<p>Autistic-like syndrome in mu opioid receptor null mice is relieved by facilitating mGluR4 activity</p><p>J.A.J. Becker, D. Clesse, C. Spiegelhalter, Y. Schwab, J. Le Merrer*, B.L. Kieffer*</p><p>Supplementary experimental procedures</p><p>Behavioral experiments</p><p>Direct social interaction, novel object recognition and novelty suppressed feeding were performed in 4 equal square arenas (50 x 50 cm) separated by 35cm-high opaque grey</p><p>Plexiglas walls over a white Plexiglas platform (View Point, Lyon, France). Stimulus mice used for social interaction (first series of experiments), social novelty and resident-intruder tests were 8-10-week-old gender-matched grouped-housed wild-type mice, socially naive to the experimental animals.</p><p>Direct social interaction. On day 1, experimental and stimulus animals were placed in each arena for a 30 min-habituation session. On day 2, a pair of experimental and stimulus mice was introduced in each arena for 10 min (15 lx). Total time spent in close contact (nose and paw contacts), number and duration of nose and paw contacts (crawling over, mounting, stepping on, pushing) and allogrooming, as well as number of following, rearing, and circling episodes were scored on video recordings . In a first series of experiments, we used naïve wild-type mice (see above) as stimulus conspecifics in this test. In a second series of experiments, the experimental design ensured that each experimental mouse met another experimental gender-, genotype- and treatment-matched naive unfamiliar (not cage mate) animal. Whatever the paradigm used, we observed no aggressive behavior (attacks, mounting, bites, tail rattling) between protagonists during this test.</p><p>Three-chamber social novelty preference test. The test apparatus consisted of a transparent acrylic box; partitions divided the box into three equal chambers (40 x 20 x 22.5 cm). Two</p><p>1 sliding doors (8 x 5 cm) allowed transitions between chambers. Cylindrical wire cages (18 x 9 cm, 0.5 cm diameter-rods spaced 1 cm apart) were used to contain the stranger mice (15 lx).</p><p>The experimental protocol was adapted from . Stimulus mice were habituated to confinement in wire cages for 2 days (20 min/day). For the sociability test, the test animal was introduced to the middle chamber for 10-min habituation (wire cages empty). Interaction phase started when an unfamiliar mouse (Stranger 1) was introduced into a wire cage in one of the side- chambers; a soft toy mouse (8 x 10 cm) was introduced in the second wire cage as a control for novelty. The dividers were raised and the experimental animal was allowed to freely explore the apparatus (10 min), and then confined in the middle-chamber while a novel stranger mouse (Stranger 2) replaced the toy mouse. During recognition phase (10 min), the experimental mouse was left to freely explore the apparatus. The number of entries in each compartment, the time spent in each chamber and in nose contact with the stimulus mouse (or wire cage, except climbing), as well as duration of each nose contact episode was scored on video recordings. The relative position of stimulus mice was counterbalanced between groups; for each experimental mouse, the location of Stranger 1 was maintained across interaction and recognition phases. In this test, four Oprm1-/- mice (males and females) displayed aggressive behaviors (bites, tail rattling) towards the restrained stimulus mice.</p><p>Novel object recognition. The novel object recognition test was performed as previously described . Briefly, on day 1, the animals were placed in an arena for 15-min habituation with two copies of an unfamiliar object. On day 2, the recognition test consisted of 3 trials of 10 min separated by 2 intertrial intervals of 5min (see Figure S2). On familiarization phase, the mice were presented with two copies of an unfamiliar object. On place phase, one of the two copies was displaced to a novel location in the arena. On object phase, the copy that had not been moved on previous trial was replaced by a novel object. Stimuli objects sized 1.5-3 x 2-3 cm. The identity of the objects and their spatial location were balanced between subjects. The</p><p>2 number of visits and the time spent to explore each object were scored on video recordings. A percentage of discrimination was calculated for number of visits and time exploring the objects as following: exploration of displaced or novel object / total exploration * 100. </p><p>Nest building