Guiard Bp
email: bruno.guiard@univ-tlse3.fr

A Citrus based

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mood disorders represent widespread and invalidating disorders, with up to 16% of the world population affected by various symptoms of the depression spectrum. Unfortunately, antidepressant drugs traditionally used to treat major depression (MD) do not provide positive treatment outcomes for all depressed patients and among responders a long delay in the onset of action is often observed [START_REF] Hamon | Monoamine neurocircuitry in depression and strategies for new treatments[END_REF]. These important shortcomings in current treatments encourage research in the field of neuropsychopharmacology to better understand the neurobiological mechanisms underlying MD and to develop new therapeutic strategies with a better profile of efficacy or to prevent the appearance of symptoms.

Growing evidence suggests that a significant proportion of individuals suffering from MD displays olfactory dysfunctions and vice-versa [START_REF] Kohli | The Association Between Olfaction and Depression: A Systematic Review[END_REF][START_REF] Taalman | Olfactory Functioning and Depression: A Systematic Review[END_REF]. In agreement with this reciprocal relationship, imaging studies have revealed the existence of an overlap between brain areas involved in MD and olfactory processing including the olfactory bulbs but also the orbitofrontal cortex, the amygdala and the hippocampus [START_REF] Hoflich | Imaging treatment effects in depression[END_REF][START_REF] Naudin | Long-term odor recognition memory in unipolar major depression and Alzheimers disease[END_REF]. Such association has also been described in rodents since impairments in olfactory acuity have been reported in an animal model of depression [START_REF] Siopi | Anxiety-and Depression-Like States Lead to Pronounced Olfactory Deficits and Impaired Adult Neurogenesis in Mice[END_REF] whereas bilateral olfactory bulbectomy induces depressive-like endophenotypes as well as endocrine, immune, and neurotransmitter dysfunctions usually observed in MD [START_REF] Song | The olfactory bulbectomised rat as a model of depression[END_REF]. Altogether, these results strongly suggest that dysfunctions seen in MD or relevant animal models could be related to alterations in the olfaction system and conversely, modulating olfactory transmission might reverberate on emotional state. In support of the latter assumption, antidepressant-like effects were observed in rats inhaling various odorants such as vanillin [START_REF] Xu | Vanillin-induced amelioration of depression-like behaviors in rats by modulating monoamine neurotransmitters in the brain[END_REF] or lemon [START_REF] Komori | Potential antidepressant effects of lemon odor in rats[END_REF] whereas bulbectomy may dampen such beneficial effects [START_REF] Xu | Vanillin-induced amelioration of depression-like behaviors in rats by modulating monoamine neurotransmitters in the brain[END_REF]. These results do not necessarily imply that loss of smell directly influences emotional-related behaviors but that an adequate connectivity between the olfactory system and limbic areas is a key determinant for this type of antidepressant responses. However, it was shown that lavender, for example, elicits anxiolytic and antidepressant-like responses even in anosmic mice [START_REF] Chioca | Anosmia does not impair the anxiolytic-like effect of lavender essential oil inhalation in mice[END_REF][START_REF] Takahashi | Anxiolytic-like effects of inhalation of essential oil from lavandula officinalis: investigation of changes in 5-HT turnover and involvement of olfactory stimulation[END_REF].

