Tareq Al Sagheer

Obelia Haida

Anais Balbous

Maureen Francheteau

Emmanuel Matas

Pierre-Olivier Fernagut

PhD Mohamed Jaber
email: mohamed.jaber@univ-poitiers.fr

Dr Sagheer

Dr Balbous

Dr Matas

Dr Fernagut

Motor Impairments Correlate with Social Deficits and Restricted Neuronal Loss in an Environmental Model of Autism

Keywords: valproic acid, cerebellum, motor cortex, gait, Purkinje cells

Background: Motor impairments are amongst the earliest and most consistent signs of autism spectrum disorders but are not used as diagnostic criteria. In addition, the relationship between motor and cognitive impairments and their respective neural substrates remain unknown. Methods: Here, we aimed at determining whether a well-acknowledged animal model of autism spectrum disorders, the valproic acid model, displays motor impairments and whether they may correlate with social deficits and neuronal loss within motor brain areas. For this, pregnant female mice (C57BL/6J) received valproic acid (450 mg/kg) at embryonic day 12.5 and offspring underwent a battery of behavioral analyses before being killed for histological correlates in motor cortex, nigrostriatal pathway, and cerebellum. Results: We show that while valproic acid male mice show both social and motor impairments, female mice only show motor impairments. Prenatal valproic acid exposure induces specific cell loss within the motor cortex and cerebellum and that is of higher magnitude in males than in females. Finally, we demonstrate that motor dysfunction correlates with reduced social behavior and that motor and social deficits both correlate with a loss of Purkinje cells within the Crus I cerebellar area. Conclusions: Our results suggest that motor dysfunction could contribute to social and communication deficits in autism spectrum disorders and that motor and social deficits may share common neuronal substrates in the cerebellum. A systematic assessment of motor function in autism spectrum disorders may potentially help the quantitative diagnosis of autism spectrum disorders and strategies aimed at improving motor behavior may provide a global therapeutic benefit.

Introduction

Autism is one of the most common and disabling neurological disorders of childhood with no commonly implemented diagnostic test or cure available. It affects 3 times more males than females with a prevalence that has been constantly increasing these last decades to reach a proportion of 1 in 68 individuals (CDC, 2014; [START_REF] Loomes | What is the male-to-female ratio in autism spectrum disorder? A systematic review and meta-analysis[END_REF]. In the current DSM-5 classification, 2 global symptoms are recognized as hallmarks of autism spectrum disorder (ASD): disorders of social communication and restricted and repetitive behaviors [START_REF] Apa | Diagnostic and statistical manual of mental disorders[END_REF]. However, in their initial reports of autistic patients, Kanner and Asperger described early motor deficits, including clumsy gait in what is now recognized as ASD [START_REF] Kanner | Autistic disturbances of affective contact[END_REF][START_REF] Asperger | Die autistisehen psychopathen im kindesalter[END_REF]. As such, motor impairments in ASD have been receiving increased attention recently, and study of their contribution to the pathophysiology of ASD is currently a growing field of research. For instance, there are a number of qualitative and quantitative reports in ASD describing impairments in visio-motor and manual dexterity tasks, limb coordination during tasks requiring balance, agility and speed as well as in gait and ataxia [START_REF] Fatemi | Consensus paper: pathological role of the cerebellum in autism[END_REF]. Furthermore, motor impairments may be amongst the earliest signs of some forms of ASD [START_REF] Ozonoff | Gross motor development, movement abnormalities, and early identification of autism[END_REF]. Accordingly, the assessment of motor disorders might help the early and quantitative diagnosis of the pathology and the identification of dysfunctional brain regions and circuits in ASD. Such approach may also pave the way for novel therapeutic interventions that might correct motor impairments and potentially the cognitive and social deficits characteristic of ASD.

With this in perspective, the cerebellum has attracted a renewed interest in ASD as a potential brain area at the crossroads of cognitive and motor symptoms characteristic of the disease. The cerebellum is not only critical for the coordination and control of movements but also implicated in higher functions such as cognition, speech, and emotion. Indeed, the cerebellum controls balance and timing and also has an important contribution in facilitating language and executive functions, all behaviors associated with ASD [START_REF] Fatemi | Consensus paper: pathological role of the cerebellum in autism[END_REF].

Environmental factors acting in utero are suspected to strongly contribute to the etiology of ASD [START_REF] Arndt | The teratology of autism[END_REF]. Among these, valproic acid (VPA) exposure during pregnancy to manage epilepsy and mood disorders has been consistently shown to be a major factor associated with developmental defects leading to ASD [START_REF] Christianson | Fetal valproate syndrome: clinical and neuro-developmental features in two sibling pairs[END_REF]. Hence, rodent models prenatally exposed to VPA have been increasingly used to model ASD with strong construct, face, and, more recently, predictive validity at the structural, behavioral, and pharmacological levels [START_REF] Nicolini | The valproic acid-induced rodent model of autism[END_REF]. However, most if not all of these studies have solely focused on the social and cognitive aspects of ASD and have neglected motor aspects of the disease.

Here, we used the VPA model of ASD to extensively determine the motor disturbances associated with this pathology, both in males and females, and to identify associated brain histopathology. We then explored correlations between behavioral and postmortem outcomes to identify their potential links and offer a new perspective on the disease by demonstrating that motor disorders are linked with social impairments and may share common neural substrates.

Materials and Methods

Animals

Animal housing and experimental procedures were performed in accordance with the European Union directive (2010/63/EU) and validated by the regional ethical committee (approval no. 2015020415093780). C57BL/6J Mice (Charles River Laboratories) were housed in ventilated cages with access to food and water ad libitum. Room temperature was maintained at 23°C on a 12-h-light/-dark cycle.

Experimental Design and Statistical Analysis

Twenty females and 10 males were used for mating. Four females were placed with a single male and left overnight. Pregnant mice received a single i.p. injection of either VPA (450 mg/kg) or NaCl 0.9% at gestational day 12,5 (E12.5) [START_REF] Nicolini | The valproic acid-induced rodent model of autism[END_REF] when the neuronal tube is closing in rodents, followed by neurogenesis [START_REF] Morriss-Kay | Mammalian neural crest and neural crest derivatives[END_REF]. Following mating, pregnant mice were left undisturbed until they gave birth. At weaning (P21), pups from different litters were allocated to the 4 experimental groups depending on prenatal treatment: VPA males (n = 34), saline males (n = 27), VPA females (n = 25), and saline females (n = 30). The experiment timeline is presented in Figure 1. The battery of tests was performed in the least stressing and challenging order to avoid potential training and learning effects. Animals were tested during their light cycle, and the experimenter was blind to the treatment until all study and analyses were completed.

Data are expressed as mean ± SEM and analyzed using GraphPad Prism-7 software. Data that followed a normal distribution (all data except grooming and raring behavior) were analyzed using 1-or 2-way ANOVAs whenever appropriate. Upon significant main effects, Tukey's or Fisher's LSD multiple comparisons were performed for behavioral or histological measures, respectively. When data did not follow a normal distribution, we conducted nonparametric tests (Kruskal-Wallis) followed by Dunn's multiple comparison tests. Pearson correlations between behavioral and neuroanatomical readouts were performed using SPSS Statistics version 21 (IBM). For all analyses, P <.05 was considered significant.