behavior. Oprm1+/+ and Oprm1-/- mice were single-housed overnight (16 hr) in a clear standard cage (21 × 11 × 17 cm) provided with a single housing-device (red plastic igloo; SDS Mazuri, Argenteuil, France) with three openings. A block of nesting material was placed in the opposite end of the cage. Each cage was scored by adding the number of openings covered (1, 2 or 3) with nesting material to the condition of this material: 1 for initiation of shredding and 2 for a totally shredded nesting block . </p><p>Motor stereotypies. To detect motor stereotypies in mutant versus wild-type animals, mice were individually placed in clear standard home cages (21×11×17 cm) filled with 3-cm deep fresh sawdust for 10 min . Light intensity was set at 30 lux. Numbers of head shakes, as well as rearing, burying, grooming, circling episodes and total time spent burying were scored by direct observation. </p><p>Y-maze exploration. Spontaneous alternation behaviour was assessed as a measure of behavioural flexibility . Each Y-maze consisted of three Plexiglas arms (40x9x16 cm) covered with distinct patterns (15 lx). Mice were placed at the center of a maze and allowed to freely explore this environment for 5 min. The pattern of entries into each arm was quoted on video- recordings. Spontaneous alternations (SPA), i.e. successive entries into each arm forming overlapping triplet sets, alternate arm returns (AAR) and same arm returns (SAR) were scored, and the percentage of SPA, AAR and SAR was calculated as following: total / (total arm entries -2) * 100. </p><p>Marble-burying. The procedure for marble burying was adapted from . Mice were introduced individually in transparent cages (21×11×17 cm) containing 20 glass marbles</p><p>(diameter: 1.5 cm) evenly spaced on 4-cm deep fresh sawdust. To prevent escapes, each cage</p><p>3 was covered with a filtering lid. Light intensity in the room was set at 40 lux. The animals were removed from the cages after 15 min, and the number of marbles buried more than half in sawdust was quoted. </p><p>Novelty-suppressed feeding. Novelty-suppressed feeding (NSF) was measured in 24-hr food-deprived mice, isolated in a standard housing cage for 30min before individual testing.</p><p>Experimental protocol was adapted from . Three pellets of ordinary lab chow were placed on a white tissue in the center of each arena, lit at 60 lx. Each mouse was placed in a corner of an arena and allowed to explore for a maximum of 15 min. Latency to feed was measured as the time necessary to bite a food pellet. Immediately after an eating event, the mouse was transferred back to home cage (free from cage-mates) and allowed to feed on lab chow for 5 min. Food consumption in the home cage was measured. </p><p>String test (traction reflex test). A metal wire was stretched horizontally 40 cm above a table. Light intensity was set at 30 lux. Three consecutive trials were performed. A trial started when the animal grasped the thread with forepaws. The latency to grab the wire with a third - hind - paw was recorded . </p><p>Muscular strength measurement. Mice grasping the grid of a dynamometer (BioSeb,</p><p>Valbonne, France) were pulled back by their tail. The maximal strength exerted by the mouse before losing grip was recorded. Muscular strength was assessed as the mean of 3 measures, separated by 30 s-recovery intervals. </p><p>Skill motor learning. Skill motor learning performance was assessed as previously described . The apparatus was a rotarod (Bioseb, Valbonne, France) accelerating from 4 to 40 rpm in 5min. The rod was covered with insulation tubing for better grip, which external perimeter was 5 cm (40 lx). On day 1, mice were habituated to rotation on the rod at 4rpm, until they were able to stay more than 180 s. From day 2 to day 5, mice were tested for three daily trials (60-s intertrial). Each trial started by placing the mice on the rod and beginning</p><p>4 rotation at constant 4 rpm-speed for 60 s. Then the accelerating program was launched, and trial ended for a particular mouse when falling off the rod. Time stayed on the rod was automatically recorded. </p><p>Foot print analysis. After coating of hind feet with nontoxic ink, mice were allowed to walk through a tunnel (50x9x6 cm) with paper lining the floor. The footprint pattern generated was scored for five parameters, as described in : step length (measure of the average distance of forward movement between alternate steps), sigma (assessment of the regularity of step length: high score indicates variable step length), gait width (measure of the average lateral distance between opposite left and right steps), alternation coefficient (assessment of the uniformity of step alternation: high score indicates altered uniformity) and linearity (measure of the average change in angle between consecutive right–right steps: high score indicates nonlinear movement).