Animal models provide instrumental in vivo tools to examine neural circuitry and molecular and cellular pathways in a controlled environment. Based on the observation that plasma cortisol is increased in a significant proportion of patients with MD [START_REF] Wong | Pronounced and sustained central hypernoradrenergic function in major depression with melancholic features: relation to hypercortisolism and corticotropin-releasing hormone[END_REF], one of these models is based on repeated corticosterone (CORT) administration. This procedure aims to mimic the effect of chronic stress notably by promoting an excessive HPA axis activation. Multiple sources of evidence demonstrate that CORT exposure in mice elicits behavioral response recapitulating the core endophenotypes of depression such as despair, anhedonia, decreased self-care and anxiety [START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF][START_REF] Orrico-Sanchez | Antidepressant efficacy of a selective organic cation transporter blocker in a mouse model of depression[END_REF] but also impairment of cognitive performances [START_REF] Darcet | Learning and memory impairments in a neuroendocrine mouse model of anxiety/depression[END_REF], sleep disruption (Le [START_REF] Dantec | NREM sleep hypersomnia and reduced sleep/wake continuity in a neuroendocrine mouse model of anxiety/depression based on chronic corticosterone administraton[END_REF] and somatic pain [START_REF] Hache | Antinociceptive effects of fluoxetine in a mouse model of anxiety/depression[END_REF]. Interestingly, the chronic administration of CORT has been also successfully used to delineate the neurobiological mechanisms associated with depression since an alteration of serotonergic neurotransmission (Rainer et al., 2012) and an impairment of adult hippocampal neurogenesis have been reported under these experimental conditions [START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF][START_REF] Quesseveur | BDNF overexpression in a mouse hippocampal astrocytes promotes local neurogenesis and elicits anxiolytic-like activities[END_REF]. Finally, treatments that show therapeutic activity in human are also effective in CORT exposed mice. For instance, it has been repeatedly demonstrated that the long-term administration of the selective serotonin reuptake inhibitor fluoxetine, the serotonin/norepinephrine reuptake inhibitor venlafaxine or the mixed melatonergic agonist/serotonergic antagonist agomelatine reverse CORT-induced anxio/depressive-like phenotype [START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF]Rainer et al., 2012;[START_REF] Mendez-David | S47445 produces antidepressant-and anxiolytic-like effects through neurogenesis dependent and independent mechanism[END_REF]. Altogether, these data strongly support the idea that CORT model displays a strong construct, face and predictive validity thereby offering a translational value to test new hypothesis and identify novel preventive or curative therapeutic strategies [START_REF] Guilloux | Integrated behavioral z-scoring increases the sensitivity and reliability of behavioral phenotyping in mice: relevance to emotionality and sex[END_REF].

D11399 contains mainly a specific essential oil of citrus sinensis which confers a sensory dimension by stimulating the sense of olfaction and referred to as "functional food ingredient" [START_REF] Griffiths | Functional food ingredient quality: opportunities to improve public health by compendial standardization[END_REF]. Hereinafter, we raised the possibility that such a compound imparts mental health-promoting effects and to address this question, we examined the effects of orally administered D11399 on emotional related-behavior in naïve mice or in the CORT animal model of depression [START_REF] Demuyser | Disruption of the HPA-axis through corticosterone-release pellets induces robust depressive-like behavior and reduced BDNF levels in mice[END_REF]. We also addressed the possibility that the behavioral effects of D11399 rely on the activation of the olfactory system. Finally, we investigated whether cellular and molecular processes commonly associated with antidepressant responses such as monoaminergic neurotransmission and neuronal maturation in the hippocampus were impacted by D11399.

Experimental procedures

In this study, 132 mice were used and all experimental procedures were conducted in accordance with ARRIVE guidelines and the European directive 2010/ 63/UE and were approved by the French Ministry of Research and local ethic committees (APAFIS#12343-2017111516096417).

Animals

Male mice (30-35 gr) were fed and watered ad libitum and lived on a 12 hours light-dark cycle. They were housed 4-5 per cage under standard conditions: room with controlled temperature (21-23°C). 3-4 months old outbred CD1 mice were used for the experiments in naïve non-stressed animals. This strain presents a genetic diversity reflecting the human population making them particularly suitable for the screening of drugs with putative antidepressant and/or anxiolytic activities in non-pathological conditions (Petit-Demoulière et al., 2005). 3-4 months old inbred C57BL/6 male mice (Janvier Labs, Saint-Berthevin, France) served for the chronic ones. This strain offers a good model for its vulnerability to stress and its sensitivity to the neurobiological impairments resulting from chronic corticosterone exposure [START_REF] Hodes | Strain differences in the effects of chronic corticosterone exposure in the hippocampus[END_REF]. Importantly, C57BL/6 mice have also been used successfully to unveil the ability of pharmacological strategies and non-pharmacological interventions to restore a normal behavioral phenotype in the CORT model [START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF][START_REF] Hache | Antinociceptive effects of fluoxetine in a mouse model of anxiety/depression[END_REF][START_REF] Dantec | NREM sleep hypersomnia and reduced sleep/wake continuity in a neuroendocrine mouse model of anxiety/depression based on chronic corticosterone administraton[END_REF].