Assessment of Developmental Milestones (P9-P16)

During early postnatal life (1-3 weeks), pups were kept in their home cage and righting reflex and eye opening were assessed. To assess righting reflex, mice were placed in the supine position and the time taken to right was monitored 3 times with a 5-minute inter-trial interval. Eye opening was assessed daily

Significance Statement

The paper's main message is that motor impairments in ASD may be indicative of this pathology and may even underlie some of its core cognitive and behavioral features. For this, we conducted a detailed analysis of motor and social behavior in both sexes of a VPA mouse model and correlated these findings to cell loss in specific brain areas such as the cerebellum and motor cortex. Our work brings evidence that motor behavior can be used as a quantitative approach for the diagnosis of ASD and is indicative of social deficits. This opens new avenues in the diagnosis and treatment of ASD targeting the affected brain areas.

SHIRPA Primary Screen (P30)

We implemented the primary SHIRPA screen that serves to identify global disturbances in gait, posture, and muscle tone as well as motor control and coordination abnormalities. Behavior was analyzed in a transparent Plexiglas arena (55 × 33 × 22 cm) with 11-× 11-cm square grid on the bottom and a 3-mm metal wire crossing diagonally on top. The transfer reaction (time to initiate movement after being placed in the arena and distance covered over 30 seconds) was first evaluated, followed by the wire maneuver test to assess motor coordination and muscle function. Negative geotaxis to assess postural stability and coordination in space was evaluated as the time taken to turn and climb a 45° inclined grid [START_REF] Rogers | Behavioral and functional analysis of mouse phenotype: SHIRPA, a proposed protocol for comprehensive phenotype assessment[END_REF].

Spontaneous Activity in the Cylinder (P30)

Spontaneous activity in the cylinder was performed as previously described [START_REF] Fleming | Assessment of sensorimotor function in mouse models of Parkinson's disease[END_REF]. Mice were put in a transparent Plexiglas cylinder (diameter: 12 cm), and their activity was videotaped for 3 minutes. Number of rearings and time spent grooming were quantified.

Assessment of Motor Coordination on the Challenging Beam (P33)

The challenging beam was performed as described previously [START_REF] Fleming | Early and progressive sensorimotor anomalies in mice overexpressing wild-type human alpha-synuclein[END_REF][START_REF] Fleming | Assessment of sensorimotor function in mouse models of Parkinson's disease[END_REF]. The beam consists of four Plexiglas sections (25 cm length) starting with a width of 3.5 cm and gradually narrowed to 0.5-by 1-cm decrements. Animals were first trained for 2 days to traverse the beam starting at the widest section and ending at the narrowest section that led into the home cage. On the test day, a mesh grid (1-cm squares) was placed over the beam surface. Animals were videotaped while traversing the grid-surfaced beam for 5 trials. Time to traverse, errors, number of steps, and errors per step made by each animal were measured and averaged.

Spatial, Temporal, and Kinetic Gait Parameters (P34)

Gait was analyzed during spontaneous walk using an automated gait analysis system (Viewpoint). The apparatus is made of a 1.5-m-long glass corridor with a dim green light beamed into the glass walkway. The light is reflected downward and a highspeed camera captures footprints' spatial and kinetic parameters. Each mouse was assessed individually for 3 consecutive runs. The following parameters were analyzed: (1) stride length: distance between 2 consecutive placements of the same paw, (2) limb base of support: distance between 2 pair prints at contact during each step cycle, and (3) pair gap: gap between the placement of the 2 trailing feet, which measures spatial coordination between the 2 pairs.

Assessment of Sociability in the Three Chambers Test (P35-P45)

Social interaction was assessed using the 3-chambers test [START_REF] Moy | Sociability and preference for social novelty in five inbred strains: an approach to assess autisticlike behavior in mice[END_REF]. The apparatus consists of a Plexiglas box (60 × 45 × 22 cm) partitioned into 3 chambers with retractable doorways. The first phase (PHASE-I) comprises 2 identical nonsocial stimuli (inverted wire cups) placed in the opposite chambers. The second phase (PHASE-II) comprises a nonsocial stimulus and a social stimulus (a naïve mouse with no previous contact with the tested animal). Each phase was of 10 minutes, during which time spent in each chamber was recorded. Subsequently, a sociability index (SI) was calculated as follows: (time exploring social chamber -time exploring nonsocial chamber) / (time exploring social chamber + time exploring nonsocial chamber).

Tissue Processing and Immunohistochemistry

After behavioral assays (P45), males and females from each group were randomly selected for histopathological analysis. Mice were deeply anesthetized with ketamine/xylazine (120/20 mg/ kg) and transcardially perfused with 0.9% saline at 37°C followed by 4% paraformaldehyde at 4°C. Brains were post-fixed in 4% paraformaldehyde at 4°C for 24 hours before cryoprotection in 30% sucrose at 4°C for 48 hours. Serial 50 µm (cerebellum) and 40 µm (striatum, cortex, and substantia nigra) free-floating sections were collected and stored at -20°C until use.

Purkinje cells (PC) within the cerebellum, dopaminergic neurons within the substantia nigra, and neurons in the striatum and the motor cortex were quantified. Every fourth cerebellar section was mounted on gelatin-coated slides, PC were identified by cresyl violet staining as previously described Pregnant dams where treated with either the antiepileptic agent valproic acid (VPA) (450 mg/kg) or NaCl 0.9% on embryonic day 12.5 (E12.5). Sex and age matched pups where separated from the dams on postnatal day 21 (P21) and raised by groups of 4 in a randomized fashion to avoid littermate effects. Comprehensive behavioral screening was performed between P30 and 45. Mice were then killed, brains harvested for histological analysis. [START_REF] Woodruff-Pak | Stereological estimation of purkinje neuron number in C57BL/6 mice and its relation to associative learning[END_REF], and their phenotype was further confirmed with calbindin immunohistochemistry (1:2500; Cb-38a, Swant). However, calbindin was not used for PC quantification, as VPA treatment can lead to reduced calbindin protein expression [START_REF] Main | Repeated prenatal exposure to valproic acid results in cerebellar hypoplasia and ataxia[END_REF]. Every 6th section throughout the entire striatum, substantia nigra, and motor cortex was selected and processed for either neuronal nuclei antigen (NeuN) to quantify neurons in the striatum and the motor cortex or for tyrosine hydroxylase (TH) immunoreactivity to quantify dopaminergic neurons in the substantia nigra pars compacta (SNc). Sections were incubated for 90 minutes in a blocking solution (3% bovine serum albumin, 0.3% Triton X-100 in PBS 1 M, pH 7.4). Rabbit anti-NeuN (1:500; Ab177487, abcam) or mouse anti-TH (1:5000; 22941, Immunostar) was applied overnight at 4°C. Biotinylated anti-rabbit or antimouse IgG was used as secondary antibody (1:250; BA-1000 and BA-9200, Vector laboratories) for 1 hour at room temperature. Signal was amplified with an ABC Elite kit and revealed with diaminobenzidine (Vector Laboratories). Sections were mounted on gelatin-coated slides and processed for cresyl violet counterstaining.