</p><p>Resident-intruder test. Inter-male aggression was assessed using the resident-intruder test .</p><p>Male mice were individually housed for 30 days before testing. The test was conducted over 4 days (15 lux). A session started when a stimulus intruder was placed in the standard home cage of the experimental resident male for 10 min. Each experimental mouse met a different intruder every day. The latency to attack, number of attacks and tail rattling, following, grooming, ptosis, and mounting episodes were scored on video recordings. </p><p>PTZ-induced convulsions. PTZ-injected mice were placed in rectangular clear plastic observation boxes (16 x 16 x 30 cm) filled with 3-cm fresh sawdust (50 lux). The number of ear and facial twitches, jerks, tonies, clonies, tonic-clonic seizures and deaths was assessed for</p><p>20 min. A convulsion score was calculated as described in . </p><p>Fos immunohistochemistry</p><p>5 Brains were removed from anesthetized and perfusion-fixed mice (0.9% NaCl followed by</p><p>4% paraformaldehyde in PB 0.1 M, pH 7.4), post-fixed for 48 hours, cryoprotected in 30% sucrose / PB overnight at 4°C and stored at -80°C until 50 µm frontal sections were cut on a cryostat. Immunohistochemistry was performed on free-floating sections using a standard avidin-biotin peroxidase method . Slides were digitized using a Hamamatsu Nanozoomer 2-</p><p>HT whole slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) at 20x magnification.</p><p>Frames were acquired using NDP View software, and Fos-positive nuclei were counted using</p><p>ImageJ software (NIH). Fos-immunoreactivity was assessed bilaterally in 27 cerebral regions on two consecutive sections per animal, and expressed as Fos-positive nuclei/mm2.</p><p>Electron microscopy</p><p>The brains were removed from anesthetized and perfusion-fixed mice (2% paraformaldehyde,</p><p>2.5% glutaraldehyde in PBS pH 7.4); the caudate putamen (CPu) or nucleus accumbens</p><p>(NAc) were punched and incubated in the same fixative overnight. Samples were post-fixed</p><p>(1% Osmium tetroxide), stained (1% Uranyl acetate) and embedded in epoxy resin. Thin sections (60nnm) were inspected with a transmitted electron microscope (CM12, Philips, FEI</p><p>Electron Optics, Eindhoven, the Netherlands) operated at 80kV. Images were acquired with an Orius 1000 CCD camera (Gatan Pleasanton, USA). Image acquisition was performed according to the systematic uniform random sampling method , frames were acquired sequentially from a random starting point with a shift of 20µm. 40 pictures were acquired per section (3 sections/animal) at 40,000x magnification. Frames focused on cell nucleus or on axon bundles were omitted. Symmetrical and asymmetrical synapses were identified based on the following criteria: clearly visible synaptic cleft, vesicles in presynaptic element, shape and size of vesicles, postsynaptic density. Measurements of the length and area of post-synaptic</p><p>6 densities were performed using ImageJ software (NIH). Only non-truncated synapses with visible synaptic cleft were retained.</p><p>Neurochemical assay for biogenic amines dosage</p><p>Brains were placed into a cooled brain matrix and sliced. Bilateral punches of the CPu and</p><p>NAc, and unilateral punches of the dorsal raphe were dissected from two consecutive brain slices. Tissues were immediately frozen on dry ice and kept at -80°C until use. HPLC dosage of biogenic amines was performed as described previously . Values were expressed using the acquisition software Azur v4.5 (Datalys, St Martin d’Hères, France). Results were given as pg/µg of total protein (Lowry method).