Drugs and protocols

D11399 (Phodé -Terssac, France) contained as active part a core (B524) mainly made up of Citrus sinensis extract (24-32 %) and was formulated with excipients based on water and plant emulsifying agents (36-48%) to obtain a liquid water-dispersible galenic form. Using gas chromatography, we determined that D11399 contains as main ingredients D-limonene (94.5%), myrcene (2.5%), alpha pinene (0.6%), sabinene (0.35%), citral (0.225%) and beta pinene (0.085%). Control solution (D12250:VEH) contained purified coco oil (24-32 %) formulated with the same excipient as D11399. Previous evidence suggests that coco oil exerts anti-stress effects [START_REF] Yeap | Antistress and antioxidant effects of virgin coconut oil in vivo[END_REF] but here it was purified and deodorized making it neutral in terms of flavor while keeping its lipophilic characteristics. In the galenic formulation, it replaces the fat-soluble Citrus extract. We conducted a pilot study to determine the total fluid intake per day and we adapted the dose of compounds to this consumption. For the first experiments in CD1 naïve mice (n=32), water consumption was controlled and replaced three times a week to adjust D11399 daily administration on the basis of 30 g live weight-LW: 0,125 mg/kg LW, 1,25 mg/kg LW, 12,5 mg/kg LW. For the second experiment in CD1 naïve mice (n=30), animals received a daily administration of D11399 (1,25 mg/kg LW)

or its VEH by gavage for one week (n=30) using a stainless-steel bulb tipped gavage needle attached to a syringe. In accordance with ethical guidelines, the volume injected corresponded to 1% of their body weight (e.g., a 30 g mouse was given 0.3 ml). For the third experiment in CD1 naïve mice (n=40), a single intraperitoneal injection of NaCl 0,9% or methimazole (1-methyl-2 mercaptoimidazole, Sigma: 100 mg/kg) was used to ablate the olfactory epithelium before administering D11399 (1,25 mg/kg LW) or (D12250: VEH) for one week (n=40). Finally, for chronic experiments in C57BL6 mice (n=30), we induced depressive-like behaviors using subcutaneously implanted CORT pellets as previously described [START_REF] Demuyser | Disruption of the HPA-axis through corticosterone-release pellets induces robust depressive-like behavior and reduced BDNF levels in mice[END_REF]. Animals were anesthetized with isoflurane and a small incision was done in order to implant CORT pellets with biodegradable carrier-binder (5mg over 90 days; Innovative Research of America -Sarasota -Florida -USA). After surgery, mice were randomly assigned to different groups receiving either the control (D12250: VEH)

or the tested (D11399; 1,25 mg/kg LW) solution. After 10 weeks of treatment, all animals were tested in behavioral paradigms while subgroups were formed for electrophysiological, neurobiochemical and immunohistochemical studies. Control group was exposed to VEH pellets (instead of CORT pellets) and were administered with (D12250: VEH).

For the neurochemical studies (ie, intracerebral microdialysis), we perfused escitalopram (1 M through the dialysis probe from Sigma-Aldrich) to specifically block the serotonin transporter.

Behavioral testing

Except for the open-field test, experimental variables were manually scored. However, to avoid interpretation biases, the operator in charge of the treatment did not perform the experiments, and the animal identification was made after the tests using ear tags.

Open field (OF)

Open field test was performed in Plexiglas setups (MED Associates, France) during 10 min in a rectangular arena (40 x 40 x 20 cm) in a room containing no conspicuous features, illuminated by a white light (30 lux) and surmounted by a video camera connected to a video recorder and a monitor. Total duration and/or the number of entries in the center were measured by an automated system (Noldus, Ethovision, The Nederland). Total ambulatory distance was also measured to ensure the absence of any locomotor effect of treatments.

