Na2PdCl4 solution was prepared by dissolving PdCl2 (0.6 mmol) in a NaCl solution (1.2 mmol in 10 mL of distillated water) under stirring and left for 12h. The product was filtered out and washed with water, yielding a yellow crystalline powder. [Cu(2pymo)2]n was produced by mixing CuCl2.2H2O (1.0 mmol) and 2-Hpymo (2.0 mmol) in 30 mL of NH4OH solution ([NH3]≈25%) and stirring at room temperature for 48h [START_REF] Barea | Mineralomimet ic Sodalite-and Muscovite-Type Coordination Frameworks. Dynamic Crystal-to-Crystal Interconversion Processes Sensitive to Ion Pair Recognition[END_REF] . The purple solid was then recovered by centrifugation and washed with water.

VOC sorption measurements using QCM

For each MOF material, VOC adsorption isotherms were recorded with QCM set-up equipped with quartz crystals (6MHz, purchased from Neyco) whose surface was covered with the selected MOF powder. Samples were prepared by depositing few drops of a suspension containing 2 mg of the MOF powder in 1 mL of methanol. The coated quartz crystals were then dried at 120°C for 2h. The deposited mass of MOF was estimated by the difference of frequencies measured by QCM before and after deposition, using the Sauerbrey equation [START_REF] Sauerbrey | The Use of Quartz Oscillators for Weighing Thin Layers and for Microweighin g[END_REF] . Before any measurement, the chamber was pumped down to limit vacuum ~10 -3 mbar in order to desorb MOF. Partial pressure was varied from 0 to 0.9 p/p0 for all four VOCs (n-hexane, cyclohexane, benzene, and toluene). The quartz holder was kept at 25°C during the whole data collection to get the mass uptake isotherm of the sorbed VOC within the MOF. The surface area (SA) and pore volume (Vp) were estimated using RASPA software [START_REF] Dubbeldam | RASPA: Molecular Simulation Software for Adsorption and Diffusion in Flexible Nanoporous Materials[END_REF] . The average occupation profile of all studied adsorbates in the MOF networks (density plot) was depicted through Grand Canonical Monte Carlo (GCMC) simulation. System sizes were selected by considering the Van der Walls cutoff (12Å) and supercells were created with lengths larger than at least twice the cutoff value. More precisely, 2x2x1 and 2x2x2 supercells for [Cu(2-pymo)2]n and [Pd(2-pymo)2]n were considered, respectively. Uncharged United-Atom TraPPE (TraPPE-UA) model [START_REF] Martin | Transferable Potentials for Phase Equilibria. 1. United-Atom Description of n-Alkanes[END_REF][START_REF] Keasler | Transferable Potentials for Phase Equilibria-United Atom Description of Five-and Six-Membered Cyclic Alkanes and Ethers[END_REF] was used for nhexane while benzene was modeled with Explicit-Atom TraPPE (TraPPE-EA) model [START_REF] Rai | Transferable Potentials for Phase Equilibria. 9. Explicit Hydrogen Description of Benzene and Five-Membered and Six-Membered Heterocyclic Aromatic Compounds[END_REF][START_REF] Rai | Transferable Potentials for Phase Equilibria. 10. Explicit-Hyd ro gen Description of Substituted Benzenes and Polycyclic Aromatic Compounds[END_REF] . Concerning MOF structures, their atoms were taken as single Lennard-Jones sites whose parameters were extracted concomitantly from UFF [START_REF] Rappé | a Full Periodic Table Force Field for Molecular Mechanics and Molecular Dynamics Simulations[END_REF] and Dreiding 42 force fields. The atomic charge assignment was ensured by the use of the classical "charge equilibration" method. Interactions between MOF structures and hydrocarbon molecules were described by a Lennard-Jones potential using Lorentz-Berthelot mixing rules. The Ewald sum technique was used to compute electrostatic interactions. The simulation was composed of 1,000,000 Monte Carlo steps for equilibration and 1,000 steps for production and the temperature was set at 25°C.