Stereology

Stereological estimates were performed using the optical fractionator method and systematic random sampling to obtain the total number of cerebellar PC, motor cortex neurons, striatal neurons, and dopaminergic nigral neurons. Each region of interest was outlined based on the mouse brain atlas [START_REF] Franklin | The mouse brain in stereotaxic coordinates[END_REF] at 2.5× objective and neurons were counted at 40× objective using Mercator Software (Explora Nova). Upper and lower guard zones of 1 µm were set at the top and bottom of the section to exclude lost profiles and each neuron or visible nucleus was counted (See Supplementary Table 1).

Results

VPA Mice Showed Delayed Postnatal Development

Delays in eye opening and righting reflex were assessed at P9-P16 as indicative of general postnatal development. For eye opening (Figure 2A), 2-way ANOVA revealed a significant effect of treatment [F(3, 560) = 22.60, P < .0001], age [F(4, 560) = 830.9, P < .0001], and treatment × age interaction [F(12, 560) = 6.623, P < .0001]. VPA males showed a significant delay at P13 (P < .001), P14 (P < .001), and P15 (P < .01). Delay appeared earlier and persisted longer in VPA males compared with VPA females, which showed a delay only at P14 (P < .001). Regarding the righting reflex, 2-way ANOVA showed a significant effect of treatment [F(3, 336) = 31.05, P < .0001], age [F(2, 336) = 323.3, P < .0001], and treatment × age interaction [F(6, 336) = 18.64, P < .0001] (Figure 2B). VPA males showed a significant latency in righting at P9 and P11 compared with saline (P < .0001), and VPA females displayed an increase in the latency only at P9 (P < .0001). Thus, VPA prenatal exposure induces significant developmental delays in males compared with saline and that are more pronounced than in VPA females.

VPA Mice Showed Altered Behavior in the SHIRPA Primary Screen

VPA mice showed altered behavior in different SHIRPA parameters tested at P30 (Figure 2C-F). Time spent immobile following transfer to the SHIRPA arena was assessed, and 2-way ANOVA revealed a significant effect of treatment [F(1, 112) = 214.2, P < .0001], sex [F(1, 112) = 136.7, P < .0001], and treatment × sex interaction [F(1, 112) = 155.6, P < .0001] (Figure 2C). Only VPA males showed a 3-fold increase in immobility compared to saline Downloaded from https://academic.oup.com/ijnp/article/21/9/871/4995459 by guest on 11 November 2020 (P < .0001). Directly after transfer to the SHIRPA arena, spontaneous locomotor activity was assessed and an effect of treatment [F(1, 112) = 129.4, P < .0001], sex [F(1, 112) = 14.64, P < .001], and treatment × sex interaction [F(1, 112) = 90.78, P < .0001] were found (Figure 2D). VPA males showed a significant decrease in locomotor activity vs saline males (-50%, P < .0001) while no difference was observed in females.

For the wire maneuver test, 2-way ANOVA revealed a significant effect of treatment [F(1, 112) = 21.40, P < .0001], and posthoc analysis showed that both VPA males and females were different from saline as they spent respectively +15% (P < .05) and +25% (P < .001) more time to climb (Figure 2E). Prenatal exposure to VPA also affected negative geotaxis with a significant effect of treatment [F(1, 112) = 16.62, P < .0001] on time needed to turn on the grid that was only observed in VPA females (P < .001). In addition, treatment [F(1, 112) = 117.0, P < .0001] and sex [F(1, 112) = 6.557, P = .0118] had an effect on the time needed to climb the grid (Figure 2F). Both VPA males and females spent significantly more time than controls to climb the grid (+78% and +59% respectively, P < .0001). This increase in time to climb was more pronounced in VPA males compared with VPA females (P < .05).

VPA Mice Showed Core ASD Symptoms

The 3-chambers test was used to assess social behavior in 5to 6-week-old male and female mice prenatally exposed to VPA or saline. None of the treatment groups, regardless the sex, showed a spontaneous preference for any of the chambers during the 10-minute habituation (PHASE I) (Figure 3A). As expected, there was a significant effect of treatment on PHASE II when a mouse was introduced to one of the chambers [F(1, 128) = 243.8, P < .0001]. Indeed, while saline males spent more time in the social chamber than in the empty chamber (69% vs 31%, P < .0001), VPA males spent similar time in both chambers (Figure 3B). This effect was not observed in females that spent more time in the social chamber than in the empty chamber, regardless of the treatment. The effect of VPA on sociability of male mice was even more striking when results were expressed as SI, with a significant effect of treatment [2-way ANOVA F(1, 64) = 6.285, P < .05], sex [F(1, 64) = 5.453, P < .05] and treatment × sex interaction [F(1, 64) = 25.75, P < .0001] (Figure 3C). Posthoc analysis showed that VPA males have a 7-fold decrease in sociability (P < .0001) compared with saline males, while females had similar SI regardless of the treatment. Repetitive and restricted behaviors are among the core symptoms and the earliest signs of ASD [START_REF] Apa | Diagnostic and statistical manual of mental disorders[END_REF]. As the grooming and rearing data do not follow a normal distribution, Kruskal-Wallis nonparametric test was conducted. Time spent grooming in the cylinder test was significantly affected by treatment [P < .0001] (Figure 3D) in both VPA males (+69%, P < .001) and females (+41%, P < .05) compared with their saline counterparts (Dunn's multiple comparison test). Additionally, Kruskal-Wallis test revealed a significant change (P < .0001) in rearing behavior (Figure 3E) as VPA males showed a significant reduction in rearing behavior compared with saline (-41%, P < .001) and with VPA females (-50%, P < .001) (Dunn's multiple comparison test).

Both VPA Males and Females Show Significant Deficits in Motor Coordination and Gait

On the challenging beam test, the time needed to traverse the beam was not affected by the treatment [F(1, 112) = 0.0045, P = .9462] (Figure 4A). However, the treatment affected errors per step [F(1, 112) = 67.04, P < .0001] as both VPA males and females showed a significant increase in errors per step compared with saline (+125% and +110%, respectively, P < .0001; Figure 4B). There was also a significant effect of beam section [F(3, 448) = 101.0, P < .0001], group [F(3, 448) = 60.61, P < .0001] and beam section × group interaction [F(9, 448) = 3.135, P < .0011] (Figure 4C). Indeed, male and female VPA mice made more errors on the second, third, and fourth beam sections compared with their saline counterparts (P < .0001 each).

Gait analysis indicated that VPA treatment did not affect paw area, speed, or regularity of the run in males or females. However, there was a significant effect of treatment [F (3, 220) = 49.86, P < .0001] on pair gap (Figure 4D). Indeed, when compared with saline, VPA males and females showed significant increase in right (+2.3-fold, P < .0001) and left pair gap (+1.62-fold, P < .01). In addition, there was a significant effect of treatment [F(3, 220) = 33.74, P < .0001] on limb base of support (Figure 4E). Posthoc analysis showed that both VPA males and females have a significant increase in both right and left limbs base of support when compared with saline counterparts (+29 and +40%, respectively, P < .0001), indicating that a significant ataxic gait is present in both males and females following prenatal exposure to VPA. Similarly, there was an effect of VPA [F(1, 109) = 46.39, P < .0001] on stride length (Figure 4F), with a decreased stride length in both VPA males and females (-6.5%, P < .0001) compared with their respective saline controls. Altogether, these results indicate major deficits in gait in both VPA males and females.