Elevated plus maze (EPM)

The apparatus (Imetronic, Pessac, France) consisted of a plus-shaped track with 2 closed and 2 open arms (30 x 10 x 20 cm) that extended from a central platform (10 x 10 cm). The apparatus was elevated 50 cm above the floor and illuminated by a white light (25 lux). Each trial began when the mouse was placed in the central zone and lasted 5 min. For each mouse, time spent in the open arms and number of entries in this compartment were calculated.

Novelty suppress feeding test (NSF)

This test was performed in a white plastic box (30 × 60 cm). Mice were food deprived for 24 h before testing and then placed in the corner of the box with a regular food pellet on a white square filter paper at the center of the arena under an intense light (~60 W) placed about 60-80 cm above the food pellet. Latency to begin eating was scored with a cut-off time of 10 minutes. Upon return to the home cage, the total amount of food intake was measured for a 5-minute period to ensure the absence of differences in hunger/motivation.

Splash test (ST)

The splash test was performed for a 5-minute period as previously described [START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF]. After squirting 200 μL of a 10% sucrose solution on the mouse's coat, grooming duration was scored as an index of self-care.

Tail suspension test (TST)

During tail suspension test, mice were both acoustically and visually isolated and suspended 50 cm above the floor by adhesive tape placed approximately 1 cm from the tip of the tail.

Mice were considered immobile only when they hung passively and completely motionless.

Immobility time was recorded during a 6-minute period [START_REF] Steru | The tail suspension test: a new method for screening antidepressants in mice[END_REF][START_REF] Steru | The tail suspension test: a new method for screening antidepressants in mice[END_REF].

Forced swim test (FST)

The forced swim test procedure was modified to enhance the sensitivity to detect the putative antidepressant-like activity of drugs [START_REF] Porsolt | Depression: a new animal model sensitive to antidepressant treatments[END_REF]. Briefly, mice were placed for 6 minutes into clear plastic buckets, 20 cm in diameter and 23 cm deep, filled up to two-thirds with water at ≈24°C. The dependent variable was the time of immobility calculated for the last 4 minutes.

Sucrose preference test (SPT)

Mice were placed and habituated into individual cages for one week with free access to two water bottles. Water consumption was measured to control for place preference. Then in order to measure baseline sucrose preference, one water bottle was removed and replaced by sucrose (1%). Sucrose and water bottles were weighted before and after experiment.

Twenty-four hours later, we compared water and sucrose consumption. Results were expressed in percentage of sucrose consumption [(Sucrose consumption / Total consumption) X 100]. This test was realized at the end of the protocol since treatments cessation was required.

Study of noradrenergic and serotonergic systems

Intracerebral in vivo microdialysis

Anesthetized mice (chloral hydrate, 400 mg/kg; s.c) were stereotaxically implanted with concentric microdialysis probes (effective membrane length 3.0mm) in the ventral hippocampus (vHPC) in order to quantify serotonin (5-HT) and norepinephrine (NE).

Coordinates from bregma, according to the mouse brain atlas [START_REF] Paxinos | The Mouse Brain in Stereotaxic Coordinates[END_REF],

were as follows (in millimeters): AP: -2.7; L: ±3; and V: -3.5. The next day, probes were connected to a microinjection pump, allowing a continuous perfusion of artificial cerebrospinal fluid (aCSF; 147 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1,2 mM CaCl2, adjusted to pH 7.4 with 2 mM sodium phosphate buffer) at a flow rate of 1.5 μL per minute. In order to measure specifically 5-HT release, citalopram (1mM) was added within the aCSF solution. Dialysate samples were collected every 15 minutes for 120 minutes and then samples were analyzed on a high-performance liquid chromatography (HPLC) system coupled with electrochemical detection (Dionex Ultimate 3000; Thermofisher, Courtaboeuf, France).