Computational Methods

Computational

Results and discussion

Description of the MOF structures

The [Pd(2-pymo)2]n framework results from the polymerization of [PdCl2(2-Hpymo)2] in alkaline medium. The latter is a square-planar complex composed of one Pd(II) atom connected to two chloride atoms and two 2-Hpymo ligands through nitrogen atoms. This Pd-N bond ensuring the connection between the metal and the ligand remains within the resulting MOF structure. The polymerization leads to the formation of a 3D microporous sodalite-type network (Figure 1). [Cu(2-pymo)2]n adopts the same topology and thus can be considered as its isoreticular form. Strictly speaking, its structure consists of sodalitic β cages (Øpore ≈ 9 Å) connected through four-and six-membered ring windows denoted 4MR and 6MR, respectively (Table 1). One may distinguish two types of 6MR windows: i) smaller and hydrophilic one decorated by carbonyl groups (6MR_CO) and ii) larger and hydrophobic one surrounded by C-H groups (6MR_H). Hence, one may consider both MOFs as amphiphilic due to the presence of pores with antagonist water adsorption behaviors. Furthermore, [Cu(2-pymo)2]n exhibits higher flexibility in comparison to its [Pd(2-pymo)2]n analog. Indeed, upon hydration, [Cu(2-pymo)2]n undergoes phase transition from cubic to rhombohedral structure [START_REF] Barea | Mineralomimet ic Sodalite-and Muscovite-Type Coordination Frameworks. Dynamic Crystal-to-Crystal Interconversion Processes Sensitive to Ion Pair Recognition[END_REF] . In order to complete the comparison between both structures and discuss their accessibility for the transport of the molecular species considered in this work, the size of pore apertures has been estimated from their crystallograph ic structures (Table 1). One may notice that the size of 6MR_CO pore aperture is larger for [Cu(2-pymo)2]n than for its analog whatever the phase, while only the rhombohedral phase exhibits larger 6MR_H pore apertures.

Synthesis and characterization of MOFs

XRD patterns of the synthesized materials allowed to identify the crystalline phases and assess their purity (Figure 2). The diffraction pattern of the synthesized [Cu(2-pymo)2]n sample corresponds to the rhombohedral phase while [Pd(2-pymo)2]n features a cubic phase. Interestingly, both MOFs seem to preserve their structural integrity when stored in air for one month (Figure S1), although [Cu(2-pymo)2]n shows slight changes in the width and intensity of the diffraction lines. This might be attributed to the higher flexibility of the Cu-based structure in comparison with its Pd-analog. Therefore, adsorbed water (air moisture) within cavities can interact with the structure and induce slight rotation of ligands, thus affecting intereticular distances. FTIR spectra do not exhibit any band corresponding to the free ligand, thus confirming the complete removal of unreacted chemicals (Figure 3). Nevertheless, the broad band at 3500 cm -1 is attributed to the presence of water molecules remaining within the MOF cavities. Overall, similar absorption spectra were obtained for both materials, essentially caused by their isostructural character. N2 adsorption-desorption isotherms were measured on outgassed samples (130°C under vacuum) and the BET method was applied to determine the experimental specific surface area (Figure S2). For both MOFs, the measured surface areas fit values reported in literature (373 vs 350 m²/g 33 and 705 vs 600 m²/g [START_REF] Navarro | Borderline Microporous-Ultramicroporous Palladium(Ii) Coordination Polymer Networks. Effect of Pore Functionalisation on Gas Adsorption Properties[END_REF][START_REF] Navarro | CO2 Sorption Properties of a Series of Robust Sodalite-Type Microporous Coordination Polymers[END_REF] for [Cu Thermogravimetric analysis of both MOFs was operated up to 600°C with a gradual temperature increase of 5°C/min under air flow (Figure S3). The TGA profile of [Cu(2-pymo)2]n encompasses three steps. A first dehydration occurs when water vapor is released from the structure between 30 and 110°C.