</p><p>Legends to Supplementary Figures</p><p>Figure S1. Oprm1-/- mice show core symptoms of autism. (a) In the direct social interaction test, Oprm1-/- mice spend more time grooming themselves, although the duration of each grooming episode was unchanged as compared to Oprm1+/+ animals. Under these conditions,</p><p>Oprm1-/- mice did not display other motor stereotypies (n=8 per genotype). (b) During the familiarization phase of the social novelty preference test, no difference is observed between control and mutant animals in the time spent in left and right compartments, nor the total number of entries in compartments (index of locomotor activity). These parameters remain similar during the interaction phase; during the recognition phase, however, Oprm1-/- mice spend less time in the compartment of the novel stimulus mouse than controls, number of entries not being modified (n=13-14 per genotype). (c) In the novel object recognition task,</p><p>Oprm1-/- mice perform better during place phase in visiting the displaced object (n=15 per genotype). (d) In the resident intruder test, male mutant animals are more aggressive, taking</p><p>7 more time to leave their nest (defensive aggressiveness) and engaging initially more</p><p>(inquisitive) nose contacts (day 1), a behavior that regresses after repeated intrusions (day 4) when the number of attacks raises to a maximum. Tail rattling, following, attack latency, mounting, mounting duration and grooming are similar across genotypes (n=8 per genotype).</p><p>(e) As regards motor stereotypies, burying behavior was unchanged in Oprm1-/- mice (n=8 per genotype). Data are presented as mean ± SEM. Solid stars: genotype effect; open stars: interaction (Genotype x Stimulus) or compartment effect. One star p<0.05, two star p<0.01; three stars p<0.001.</p><p>Figure S2. Oprm1-/- mice display several secondary symptoms of autism. </p><p>(a) Fos-immunoreactivity induced by exposure to a conflict task (novelty-suppressed feeding test) was assessed across 27 brain regions in control and mutant mice (n=10 per genotype).</p><p>Schematic diagrams of coronal sections adapted from the mouse brain atlas indicate the approximate antero-posterior levels where regions of interest were sampled for immunostaining quantification. Coordinates refer to Bregma. (b) Oprm1-/- mice show impaired locomotor coordination, as indicated by increased latency to grasp the string with a third paw in the grip test (n=14-16 per genotype). (c) No difference in muscle strength of mutants is measured in the grip test (n=9-10 per genotype). (d) Oprm1-/- mice have longer paces than controls, but display no modification of gait width, linearity and alternation coefficient in the foot prints analysis (n=15-16 per genotype). (e) Mutant animals are more sensitive than controls to pentylenetetrazole (PTZ)-induced seizures (n=8 per genotype), as indicated by increased number of clonies and tonic-clonic crises at the dose of 50 mg/kg.</p><p>Differences in numbers of twitches, jerks and tonies fail to reach statistical significance. Data are presented as mean ± SEM. Genotype effect: one star p<0.05, two stars p<0.01; three stars p<0.001. Abbreviations: AcbC: core of the nucleus accumbens, AcbS: shell of the nucleus</p><p>8 accumbens, BLA: basolateral nucleus of the amygdala, BNST: bed nucleus of the stria terminalis, CA1: CA1 region of the hippocampus, CA3: CA3 region of the hippocampus,</p><p>CeA: central nucleus of the amygdala, Cg1: anterior cingulate cortex, Cg2: posterior cingulate cortex, CPu: caudate putamen, DG: dentate gyrus of the hippocampus, DR: dorsal raphe, IC: insular cortex, IL: infralimbic cortex, LH: lateral hypothalamus, LS: lateral septum, M1: primary motor cortex, MD: mediodorsal nucleus of the thalamus, MeA: medial nucleus of the amygdala, MPA: medial preoptic area, PAG: periaqueductal gray, PH: posterior hypothalamus, Pir: piriform cortex, PrL: prelimbic cortex, VP: ventral pallidum, V1: primary visual cortex, VTA: ventral tegmental area.</p><p>Figure S3. Behavioral deficits in Oprm1-/- mice have a genetic origin. (a) Cross-fostering behavioral experiments were performed successively in the same cohorts (n=8 per genotype).</p><p>(b) In the direct social interaction test, cross-fostered Oprm1-/- mice show decreased time spent in close contact, decreased frequency and duration of nose contacts and paw contacts and decreased number of following episodes, but self-groom more often, especially after social contact, and for longer than controls. Duration of grooming episodes, as well as number of rearings and circlings are not modified. (c) Cross-fostered knockout mice build a nest normally, while Oprm1+/+ animals raised by Oprm1-/- parents tended to show low nesting abilities, indicating a genetic rather than parental origin of their alteration in Oprm1-/- animals.