In vivo electrophysiological recordings of 5-HT neurons in the dorsal raphe

Mice were anaesthetized with chloral hydrate (400 mg/kg i.p.) and placed into a stereotaxic frame. The extracellular recordings of 5-HT neurons in the dorsal raphe (DR) were performed using single-barreled glass micropipettes (Stoelting, USA). Their impedance typically ranged between 5-8 M. The central barrel used for extracellular unitary recording was filled with 2 M NaCl solution. Glass micropipette was positioned using the following coordinates (in mm from lambda): AP: +1.0 to 1.2; L: 0 to 0.1; V: 5 to 7. The presumed 5-HT neurons were then identified using the following criteria: a slow (0.5-2.5 Hz) and regular firing rate and longduration (2-5 ms) bi-or triphasic extracellular waveform [START_REF] Aghajanian | Intracellular recordings from serotonergic dorsal raphe neurons: pacemaker potentials and the effect of LSD[END_REF].

Quantification of immature neurons

Slices preparation

Mice were deeply anesthetized with Pentobarbital and perfused trans-cardially with saline phosphate buffer (PBS) for 2 min followed by 4% paraformaldehyde in phosphate buffer 0.1 M (PB). Brains were removed and post-fixed overnight in 4% paraformaldehyde at 4 °C and cryoprotected in 30% sucrose solution. Coronal sections (30-μm thick) were cut thanks to a microtome (Leica SM2010R) through the entire rostro-caudal axis of the hippocampus.

Sections were stored in cryoprotectant at -20 °C until processed for immunochemistry.

Doublecortin (DCX) immunochemistry

Free-floating brain sections were washed several times with PBS containing 0.25% Triton-X100 (PBST) before quenching endogenous peroxidase with 3% H 2 O 2 and 10% methanol in PBST. Sections were then incubated in a solution containing 10 mM Sodium Citrate pH 6 + 0.05% Tween 20 for 10 minutes at 100°C for antigen retrieval. Sections were then blocked in previously described [START_REF] Verret | Hippocampal neurogenesis during normal and pathological aging[END_REF].

Statistical analysis

Results were expressed as mean ± SEM. A one-way analysis of variance (ANOVA) for treatment factor was applied to compare effect of treatments. When a main effect was significant, comparisons were analyzed using a Tukey post-hoc test (Fig. 1, Fig. S1, Fig. 4, Fig. 6, Fig. 7 and Table 1). For the analysis of treatments effects on behavioral performances in the NSF and FST in presence (or not) of methimazole, a two-way ANOVA with time and treatment factors was used (Fig. S2, Fig. 3). When a main effect of pre-treatment, treatment or interaction pre-treatment x treatment was significant, comparisons were analyzed using a Tukey post-hoc test. In the NSF test, a Kaplan-Meier survival analysis was applied due to the non-normal distribution of data. The Mantel-Cox log-rank test was used to evaluate differences between experimental groups. For the analysis of treatment effects on 5-HT and NE extracellular levels (kinetics), a two-way ANOVA with time and treatment factors was used. When a main effect of time or interaction time x treatment was significant, comparisons were analyzed using a Tukey post-hoc test (Fig. 5). For sucrose preference test, the percentage of sucrose preference was analyzed using a one sample t-test (comparison to the 50% chance value) to reveal sucrose preference (Fig. 5). Cell counts were evaluated by Kruskal-Wallis test followed by post hoc analysis to identify significant pairwise differences.

GraphPad Prism7 software was used for these statistical analysis (significance level was set at p<0.05) and to prepare the graphs.