Lately, a second dehydration step takes place up to 250°C after which the structure collapses slowly and decomposes completely above 500°C. Note that a similar TGA profile was reported for HKUST-1

(copper based-MOF containing open metal sites), and the second desorption step corresponded to the release of water molecules strongly coordinated to copper sites [START_REF] Lestari | Solvothermal and Electrochemical Synthetic Method of HKUST-1 and Its Methane Storage Capacity[END_REF][START_REF] Chen | High Efficiency Synthesis of HKUST -1 under Mild Conditions with High BET Surface Area and CO2 Uptake Capacity[END_REF] . However, unlike HKUST-1, both Adsorption isotherms derived from QCM show an increase of adsorbed amount at p/p0<0.2 followed by a monotonous slight loading. More specifically for [Cu(2-pymo)2]n, the VOC uptake can be ranked as follows: n-hexane > benzene >> toluene ≈ cyclohexane (Figure 5.a). This trend is consistent with literature findings regarding hydrocarbon selectivity toward apolar type-MOFs. Pirngruber et al. [START_REF] Peralta | Comparison of the Behavior of Metal-Organic Frameworks and Zeolites for Hydrocarbon Separations[END_REF] showed that MOFs exhibiting apolar character such as ZIF-8, tend to adsorb alkanes over alkenes or aromatics, while ionic MOFs such as CPO-27 (cationic) or RHO-ZMOF (anionic), tend to favor conjugated molecules over alkanes. Concerning [Cu(2-pymo)2]n, different factors can explain its sorption selectivity (nBenzene/nn-hexane= 0.7 at 0.5 p/p0) (Figure S4). The high loading of n-hexane is attributed concomitantly to its smaller kinetic diameter (4.3 Å) and higher flexibility which enhance its diffusion within the structure and thus allow its accommodation efficiently within pores. In contrast, the adsorbed amount of toluene is the lowest in the series, in part due to its larger kinetic diameter (5.8 Å)

and its more rigid structure. However, despite their similar kinetic diameters, benzene and toluene clearly differ in their adsorbed amounts. This phenomenon might be explained by the particularity of aromatic molecules to organize themselves through π interactions (e.g. π-π stacking) [START_REF] Headen | Structure of π-π Interactions in Aromatic Liquids[END_REF] . Indeed, packing configurations of benzene and toluene strongly differ due to the steric hindrance generated by the -CH3 group in toluene [START_REF] Chipot | Benzene Dimer: A Good Model for π-π Interactions in Proteins? A Comparison between the Benzene and the Toluene Dimers in the Gas Phase and in an Aqueous Solution[END_REF][START_REF] Law | Dimers of Aromatic Molecules: (Benzene)2, (Toluene)2, and Benzene-Toluene[END_REF] . The higher content of adsorbed benzene compared to toluene results most likely from better pore filling caused by higher packing in MOF cages.

Computational study of the VOC adsorption

As described in the experimental part, GCMC simulations were carried out to investigate VOC adsorption within the MOF structures. Supercells were created from Crystallographic Information File (CIF) available in the Cambridge Crystallographic Data Center (CCDC) database. As evoked previously, [Cu(2-pymo)2]n can adopt either cubic or rhombohedral structure depending on its exposure to the guest molecule (e.g. water). However, only the rhombohedral topology is observed once in direct contact with the studied series of VOCs. Hence, only this particular structure is exclusively taken into consideration for the simulations. The [Pd(2-pymo)2]n structure keeps the same cubic topology whatever it is solvated or not. As we focused our utmost attention on benzene and n-hexane, the preferential location of these adsorbates was deduced for each MOF by injecting a single molecule into the supercell.

GCMC simulations clearly show that benzene and n-hexane are not localized and distributed equally in both structures (Figure 6). It is noteworthy that n-hexane does not pass through the 6MR_CO windows, probably because of their hydrophilicity.

Beyond the great value knowledge acquired on adsorption processes, information obtained from GCMC simulations also help to improve these MOFs through rational design for VOC capture purposes.

Since steric effect prevents the penetration of toluene in the [Pd(2-pymo)2]n structure, a very precise increase of the ligand length may be a straightforward option if a capture of both benzene and toluene is required. On the other hand, the selectivity toward benzene could also be improved by further repellin g other compounds such as n-hexane. As mentioned, n-hexane is only allowed to pass through 6MR_H due to its sufficient hydrophobicity (6MR_CO windows are forbidden). Moreover, replacing the _H group with hydrophilic functions such as carbonyl, hydroxyl or amine, should repel n-hexane and thus allow only benzene accommodation within the pores.