</p><p>(d) Motor stereotypies are observed in cross-fostered Oprm1-/- mice, with increased numbers of rearing (females), grooming, circling episodes and head shakes; number of burying episodes is not modified in mutants, but duration of each episode is shortened. (e) and (f)</p><p>Anxiety of cross-fostered Oprm1-/- animals is exacerbated in marble burying and novelty- suppressed feeding tests. In the later, however, food intake in the home cage is comparable</p><p>9 between Oprm1-/- and Oprm1+/+ mice. Data are presented as mean ± SEM. Genotype effect: one star p<0.05, two star p<0.01; three stars p<0.001.</p><p>Figure S4. Oprm1-/- mice display abnormal striatal asymmetrical synapses and transcriptional modifications across four brain regions. (a) Caudate putamen (CPu) and nucleus accumbens (NAc) were punched bilaterally; prefrontal cortex (PFC) was dissected from two consecutive slices . (b) Representative microphotographs from NAc brain samples in electron microscopy illustrate decreased surface of postsynaptic densities in mutant animals. (c) Length of postsynaptic densities in the CPu and NAc is similar in Oprm1+/+ and</p><p>Oprm1-/- mice (CPu: Oprm1+/+ - n=710 asymmetrical synapses, Oprm1-/- - n=799; NAc:</p><p>Oprm1+/+ - n=605, Oprm1-/- - n=605). Scale bar: 200 nm. Asterisk: presynaptic element. (d)</p><p>Clustering analysis of all gene expression data, including candidate genes for autism and genes encoding major players of GABAregic and glutamatergic neurotransmission, classifies genes in four main clusters (a-d). Sixteen genes in clusters (a) and (c) encode GABAergic receptors/receptor subunits and transporters (blue). Cluster (d) gathers genes mostly involved in glutamatergic signaling (orange) that show lowered mRNA levels in the striatum of</p><p>Oprm1-/- mice. Gene names are displayed in Table S2. CeA: Central nucleus of the amygdala;</p><p>CPF: prefrontal cortex; CPu: Caudate putamen; NAc: nucleus accumbens.</p><p>Figure S5. VU0155041, a positive allosteric modulator of mGluR4 glutamate receptors, alleviates autistic-like symptoms in Oprm1-/- mice more efficiently than the antipsychotic risperidone. (a) In a first series of experiments using wild-type naïve stimulus mice, risperidone (0.2 mg/kg), VU0155041 (5 mg/kg) or their vehicle (saline) were injected daily 17</p><p>(risperidone experiments: n=14 per genotype and treatment) or 16 (VU0155041 experiments: n=9-10 per genotype and treatment) consecutive days and behavioral tests were performed on</p><p>10 days 8, 9, 10, 14 and 16, on which mice were injected 60 min (risperidone experiments) or</p><p>120 min (VU0155041 experiments) prior to testing. (b) Chronic risperidone treatment (first series) decreases locomotor activity in Oprm+/+ and Oprm1-/- animals. (c) Consistent with this, risperidone decreases dopamine levels in the CPu and NAc of all mice (more data in Table</p><p>S5). (d) In contrast, VU0155041 (first series) increases locomotor activity in control and mutant animals. (e) Both risperidone and VU0155041 treatments fail to restore nest building in mutant animals. (f) As regards motor stereotypies, risperidone decreases rearing activity in both mouse lines, but does not relieve excessive burying in Oprm1-/- mice; VU0155041 reduces rearing behavior in mutant animals whereas increasing this activity in controls, and has no detectable effect on burying behavior. (g) In a second series of experiments, each experimental mouse met a genotype- and treatment-matched mouse; risperidone (0.2 mg/kg),</p><p>VU0155041 (5 mg/kg) or vehicle (saline) were injected daily for 8 consecutive days and direct social interaction was assessed on day 8 (n=8 per genotype and treatment). (h) Using such paradigm reveals the suppressive effects of risperidone on social initiative in both</p><p>Oprm1+/+ and Oprm1-/- lines, while VU0155041 normalizes interaction in mutant animals</p><p>(n=8 per genotype and treatment). Data are presented as mean ± SEM. Open stars: Treatment effect; solid stars: Genotype x Treatment interaction (post-hoc: Newman-Keules test). One star p<0.05, two star p<0.01; three stars p<0.001.</p><p>Legends to Supplementary Tables</p><p>Table S1. List of primers used for qRT-PCR.