Results

Behavioral tests in non-stressed CD1 mice given D11399 in the drinking water

In an attempt to examine the putative anxiolytic-and antidepressant-like activities of D11399, we first tested its effects in various behavioral paradigms such as the open field (OF), the elevated plus maze (EPM), the novelty suppressed feeding (NSF) and the forced swimming test (FST) in naïve mice. In the OF and EPM, one-way ANOVA did not reveal any significant effect of treatment factor (F 3,28 =0.7, p=0.53 for the time spent in the center zone of the arena, F 3,28 =1.4, p=0.24 and F 3,28 =1.5, p=0.22 for the time spent and number of entries in the open arms; respectively) (Fig. 1A-D). In a marked contrast, a significant effect of treatment factor was unveiled on the latency to feed in the NSF (F 3,28 =2.9, p=0.04). In particular, D11399 administered at the dose of 1,25 mg/kg/day LW significantly reduced the latency to feed (p=0.04) compared to control VEH mice while the lower (0.125 mg/kg/day LW) and the higher (12.5 mg/kg/day LW) doses failed to do so (p=0.98 and p=0.97; respectively) (Fig. 1E). In the FST, a significant effect of treatment factor was also detected on the immobility time (F 3,28 =4.3, p=0.012). Again, the dose of 1.25 mg/kg/day LW of D11399 significantly reduced this parameter (p=0.02) but not the doses of 0.125 and 12.5 mg/kg/day LW (p=0.21 and p=0.99; respectively) (Fig. 1F).

Because the behavioral effects of D11399 could depend on how it is perceived in mice (ie, rewarding or aversive stimulus), we performed a drinking preference test. Our results indicate that in the early days of treatment (D1 -D3), mice displayed a slight preference for D11399 with a statistically significant effect after 3 days of exposure (p=0.015). In a marked contrast, no preference for D11399 was detected in the following days (D4 -D7), ruling out the possibility that a rewarding effect of D11399 interfered with the mice behavior evaluated at D7 (Fig. S1).

Behavioral tests in non-stressed CD1 mice given D11399 by oral gavage

To determine whether the behavioral effects of D11399 recruit the olfactory system, we then conducted the same battery of tests using a daily oral gastric gavage. As observed after its continuous administration in the drinking water, D11399 did not modify anxiety in the OF (time spent in the center zone of the arena: t 28 =0.14, p=0.88; Fig. 2A) and the EPM (time spent and number of entries in open arms: t 28 =0.38, p= 0.71 and t 28 =2.01, p=0.06 respectively; Fig. 2C-D). Interestingly, this lack of effect was also observed in the NSF (latency to feed in the NSF: t 28 =0,24, p=0.31; Fig. 2E) and the FST (immobility time: t 28 =1.81, p=0.07; Fig. 2F).

Behavioral tests in non-stressed CD1 mice given D11399 while displaying loss of olfaction

In a new set of experiments, we sought to determine to what extent olfaction contributed to the beneficial behavioral effects of D11399 at the active dose of 1.25 mg/kg/day LW. To this end, we first validated the ability of a single administration of methimazole, an antithyroid drug, to induce long-lasting (i.e. 10 days) loss of olfaction in naïve mice as previously reported [START_REF] Blanco-Hernandez | Odor memory stability after reinnervation of the olfactory bulb[END_REF]. Our results indicated that CD1 mice displayed a significant reduction and increase; respectively, of time spent and latency to sniff a piece of paper soaked in female urine 2, 5 or 10 days after the injection of methimazole (Fig. S2). We then pre-treated VEH-or D11399-administered mice with a single administration of NaCl or methimazole and evaluated their behavior one week after the initiation of the treatments.

In the NSF, two-way ANOVA revealed a significant effect of pre-treatment (F 1,36 =4,4; p=0.04)

but not of treatment (F 1,36 =1,5; p=0.22) or the interaction between these main factors (F 1,36 =0.2; p=0.62) (Fig. 3A). Unfortunately, pairwise comparisons did not allow us to unveil significant differences between groups. We thus decided to analyze our data using the survival curve. Doing so, in VEH-administered mice we showed that methimazole failed to modify the latency to feed (p>0.05) but it significantly increased this parameter in D11399treated mice (p<0.05) (Kaplan-Meier survival analysis, Mantel-Cox long-rank test; p=0.007; Fig. 3B).