Conclusion

This study aims to explore the notorious affinity of aromatics to palladium based-materials and more

[PdCl2( 2 -

 2 Hpymo)2] was synthesized by dissolving 2-Hpymo (1.2 mmol) in the freshly prepared Na2PdCl4 solution, for 4h at room temperature. The resulting solid was then filtered out and washed with water. The preparation of [Pd(2-pymo)2]n was carried out by refluxing a solution containing [PdCl2(2-Hpymo)2] (0.4 mmol) and KOH (0.4 mmol) dissolved in 10 mL of distillated water for 20h.

  methods were used to determine the theoretical microstructural characteristics of solvent-free [Pd(2-pymo)2]n, [Cu(2-pymo)2]n (rhombohedral) and [Cu(2-pymo)2]n (cubic) structures.

Figure 1 .

 1 Figure 1. Structural description of a) [Pd(2-pymo)2]n, b) [Cu(2-pymo)2]n (cubic) and c) [Cu(2-pymo)2]n

( 2 -

 2 pymo)2]n and [Pd(2-pymo)2]n, respectively), although being slightly higher, possibly caused by the reduced amount of defects, PdO and/or better cleaning. Interestingly, these experimental values also fit theoretical ones obtained by simulation (373 vs 406 and 705 vs 694 m²/g for [Cu(2-pymo)2]n and [Pd(2-pymo)2]n, respectively. This observation tends to indicate that the MOF activation treatment (outgassing) was efficient as pores are free of any adsorbed species.

[Cu( 2 -

 2 pymo)2]n and [Pd(2-pymo)2]n do not contain open metal sites. Therefore, in the present case, one may assume that residual water is still present after the first dehydration step due to strong interactions between Cu 2+ sites and water molecules. Regarding [Pd(2-pymo)2]n, the TGA curve exhibits two main steps including desorption of water molecules up to 110°C followed by the structure collapse starting already at 330°C. The absence of the second dehydration step reveals weaker interactions between palladium sites and water, thus resulting in different adsorption behavior compared to its copper basedanalog.

Figure 2 .

 2 Figure 2. Comparison of experimental XRD patterns of the synthesized MOF powders (red) with the

Figure 3 .

 3 Figure 3. FTIR spectra of a) [Cu(2-pymo)2]n and b) [Pd(2-pymo)2]n.

Figure 4 .

 4 Figure 4. PXRD patterns of [Cu(2-pymo)2]n exposed to VOCs.

  Some differences are noticed when comparing adsorption isotherms of both isostructural MOFs. The trend observed for [Pd(2-pymo)2]n shows deviations from its Cu-based analog and the VOC adsorption preferences can be ranked as follows: benzene >> cyclohexane > toluene > n-hexane (Figure5.b). As expected, benzene is clearly the dominant adsorbate within the series. Interestingly, n-hexane and cyclohexane loadings are lower but very similar, indicating a potential weaker affinity of these molecules toward [Pd(2-pymo)2]n (nBenzene/nn-hexane= 1.8 at 0.5 p/p0) (FigureS4), reaching typically the absorption capacity of benzene ~0.42 molBenzene/mol[Pd(2-pymo)2]n. This hypothesis is reinforced by the differences in adsorption enthalpies for benzene and cyclohexane onto PdCl2 (ΔHbenzene: 39-46 kJ/mol, ΔHcyclohexane: 21-29 kJ/mol)[START_REF] Takahashi | Aromatics/Aliphatics Separation by Adsorption: New Sorbents for Selective Aromatics Adsorption by π-Complexation[END_REF] . The lower affinity of cyclohexane can be clearly explained by the lack of π-electrons in the molecule. Similar to [Cu(2-pymo)2]n, benzene and toluene do not adsorb in the same manner in [Pd(2-pymo)2]n (nBenzene/nToluene= 1.7 at 0.5 p/p0) (FigureS4). Such difference could again result from steric effect, limiting accommodation of toluene molecules in the MOF porous structure and their access to palladium sites. Therefore, one may conclude that the affinity of [Pd(2-pymo)2]n is unambiguously turned to aromatics but more importantly, this particular structure displays significan t preference for benzene over toluene. First assumptions might be proposed regarding the benzene sorption mechanisms within [Pd(2-pymo)2]n. We may hypothesize that first, benzene chemisorption occurs at low pressures due to the strong interactions with Pd nodes. Then at higher pressure physisorption may be invoked, involving π-π interactions between the "free" and the already chemisorbed benzene molecules. The results indicate that the difference of adsorption uptake is strongly affected by three main characteristics of the VOC molecule: kinetic diameter, flexibility/rigidity and presence/absence of π electrons.It should be highlighted that the overall lower adsorption capacity of [Pd(2-pymo)2]n compared to [Cu(2-pymo)2]n is counterbalanced by its remarkably high selectivity for benzene in the tested VOC series. This is particularly noticeable when comparing the adsorption isotherms per mole of adsorbent (FigureS4) but also when VOCs are considered as liquid (in mL) accommodated within the MOF porous structure (FigureS5). This finding emphasizes the interest of [Pd(2-pymo)2]n as a preferential benzene sorbent applicable for its favored removal or sensing. In comparison, the Cu-based analog can be used for the simple capture of a broad spectrum of hydrocarbon molecules when no selectivity is required. It is also noteworthy to realize that the high atomic mass of Pd (106.4 g.mol -1 ) compared to Cu (63.5 g.mol -1 ) contributes to lower the apparent adsorption capacity of [Pd(2-pymo)2]n measured in mmoladsorbate/gMOF. Hence, for a given volume of sorbent, the adsorption capacities of Pd and Cu-based MOFs are more comparable, and [Pd(2-pymo)2]n appears as a competitive material for the design of devices for the specific capture or detection of benzene.