</p><p>Table S2. Levels of Fos expression across 17 brain regions in Oprm+/+ and Oprm1-/- mice</p><p>90 min after the novelty-suppressed feeding test</p><p>11 Results are expressed as mean number of Fos-positive nuclei per mm2 ±S.E.M. counted in both hemispheres. AcbC: core of the nucleus accumbens, AcbS: shell of the nucleus accumbens, BLA: basolateral nucleus of the amygdala, BNST: bed nucleus of the stria terminalis, CA1: CA1 region of the hippocampus, CA3: CA3 region of the hippocampus,</p><p>CeA: central nucleus of the amygdala, Cg1: anterior cingulate cortex, Cg2: posterior cingulate cortex, CPu: caudate putamen, DG: dentate gyrus of the hippocampus, DR: dorsal raphe, IC: insular cortex, IL: infralimbic cortex, LH: lateral hypothalamus, LS: lateral septum, M1: primary motor cortex, MD: mediodorsal nucleus of the thalamus, MeA: medial nucleus of the amygdala, MPA: medial preoptic area, PAG: periaqueductal gray, PH: posterior hypothalamus, Pir: piriform cortex, PrL: prelimbic cortex, VP: ventral pallidum, V1: primary visual cortex, VTA: ventral tegmental area.</p><p>Table S3. Principal component analysis performed on cFos staining data after the novelty-suppressed feeding test: component loadings for 17 brain regions and two behavioral parameters in variables' and subjects' spaces</p><p>Component loadings over the absolute value of 0.7 (variable's space) or 1.5 (subjects' space), considered as most significant, are displayed in bold. Percent of total variance explained by each component is shown at the bottom for each condition. Data for PC1 and PC2 are plotted in Fig. 2B (right panel). AcbC: core of the nucleus accumbens, AcbS: shell of the nucleus accumbens, BLA: basolateral nucleus of the amygdala, BNST: bed nucleus of the stria terminalis, CA1: CA1 region of the hippocampus, CeA: central nucleus of the amygdala, Cg2: posterior cingulate cortex, CPu: caudate putamen, DR: dorsal raphe, IC: insular cortex, LH: lateral hypothalamus, LS: lateral septum, M1: primary motor cortex, MeA: medial nucleus of the amygdala, PAG: periaqueductal gray, VP: ventral pallidum, VTA: ventral tegmental area.</p><p>F: female; M: male.</p><p>12 Table S4. Transcription levels of a set of 85 genes across four brain regions in Oprm1-/- versus Oprm1+/+ mice</p><p>Data are presented as fold-change in Oprm1-/- versus Oprm1+/+ mice (median ± SEM).</p><p>Student’s t-tests were performed on transformed data (see Supplementary Methods) to determine whether fold changes differed from 0 (no regulation: corresponds to ±1 in table).</p><p>Significant regulations are highlighted in bold. n.d.: not detected.</p><p>Table S5. Statistical analysis: risperidone experiment</p><p>Risperidone treatment (0.2 mg/kg, IP) was administered for 7-17 consecutive days. On testing days, injections were performed 60 min before behavioral assay. In the social interaction test, each experimental animal met a naïve gender and aged-matched wild-type animal. F: female,</p><p>Gend: gender, Geno: genotype, M: male, Treat: treatment. See Figures 4, 5 and S5.</p><p>Table S6. Statistical analysis: VU0155041 experiment</p><p>VU0155041 treatment (5mg/kg, IP) was administered for 7-17 consecutive days. On testing days, injections were performed 120 min before behavioral assay. In the social interaction test, each experimental animal met a naïve gender and aged-matched wild-type animal. F: female, Gend: gender, Geno: genotype, M: male, Treat: treatment. See Figures 4, 5 and S5</p><p>Table S7. Bioamine concentrations in the caudate putamen, nucleus accumbens and dorsal raphe of Oprm1+/+ and Oprm1-/- animals after chronic risperidone</p><p>Serotonine (5HT), dopamine (DA), norepinephrine (NE), 3,4-dihydroxyphenylalanine acid</p><p>(DOPAC) and 5-hydroxyindoleacetic acid (5HIAA) levels (pmol/µg protein) were determined</p><p>60 min after risperidone or saline injection. Data are presented as mean ± SEM.</p><p>13 Table S8. Statistical analysis: Comparison of risperidone and VU0155041 effects in social interaction</p><p>VU0155041 (5 mg/kg, IP) and risperidone (0.2 mg/kg) treatments were administered for 8 consecutive days. On testing day, injections of VU0155041 were performed 120 min before behavioral assay, whereas risperidone and vehicle (saline 0.9%) were injected 60 min before testing. In this experiment, each experimental animal met a naïve gender-, genotype- and aged-matched animal. F: female, Gend: gender, Geno: genotype, M: male, Treat: treatment.</p><p>See Figure 4.</p><p>Table S9. Statistical analysis: Social interaction under VU0155041 treatment and immediate early genes expression</p><p>VU0155041 (5 mg/kg, IP) or saline (0.9 %) were administered for 8 consecutive days. On testing day, injections (VU0155041 or saline) were performed 120 min before behavioral assay. In this experiment, each experimental mouse met a gender-, genotype-, aged- and treatment - matched animal. 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