Cell Loss in Motor Brain Areas of VPA Mice

In the cerebellum, we focused our analyses on lobules VI and VII that were found to be the most affected in ASD [START_REF] Courchesne | Abnormality of cerebellar vermian lobules VI and VII in patients with infantile autism: identification of hypoplastic and hyperplastic subgroups with MR imaging[END_REF][START_REF] Skefos | Regional alterations in purkinje cell density in patients with autism[END_REF] (Figure 5). In lobule VI (SIM, 6cb), there was no effect of treatment on PC cell number (Figure 5A-B). However, there was an effect of treatment in lobule VII depending on the sub-lobules (Crus I, Crus II, PM, and 7cb, Figure 5J-K) and sex of the VPA-treated mice. Two-way ANOVA revealed a significant effect of treatment [F(1, 34) = 16.28, P < .001] and sex [F(1, 34) = 25.3, P < .0001] in Crus I. Posthoc analysis revealed a significant PC loss in VPA males in Crus I (-28%, P < .001) and a trend in VPA females (P = .0594) compared with saline (Figure 5C). There was also a significant effect of treatment in Crus II [F (1, 34) = 8.502, P < .01] where VPA females, but not males, showed a significant reduction of the number of PC (-20%, P < .01) (Figure 5D). These findings indicate that prenatal VPA treatment caused sex-dependent and specific regional loss of PC in the hemisphere part of the lobule VII.

To determine whether deficits in motor behavior may be related to a loss of cortical neurons, we quantified NeuN immunoreactive neurons in the primary and secondary motor cortex. Two-way ANOVA revealed a significant sex × treatment interaction [F(1, 37) = 17.61, P < .001], and posthoc analysis showed a significant cortical cell loss in VPA males (-17%, P < .001; Figure 5G). This loss was not observed in females (P = .0916), indicating that VPA treatment induces sex-dependent alterations in number of neurons in the motor cortex.

We then explored the effect of VPA treatment on the nigrostriatal pathway, known to be involved in motor control and production of movement. Prenatal VPA exposure did not affect the number of striatal neurons (Figure 5H) or the number of dopaminergic neurons in the SNc (Figure 5I), indicating that motor deficits cannot be attributed to striatal or nigral neuronal loss.

Relationships between Behavioral and Histopathological Findings

Correlation analyses were performed to determine potential links between behavioral and histological readouts that may be affected by VPA exposure (Table 1). Interestingly, more correlations were observed in males (46/136) than in females (17/136) (Fisher exact test: P < .0001). In males, developmental delays observed at P9-16 correlated with motor impairments at a more advanced age (P30-34). For example, the delay of open eyes correlated with the increase of the time spent to climb the grid and with the increase of the left pair gap. The longer righting reflex correlated with the increase of the right pair gap and both delay of open eyes and righting reflex correlated with an increased immobility time in the transfer reaction and a decreased locomotor activity. Moreover, the righting reflex also correlated with the SI. Interestingly, the SI correlated with several motor impairments such as the number of errors per step, time to climb the grid, right pair gap, immobility time, and locomotor activity. Altogether, these data indicate that in VPA-exposed males, the severity of motor impairments correlates with decreased sociability. In addition, neuronal loss in males appears to be linked to a number of behavioral parameters. Indeed, the number of PC in Crus I correlated with motor impairments (errors per step, stride length, distance between the right pair print) and social deficits. The number of neurons in the motor cortex correlated with the time of righting reflex and with the distance of right and left pair gap. These two histological parameters also both correlated with the delay of eye opening, time of immobility and to climb the grid, and finally with the SI.

Collectively, this analysis revealed that, in males: (1) developmental delays correlate with motor impairments later assessed, with social interaction deficits and with neuronal loss in Crus I and motor cortex; (2) motor disturbances correlate with social interactions deficits and neuronal loss; (3) neuronal loss also correlate with the decrease of the SI. Finally, (4) developmental delays, motor impairments, sociability and neuronal loss are all inter-related in males.

In females, the time to turn and climb the grid and time to climb the wire are all correlated. Performances on the grid are also correlated with the distance between the right pair print, which also correlates with the right pair gap. PC loss in Crus II correlated with the time to climb the wire and with the stride length. Interestingly, the number of PC in Crus I also correlated with stride length and developmental delay observed with the righting reflex test. However, there was no correlation between PC counts in Crus I and II and the number of errors per step.

Discussion

Increasing evidence indicates that motor impairments are present in children with ASD [START_REF] Ming | Prevalence of motor impairment in autism spectrum disorders[END_REF]. However, their contribution to the overall clinical expression of the disease remains undetermined. Standardized and quantifiable procedures to explore motor disorders may permit early and reproducible diagnosis of this disease. For instance, 5-year-old ASD children show lower gross and fine motor scores, greater praxis errors, lower movement rates, and greater movement variability [START_REF] Kaur | Comparing motor performance, praxis, coordination, and interpersonal synchrony between children with and without autism spectrum disorder (ASD)[END_REF]. Focusing on motor behavior may also allow a better identification of the underlying brain circuitry involved and that may be common to behavioral hallmarks of ASD [START_REF] Stoodley | Altered cerebellar connectivity in autism and cerebellar-mediated rescue of autism-related behaviors in mice[END_REF].

Here, we show that prenatal exposure to VPA (1) provoked early developmental deficits and delayed central nervous system maturation; (2) produced ASD social core features in males but not in females; (3) resulted in deficits in motor coordination and gait in males and, to a lower extent, in females; (4) induced cerebellar PC loss in specific lobules and in relation to sex; and (5) decreased the number of motor cortex neurons only in males. In addition, we show that motor behavior is directly correlated with social deficits and is indicative of the extent of cell loss within the cerebellum and the motor cortex.

The VPA rodent model of ASD is widely used due to its proven construct, face, and, more recently, predictive validity [START_REF] Nicolini | The valproic acid-induced rodent model of autism[END_REF]. This model stems directly from clinical observations showing that prenatal VPA exposure increases up to 10-fold the risk to develop ASD [START_REF] Moore | A clinical study of 57 children with fetal anticonvulsant syndromes[END_REF][START_REF] Rasalam | Characteristics of fetal anticonvulsant syndrome associated autistic disorder[END_REF]. Injection of VPA to pregnant rodent females systematically induces ASD-like syndromes in the offspring [START_REF] Rodier | Embryological origin for autism: developmental anomalies of the cranial nerve motor nuclei[END_REF] such as impaired social interaction [START_REF] Schneider | Behavioral alterations in rats prenatally exposed to valproic acid: animal model of autism[END_REF]JW Kim et al., 2014;KC Kim 2014), repetitive behavior [START_REF] Gandal | Validating γ oscillations and delayed auditory responses as translational biomarkers of autism[END_REF]JW Kim et al., 2014), and delayed motor development [START_REF] Schneider | Behavioral alterations in rats prenatally exposed to valproic acid: animal model of autism[END_REF][START_REF] Main | Repeated prenatal exposure to valproic acid results in cerebellar hypoplasia and ataxia[END_REF]. Few studies explored motor performances in this model and reported impaired swimming performance [START_REF] Schneider | Behavioral alterations in rats prenatally exposed to valproic acid: animal model of autism[END_REF] and righting reactions [START_REF] Wagner | A new neurobehavioral model of autism in mice: pre-and postnatal exposure to sodium valproate[END_REF].