Figure 5 .

 5 Figure 5. Experimental adsorption isotherms of selected VOCs for a) [Cu(2-pymo)2]n and b) [Pd(2-

Figure 6 .

 6 Figure 6. Density plots for a) n-hexane and b) benzene in [Cu(2-pymo)2]n. Density plots for c) n-hexane

  specifically to MOFs with Pd nodes. In this respect, this work is a valuable contribution to the current state of the art through both experimental and simulation investigations of the aromatic VOCs sorption in a Pd-based MOF. Four VOCs (n-hexane, cyclohexane, benzene and toluene) were examined to assess the sorption properties of two isostructural MOFs: [Cu(2-pymo)2]n and [Pd(2-pymo)2]n. QCM measurements showed that benzene exhibits higher affinity to Pd-based MOF while n-hexane has a preference for the Cu-based MOF. Toluene and cyclohexane are adsorbed in lower amounts due to their larger size and/or lower flexibility. GCMC simulations were useful to compare the localization of the adsorbed VOCs in both MOF structures. Benzene and n-hexane occupy hexagonal channels in [Cu(2pymo)2]n, while for [Pd(2-pymo)2]n, benzene is localized in the β cages and n-hexane passes through the pore aperture perpendicularly to the 6MR-H. Attractively, the [Pd(2-pymo)2]n MOF clearly exhibits higher selectivity to benzene over other hydrocarbons, with a consistent adsorption capacity in comparison with the same volume of [Cu(2-pymo)2]n. Overall, this study validates the highly favorable role of palladium as a metal node in MOF structure for the selective capture of aromatics. In addition, it also demonstrates that the involved charge interactions can be judiciously coupled with steric hindrance effects in the [Pd(2-pymo)2]n MOF network to favor the preferential capture and detection of benzene

Table 1 .

 1 Measured values of pore apertures, theoretical surface area, and pore volume for [Pd(2-

	pymo)2]n and [Cu(2-pymo)2]n.
	Phase	4MR	6MR_CO	6MR_H	SA	Vp
		(Å)	(Å)	(Å)	(m²/g)	(cm 3 /g)
	[Pd(2-pymo)2]n Cubic	2.82	7.86	5.00/5.00/5.00 694	0.27
	[Cu(2-pymo)2]n Cubic	3.17	8.08	3.75/3.75/3.75 571	0.21
	[Cu(2-pymo)2]n Rhombohedral 2.48	8.52	5.40/3.94/3.94 406	0.16

Table 2 .

 2 List of adsorbates, characteristics and kinetic diameters.

	Adsorbate	Type	Shape	Flexibility Kinetic	Saturation vapor
					diameter (Å)	pressure at 25°C
						(mbar)
	n-hexane	Alkane	Linear	Flexible	4.3	199.4
	Cyclohexane	Alkane	Cyclic	Flexible	6.0	130.7
	Benzene	Aromatics Cyclic	Rigid	5.8	126.9
	Toluene	Aromatics Cyclic	Rigid	5.8	37.1

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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