Here, and in agreement with previous results, VPA prenatally exposed males and females displayed a delay in eye opening [START_REF] Roullet | Behavioral and molecular changes in the mouse in response to prenatal exposure to the anti-epileptic drug valproic acid[END_REF] and an increased latency to righting [START_REF] Iijima | Distinct defects in synaptic differentiation of neocortical neurons in response to prenatal valproate exposure[END_REF], indicating that postnatal development and motor reflexes are affected. VPA males displayed reduced rearing in the cylinder test, increased immobility, and reduced locomotor activity in the SHIRPA arena. This is in line with previous findings obtained in older animals showing reduced locomotor activity following prenatal VPA treatment [START_REF] Kataoka | Autism-like behaviours with transient histone hyperacetylation in mice treated prenatally with valproic acid[END_REF][START_REF] Yamaguchi | Environmental enrichment attenuates behavioral abnormalities in valproic acid-exposed autism model mice[END_REF] and with clinical studies showing that children with ASD spend less time actively exploring their environment [START_REF] Pierce | Evidence for a cerebellar role in reduced exploration and stereotyped behavior in autism[END_REF].

Motor stereotypies, repetitive and incontrollable motor behaviors, are among the core symptoms of ASD (APA, 2013). In rodents, grooming behavior is often suggested to reflect repetitive/stereotypic behavior [START_REF] Kalueff | Neurobiology of rodent self-grooming and its value for translational neuroscience[END_REF]. In our study, VPA mice show increased grooming, confirming previous reports with the same animal model [START_REF] Gandal | Validating γ oscillations and delayed auditory responses as translational biomarkers of autism[END_REF][START_REF] Castro | The effect of ketogenic diet in an animal model of autism induced by prenatal exposure to valproic acid[END_REF]. Motor coordination was further explored using the challenging beam test, usually performed in animal models of Parkinson's disease [START_REF] Fleming | Early and progressive sensorimotor anomalies in mice overexpressing wild-type human alpha-synuclein[END_REF][START_REF] Fleming | Assessment of sensorimotor function in mouse models of Parkinson's disease[END_REF]. Both VPA males and females showed a significant increase in the number of errors made during the beam crossing, indicating major motor coordination deficits. This is line with recent findings in chronically treated rats with VPA showing altered beam walking and motor coordination on a rotarod [START_REF] Main | Repeated prenatal exposure to valproic acid results in cerebellar hypoplasia and ataxia[END_REF].

Gait disturbances and motor development delays were reported in ASD patients [START_REF] Yirmiya | The prodrome of autism: early behavioral and biological signs, regression, peri-and postnatal development and genetics[END_REF]. We found altered stride length, pair base of support, and pair gap in our VPA mice models, in line with recent findings with mice bearing a TSC1 genetic knock-out [START_REF] Tsai | Autistic-like behaviour and cerebellar dysfunction in purkinje cell tsc1 mutant mice[END_REF] duplication [START_REF] Piochon | Cerebellar plasticity and motor learning deficits in a copy-number variation mouse model of autism[END_REF] they also in line with recent findings reported in rats exposed to VPA [START_REF] Main | Repeated prenatal exposure to valproic acid results in cerebellar hypoplasia and ataxia[END_REF]. Interestingly, analyses of family videos have shown that, within a year, movements and postures of children that will later be diagnosed with ASD were asymmetric [START_REF] Esposito | An exploration of symmetry in early autism spectrum disorders: analysis of lying[END_REF]. [START_REF] Karmel | Early medical and behavioral characteristics of NICU infants later classified with ASD[END_REF] showed that a decrease in arm tone is present at the age of 1 month in infants who will later develop ASD [START_REF] Karmel | Early medical and behavioral characteristics of NICU infants later classified with ASD[END_REF]. Furthermore, the presence or absence of fine motor delay at 14 months could predict the developmental trajectory of children who develop ASD [START_REF] Landa | Developmental trajectories in children with and without autism spectrum disorders: the first 3 years[END_REF], and 70% of high-risk ASD babies who showed early motor retardation were found to later develop a communication deficit [START_REF] Bhat | Relation between early motor delay and later communication delay in infants at risk for autism[END_REF]. Motor deficits may even aggravate social deficits via an impaired ability to properly interact with the environment and with peers. For instance, Crus I, which is affected in ASD and in the VPA animal model as described below, is involved in visuospatial, motor, and cognitive processing and is thus critical to interpreting the gestures of others but also to guide skilled behavior and imitation, all essential for normal social interaction and all affected in ASD [START_REF] Marko | Behavioural and neural basis of anomalous motor learning in children with autism[END_REF][START_REF] Nebel | Intrinsic visual-motor synchrony correlates with social deficits in autism[END_REF].

Beyond motor behavior, the present study points to a major sex-difference in the effect of VPA exposure on social behavior. We confirm previous findings in male mice prenatally exposed to VPA showing deficits in social behavior [START_REF] Roullet | Behavioral and molecular changes in the mouse in response to prenatal exposure to the anti-epileptic drug valproic acid[END_REF]JW Kim et al., 2014) and we further report that, in contrast, VPAexposed females developed normal social interaction skills. This suggests that clinical and preclinical exploration based exclusively on social and cognitive readouts may miss some of the neurodevelopmental consequences of VPA exposure in females.

Postmortem and brain imaging studies have consistently identified the cerebellum as one of the most abnormal brain region associated with ASD, and postmortem brains analyses showed a specific loss of cerebellar PC [START_REF] Bailey | A clinicopathological study of autism[END_REF][START_REF] Wegiel | Stereological study of the neuronal number and volume of 38 brain subdivisions of subjects diagnosed with autism reveals significant alterations restricted to the striatum, amygdala and cerebellum[END_REF]. Here we show a loss of PC within the hemispheric part of lobule VII, in Crus I in males and in Crus II in females. The extent and regionally restricted loss is comparable to those found in postmortem ASD brains [START_REF] Skefos | Regional alterations in purkinje cell density in patients with autism[END_REF][START_REF] Wegiel | Stereological study of the neuronal number and volume of 38 brain subdivisions of subjects diagnosed with autism reveals significant alterations restricted to the striatum, amygdala and cerebellum[END_REF]. Interestingly, PC loss in Crus I was found to be correlated with decreased social interaction in VPA males in accordance with the known cognitive role of this sub-lobule in communication and speech in human, both altered in ASD [START_REF] Stoodley | Functional topography of the cerebellum for motor and cognitive tasks: an fmri study[END_REF].

The cerebellum is reciprocally connected to areas involved in motor control such as the striatum, the motor, and premotor cortex [START_REF] Kelly | Cerebellar loops with motor cortex and prefrontal cortex of a nonhuman primate[END_REF][START_REF] Bostan | The basal ganglia communicate with the cerebellum[END_REF]. Our results show no change in the number of striatal or dopamine nigral neurons of VPA mice, in accordance with postmortem analysis in the striatum of ASD patients [START_REF] Wegiel | Stereological study of the neuronal number and volume of 38 brain subdivisions of subjects diagnosed with autism reveals significant alterations restricted to the striatum, amygdala and cerebellum[END_REF]. In contrast, the number of cortical neurons was decreased in VPA males. This resonates with a previous study showing altered cellular morphology in the motor cortex neurons of a VPA rat model of autism [START_REF] Snow | Altered morphology of motor cortex neurons in the VPA rat model of autism[END_REF]. All together our study indicates that the teratogenic effects of VPA on ASD symptoms extend beyond expression of social skills deficits, mainly observed in males, to reach motor deficits and specific cell loss that are observed in both males and females. As such, some forms of ASD may be equally expressed in both sexes but underdiagnosed in women who develop better social coping strategies than male ASD patients [START_REF] Tierney | Looking behind the mask: social coping strategies of girls on the autistic spectrum[END_REF]. This is the first study showing strong interrelationships among development, sociability, motor performances, and neuronal loss in any mouse model of ASD. Our study indicates that motor impairments are directly correlated to social interaction deficits and to cell loss. Moreover, the more profound cell loss, the more deficits are observed both in social interactions and motor behavior.

Thus, we point out here that motor impairment is a relevant and easily implementable biomarker of ASD severity and suggest that a biomechanical assessment of the children's motor abilities may be a precious adjunct tool for the diagnosis of the pathology. Our findings also suggest that focusing on motor behavior and its histopathological correlates may pave the way towards the development of therapeutic strategies aimed at specific brain areas such as the cerebellum and the motor cortex.

  at postnatal day (P12) to P16 and scored as either 0 = both eyes closed, 1 = one eye open, or 2 = both eyes open.

Figure 1 .

 1 Figure 1. Behavioral experimental plan. For mating, 3 females were transferred to the male cage and the presence of vaginal plug designated gestational day 0 (GD 0).

Figure 2 .

 2 Figure 2. Valproic acid (VPA) prenatal treatment affects several developmental and behavioral paradigms in young pups and that are more pronounced in males than females. (A) VPA mice displayed significant delayed eye opening and (B) latency to righting in comparison with saline. Eye opening was assessed daily at postnatal day (P12) to P16 and scored as 0 = both eyes closed, 1 = one eye open, or 2 = both eyes open. (C) On P30, only VPA males showed a significant increase in immobility (freezing) when transferred to the SHIRPA arena as well as (D) a significant decrease in their locomotor activity. (E) VPA mice show a significant increase in the time climbing the wire and (F) time to climb the grid during the negative geotaxis test in comparison with saline. VPA males (n = 34/34), saline males (n = 27/27), VPA females (n = 25/25), and saline females (n = 30/30). Data are expressed as mean ± SEM; 2-way ANOVA followed by Tukey's posthoc were performed (*P < .05, **P < .01, ***P < .001).

Figure 3 .

 3 Figure 3. Valproic acid (VPA) prenatal treatment induced core symptoms of autism spectrum disorder (ASD) mainly in males. (A-C) Social behavior analysis. VPA males (n = 27/34), saline males (n = 10/27), VPA females (n = 16/25), and saline females (n = 15/30). Data are expressed as mean ± SEM; 2-way ANOVA followed by Tukey's posthoc were performed (*P < .05, **P < .01, ***P < .001). (A) Social interaction was assessed using the 3-chambers test. None of the treatment groups showed preference to any of the chambers during the 10-minute habituation (PHASE I). (B) VPA prenatally treated male mice spent less time in the social chamber and more time in the nonsocial chamber relative to controls, (C) they also show a significant decrease in sociability index. (D-E) Grooming and rearing behavior. VPA males (n = 34/34), saline males (n = 27/27), VPA females (n = 25/25), and saline females (n = 30/30). (D) VPA mice presented repetitive behavior and (E) a significant decrease in rearing exploratory behavior. Data did not follow a normal distribution and was analyzed using a non-parametric test (Kruskal-Wallis) followed by Dunn's multiple comparison test.

Figure 4 .

 4 Figure 4. Valproic acid (VPA) prenatal treatment induced motor coordination deficits and abnormal gait in both males and females. (A) VPA treatment did not affect the time needed to traverse the beam for any group of mice. (B) VPA males and females showed similar and significant increase in the number of errors per steps in comparison to saline. (C) VPA mice showed significant deficits on the second, third, and fourth sections of the beam. (D) Gait analysis indicated that VPA males and females have a significant increase in both right and left pair gap, (E) right and left limb base of support as well as (F) a significant decrease in stride length in comparison with saline controls. For challenging beam: VPA males (n = 34/34), saline males (n = 27/27), VPA females (n = 25/25), and saline females (n = 30/30). For the gait analysis: For the challenging beam, VPA males (n = 32/34), saline males (n = 27/27), VPA females (n = 25/25), and saline females (n = 30/30). Data are expressed as mean of 5 trials ± SEM, 2-way ANOVA followed by Tukey's posthoc were performed (*P < .05, **P < .01, ***P < .001).

Figure 5 .

 5 Figure 5. Loss of neurons in mice prenatally exposed to VPA is sex and region specific. (A-F) Stereological Purkinje cell (PC) count after Nissl staining on coronal section of cerebellum. (J-K) Photos of the different sub-lobules of the lobule VII, scale bars = 1 mm. (L) Illustration of the monolayer organization of PC in the cerebellar cortex after DAB-calbindin immunolabeling, scale bars = 200 µm. No difference in PC number was found within the hemispheric part of the lobule VI, SIM (A), or in the vermal part of the lobule VI, 6cb (B). Significant loss of PC was found in the sub-lobule Crus I of the lobule VII in VPA males (C), whereas VPA females showed PC cell loss in the sub-lobule Crus II (D) of the lobule VII. (E) No effect of treatment on the number of PC in the hemispheric sub-lobule PM, or (F) in the vermal sub-lobule 7cb of the lobule VII was found. (G) A decrease in the number of NeuN-stained neurons in the M1/M2 motor cortex was found in VPA males (outlined area on N, scale bars = 400 µm). (H) There was no effect of treatment on number of NeuN-stained neurons in the striatum. (I) No change was found in the number of neurons expressing tyrosine hydroxylase (TH) in the substantia nigra pars compacta (SNc) after DAB-TH immunolabeling on ventral mesencephalic coronal sections (M scale bars = 400 µm). n(A) = (saline males/VPA males/saline females/VPA females) = 9/8/10/9; n(B) = 11/8/10/10; n(C) = 11/8/9/10; n(D) = 11/8/9/10; n(E) = 10/7/9/8; n(F) = 9/6/8/9; n(G) = 12/8/11/10; n(H) = 12/8/10/9; n(I) = 12/8/10/10. Statistical test: 2-way ANOVA with posthoc Fisher's LSD test. *P < .05, **P < .01, ***P < .001

Table 1 .

 1 Interrelations of the Screened Shifts Behavior and the Anatomical Changes Crus II, number of PC in each sub-lobule of the cerebellum; EPS, errors per step on the challenging beam; LA, locomotor activity: number of crossed squares; LLBS, left limbs base of support; LPG, left pair gap; M1/M2, number of neurons in the motor cortex; NG1/2, negative geotaxis, 1: time to turn, 2: time to climb; OE, open eyes: age when both eyes are open; RR, mean of time of the righting reflex on P9 and P11; RLBS, right limbs base of support; RPG, right pair gap; SI, social index: calculated from the 3-chambers test; SL, stride length on the gait lab; TR, transfer reaction: time of immobility; WM, wire maneuver: time to climb.Correlation analyses using Pearson's method. Cases filled with dark grey or light grey, respectively, for males or females, and indicated in bold, represent the significant correlations between 2 variates. Note the strong correlations among developmental disorders, motor impairments, social interaction deficits, and neuronal loss in males, whereas in females, no relevant correlations were observed.

	or 15q11-13

*P < .05, **P < .01; coeff. gives the correlation coefficient r.

Acknowledgments

We thank the staff of the PREBIOS animal facility (University of Poitiers-France), Marcello Solinas, Marianne Benoit-Marand, Afsaneh Gaillard, and Laurie Galvan for kind advice and Denis Couratin for technical support.

Statement of Interest

None.

Funding

This work was supported by grants from the Fondation pour la Recherche Médicale. T.A.S. was awarded a scholarship from the Association of Specialization and Scientific Guidance (Lebanon). O.H. was supported by a fellowship from the Institut National de la Santé et de la Recherche Médicale (INSERM) and the region Poitou-Charentes. The University of Poitiers and INSERM provided infrastructural support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supplementary Material

Supplementary data are available at International Journal of Neuropsychopharmacology online.


Mathieu Xemard

Marie Cordier

Elisa Louyriac

Laurent Maron

Carine Clavaguera
email: carine.clavaguera@u-psud.fr.

Grégory Nocton
email: greg.nocton@polytechnique.edu.

Mathieu Xémard

Carine Clavaguéra

Small molecule activation with divalent samarium triflate: a synergistic

come

The divalent samarium triflate salt does not react with CO 2 or water but does with traces O 2 or N 2 O to form a tetrameric bis-oxo samarium motif. The reaction with O 2 is a 4ereductive cleavage whose electrons are coming from four different samarium centers. This highlights a rare synergistic effect to cleave O 2 , which has no precedent with divalent lanthanides complexes. Additionally, the addition of CO 2 on the tetrameric bis-oxo intermediate leads to the formation of a tetrameric bis-carbonate samarium triflate. Thus, the concomitant reaction of CO 2 with traces of O 2 leads to the same bis-carbonate tetrameric assembly.

The chemistry of low-valent coordination compounds has multiple assets in which the small molecules activation is prominent because of the growing importance of transforming abundant, such as N 2 , H 2 O and O 2 . [1][2][3][4][5][6] and/or pollutant molecules, such as N 2 O, 7 NO x , 8 CO 9, 10 and CO 2 , 11 at low economical and environmental cost. In this context, electron rich transition metals complexes or low-valent uranium are often used since they allow multiple electron-transfer steps leading to the complete reductive cleavage of the considered small molecule.

12-17

On the other hand divalent lanthanides do not allow multiple electron transfers The mechanism of the carbonate formation is particularly interesting since it involves a bent CO 2 2-fragment, formed in between two lanthanide metal centers, which further reacts with another CO 2 yielding CO and CO 3 2-. 31 The overall reaction is a two-electron disproportionation of CO 2 and has the important drawback of releasing CO, also a pollutant. In a recent report, we have detailed the reaction of bulky samarium complexes with CO 2 forming carbonate species. 30 In this precedent work, the use of a low quality CO allowed the serendipitous formation of a rare peroxo samarium dimer from the reaction with traces of oxygen. 30 In an opposite strategy to the typical one, for which the bulk increase allows cleaner reactions by kinetic control, we reasoned that the decrease of the bulk of the ligand set on the samarium center would possibly allow the involvement of more than two samarium centers, i.e. more than two electrons. F NMR. This contrasts with the many other reports in the literature, including ours, which describe a fast reactivity of CO 2 with divalent samarium complexes at room temperature. However, the redox potential of samarium is known to be extremely dependent of the ligand. 34 Additionally, the addition of several equivalents of water does not modify the fate of the reactions. However, when CO Reacting a degassed deep purple thf solution of Sm(OTf) 2 (dme) 2 with N 2 O leads to the complete discoloration of the solution in approximately 15 hours and an intractable white precipitate crashes out. Filtration and slow evaporation of the solvents allowed the crystallization of the bis-oxo tetramer [Sm 4 (µ 3 -O) 2 (µ 2 -OTf) 6 (µ 3 -OTf) 2 (thf) 4 (dme) 2 ] (1) in good yields (85%) a pure analytical form. On the other hand, the reaction of Sm(OTf) 2 (dme) 2 with one atmosphere of dry O 2 in thf is not clean and leads to an intractable clear powder and trivalent samarium triflate. X-Ray suitable crystals of 1 were grown by slow diffusion of pentane in a thf solution giving access to the topology of the oxo complex (Fig. 1a). It crystallizes in the monoclinic P2 1 /n space group (Table S1). The [Sm 2 (µ 3 -O)] 2 core of the tetramer is composed of four samarium centers bridged by two µ 3 -oxo anions (Fig. 1a). In the asymmetric unit (half of the molecule) one samarium center is 8-coordinated bearing only one coordinated thf molecule while the second one is 8coordinated and bears one coordinated thf molecule and one dme molecule. The Sm-O distances on the O (2-py) as large colorless crystals (Fig. 1b). The 2-thf cluster can also be obtained in good yield (80%) by direct reaction of the raw suspension obtained from the reaction of Sm(OTf) 2 (dme) 2 with N 2 O. X-Ray diffraction analysis shows that 2-thf crystallize in a P-1 triclinic space-group and 2-py in a P2 1 /n monoclinic one. For both assemblies, half the molecule is obtained as the image of the other half by an inversion centre and the roughly planar core can be described as [Sm 2 (μ 3 -η 2 (O,O′):η 1 (O):η 1 (O′)-CO 3 )] 2 with four 8-coordinated Sm surrounding two bridging carbonate anions. The carbonate coordination mode is rather unusual but has already been observed with only few lanthanides clusters, mainly Dy cluster, some of them obtained via atmospheric fixation of CO 2 in aqueous media, often implying poor control of the geometry of the cluster and long synthetic procedure. [37][38][39][40] The C-O distances in the CO ), a tetrametallic assembly has also been isolated (Fig. S16, Table S5). The assemblies, [Sm 2 (µ 3 -OTf) 2 (µ 3 -OTf)(OTf)(thf) 6 ] 2 (3) is featuring relatively long Sm-O distances on TfO -anions (average of 2.63(8) Å) compared to 2.54 Å for [Sm(OTf) 2 (thf) 1.5 ] n . 36 Although the 19 F solution NMR of Sm(OTf) 2 (dme) 2 shows only one broad signal at δ = -70.33 ppm, indicating strong exchange, the isolation of 3 as a tetrameric assembly is an insight into the mechanism involved in the formation of 1: the topology of the assembly may be prior to the reactivity with N 2 O and/or may lead to the formation of 1 by a concerted mechanism. Moreover the strong similarity between 2-thf and 2-py is also good evidence that the core of the cluster, once formed, can be retained in solution and that only solvents molecules are exchanging in solution phase. In order to obtain better insight in the dynamics in solution, 2-thf was recrystallized from a pyridine solution yielding 2-py, while the fate of the triflate anions in 1 and 2 was monitored by 19 F NMR. For 1, only one broad signal is observed at δ = -79.43 ppm in thf (Fig. S1), indicating a fast exchange between the two bridging modes on triflates. On the other hand, for 2-thf, two broad signals are observed on the 19 F NMR in thf (Fig. S2) at δ = -79.20 ppm and δ = -80.19 ppm. These two signals should correspond to the two available coordination modes on triflate anions. The range of chemical shifts observed for both 1 and 2-thf is close to the chemical shift observed for Sm(OTf) 3 in thf (δ = -79.30 ppm and δ = -80.10 ppm) but are strongly shifted from the one of the Sm(OTf) 2 (dme) 2 starting material which is in agreement with triflates anions on Sm III centres.

The formation of 2 from 1 validates the oxo intermediate mechanism and is not surprising considering the recent work from Roesky 42, 43 and the seminal study of Meyer on uranium bridged oxo complexes. [44][45][46] The reaction of dry air on 1 is not clean but has led to the re-crystallization of 2, which is indicative of selective CO 2 abstraction from air. Yet, the presence of humidity in air complicates the reactivity, yielding mostly to Sm(OTf) 3 . Such a selective atmospheric fixation of CO 2 on lanthanide hydroxo complexes has already been described by Mazzanti. Please do not adjust margins Please do not adjust margins two more divalent samarium centers to cleave O 2 and form the bis-oxo tetrameric samarium complex 1. As shown in Figure 2, 1 lies -170.7 kcal/mol below the starting materials, which explains its easy formation. Then, the CO 2 insertion on the bisoxo complex 1 has been computed and indicates that the transition state is only 11.8 kcal/mol while the reaction is exergonic by 19 kcal/mol (Fig. 2). At the transition state, the CO 2 molecule is strongly activated by two samarium atoms (O-C-O angle around 157°), i.e. a double nucleophilic assistance (Sm 1 -O 1 : 2.60 Å, Sm 2 -O 1 : 2.55 Å). The oxo group O 1 adopts a pyramidal shape and is lying 0.58 Å above the Sm 1 -Sm 2 -Sm 3 plane (compared to 0.25 Å in complex 1). This situation is due 3 -coordination mode of the oxo O 1 that enforces its remaining p lone pair to point out of the plane inducing an out-of-plane attack of CO This indicates that the presence of traces of O 2 allows the formation the bis-oxo species from synergistic action of four samarium centers, each providing one electron to cleave O 2 . In this example, the low bulk and the dynamics in solution of the triflate anions allow this synergy. This opens new possibilities for small molecules activation with divalent lanthanides, at the opposite of the typical strategy, which consists in increasing the steric bulk for a better stabilization of the divalent lanthanide center. This example also comes as a warning for the quality of the CO 2 and the rigor of the experimental protocol that should be used for these small activation mechanistic studies.

In conclusion, this article reports the reaction of N 2 O and O 2 with the Sm(OTf) 2 (dme) 2 divalent samarium precursor to yield a rare tetrametallic bis-oxo complex of Sm III . The reaction with O 2 is, to our knowledge, a unique example of synergic four electrons reductive cleavage O 2 with divalent lanthanide complexes. The subsequent reaction of CO 2 on this bis-oxo complex yields the tetrameric bis-carbonate samarium complex from CO 2 insertion in the oxo-bridge. This work shows that the use of less bulky divalent lanthanide complexes opens new routes for small molecules activation using the synergistic effort of multiple metal centers to cleave molecules that need more than 2e-such as O 2 , CO or N 2 . Further studies are conducted in this direction.

Notes and references ‡ This project was financed by the French National Agency with grant number ANR-15-CE29-0019. We thank CNRS and Ecole polytechnique for funding. MX is grateful to the DGA for funding. LM is member of the Institut Universitaire de France.

of 2 .BFigure 1 .

 21 Figure 1. A) ORTEP of 1; hydrogen, carbon and fluorine atoms are removed for clarity; Sm green, O red, S yellow. Selected distances (Å) and angles (°): Sm01-O13 2.271(7), Sm02-O13 2.207(7), Sm01-O13#3 2.250(8), Sm01-SM01#3 3.497(1), Sm01-SM02#3 3.940(1), Sm01-O13-Sm02 133.2(4), Sm01-O13-Sm01#3 101.3(3), Sm01#3-O13-Sm01 124.2(3). B) ORTEP of 2-thf; hydrogen, carbon (excepted C1) and fluorine atoms are removed for clarity; Sm green, O red, S yellow. Selected distances (Å) and angles (°): Sm1-Sm2 4.580, Sm1-Sm1#2 10.102, Sm2-Sm2#2 4.522, Sm1-O1 2.379(2), Sm1-O3 2.392(2), Sm2-O2#2 2.295(2), Sm2-O3 2.362(2), O1-C1-O2 125.1(2), O1-C1-O3 115.8(2).

Figure 2 .complex 1

 21 Figure 2. Computed enthalpy profile for the O2 reductive cleavage by four Sm(OTf)2 to form 1 and subsequent reaction with CO2.Theoretical calculations have been performed on the formation of 1 and 2. The addition of O 2 between two samarium centers is very favorable in good agreement with our precedent work. The key point here is that the flexibility and low steric bulk of the triflate anions allow the addition of

  2 of lower quality is used or when traces of O 2 are allowed in by the experimental protocol, the purple thf This journal is © The Royal Society of Chemistry 20xx Sm 4 (µ 3 -CO 3 -κ 4 O,O',O'') 2 (µ 2 -OTf) 6 (OTf) 2 Sm(thf) 10 ], (2) (Scheme 1). The formation of carbonate dianion from CO 2 with samarium complexes has precedent in the literature and several mechanisms are proposed: i) the oxo formation from CO 2 with CO release and following CO 2 insertion or ii) the reductive disproportionation of two CO 2 with CO release. The key point here is that a clean CO 2 in a rigorous experimental setup does not react with Sm II (OTf) 2 (dme) 2 . It means that CO 2 is not the unique reactant in this reaction and the oxo intermediate mechanism should be privileged. Therefore, we started to investigate the formation mechanism of this tetrameric assembly using N 2 O but also O 2 , a more challenging way of forming oxo compounds with divalent lanthanides complexes.

		Please do not adjust margins
	solution of Sm	II (OTf) 2 slowly fades until it gets clear. After the
	filtration of the solution, pentane layering allowed the
	formation of transparent crystals that were analyzed as a
	tetrameric assembly of samarium containing two carbonate
	dianion, [
		Please do not adjust margins

Scheme 1. Synthetic scheme for 1 and 2.

  Please do not adjust margins Please do not adjust margins Reacting a thf suspension or a thf/pyridine solution of 1 with CO 2 leads respectively after filtration and layering with pentane to [Sm 4 (µ 3 -CO 3 -κ 4 O,O',O'') 2 (µ 2 -OTf) 6 (OTf) 2 Sm(thf) 10 ] (2-thf) and to [Sm 4 (µ 3 -CO 3 -κ 4 O,O',O'') 2 (µ 2 -OTf) 6 (OTf) 2 Sm(py) 10 ]

  2 . Since the reaction of 1 atmosphere of dry O 2 with the Sm II (OTf) 2 (dme) 2 complex in thf was not conclusive, low amount of O 2 was used to mimic what may happen when using a lower quality CO 2 (i.e. containing traces amounts of O 2 ). As such, the deep purple thf solution fades slowly and storage of the colorless solution at -35 °C afforded few crystals of 1 (the yield could not be calculated). The 19 F NMR indicates the formation a broad signal at δ = -79.81 ppm, characteristic of 1.