In the FST, two-way ANOVA revealed a significant effect of pre-treatment (F 1,36 =4,2; p=0.04)

but not of treatment (F 1,36 =1,8; p=0.18) or the interaction between these main factors (F 1,36 =3.3; p=0.07). Interestingly, although methimazole had no effect in VEH-administered mice compared to mice pre-treated NaCl, it significantly reversed D11399-induced decrease of immobility time (Fig. 3C).

Behavioral tests in C57BL6j mice exposed to CORT

Based on the latter results, we then evaluated the effects of D11399 (1.25 mg/kg/day LW) in a mouse model of depression based on chronic exposure to subcutaneous CORT or its vehicle (VEH) for 10 weeks (Fig. 4A). In the OF, one-way ANOVA revealed a significant effect of treatment factor on time and entries in the center zone of the arena (F 2,27 =3.6, p=0.03 and F 2,27 =4.2, p=0.02; respectively) while no significant effect was unveiled regarding total distance travelled (F 2,27 =2,3, p=0.11). Post-hoc analysis showed a significant decrease of time spent in center of arena (p=0.04) and in the number of entries in the center (p=0.03) in CORT-VEH treated mice compared to VEH-VEH (Fig. 4B-C). Interestingly, the decrease in time spent in the center of arena was no longer evident in CORT-D11399 mice compared to VEH-VEH mice (p=0.91) (Fig. 4B). However, this parameter was not significantly different between CORT-VEH and CORT-D11399 mice (p=0.11) thereby suggesting a partial reversal of anxiety by D11399 (Fig. 4B). In the EPM test, one-way ANOVA did not show significant effects of treatment factor for the time spent (F 2,26 =1.6, p=0.22) and the number of entries in open arms (F 2,26 =1.04, p=0.36) (Fig. 4E-F). In the NSF, one-way ANOVA revealed a significant effect of treatment factor (F 2,26 =3.5, p=0.04). Although an increase in the latency to feed was observed in CORT-VEH compared to VEH-VEH, this effect did not reach statistical significance (p=0.09). Interestingly this latency to feed did not differ between VEH-VEH and CORT-D11399 mice (p=0.96) (Fig. 4G). Importantly, home cage consumption was not different between groups (F 2,26 =1.2, p=0.29) thereby ruling out the possibility that changes observed in the latency to feed were related to changes in hunger.

In the TST, one-way ANOVA indicated a significant effect of treatment factor (F 2,26 =4.6, p=0.01). This was reflected by a significant increase in immobility time in CORT-VEH compared to VEH-VEH mice (p=0.01) whereas this difference was abolished in CORT-D11399 mice (p=0.80) (Fig. 4H). Immobility time was not significantly different between CORT-VEH and CORT-D11399 mice (p=0.07) emphasizing a partial reversal of despair by D11399 (Fig. 4H). In the splash test, used for assessing self-care, a significant effect of treatment was found (F 2,26 =18.4, p<0.001). CORT-VEH treated mice showed a significant decrease in time of grooming in ST (p<0.001) compared to VEH-VEH mice which was not present in CORT-D11399 mice (p=0.22) (Fig. 4I). Interestingly, a significant difference on time of grooming was observed between CORT-VEH and CORT-D11399 mice (p<0.001).

Finally, in sucrose preference test, one-way ANOVA showed a significant effect of treatment factor (F 2,26 =6.1, p=0.007). Sucrose preference in CORT-VEH mice was significantly lower than in VEH-VEH group (p=0.005) but not in CORT-D11399 (p=0.23) compared to VEH-VEH. It is also noteworthy that sucrose preference was not different from the chance level in CORT-VEH treated mice (p=0.20) whereas a significant preference was detected in both VEH-VEH (p<0.001) and CORT-D11399 (p=0.002) (Fig. 4J).

Altogether, these behavioral data emphasize that CORT caused an anxio-depressive-like phenotype which is attenuated by D11399.

Study of the monoaminergic systems in C57BL6j mice exposed to CORT

Because an increase in noradrenergic and serotonergic neurotransmissions has been associated to antidepressant-like response, we then determined to what extent these monoaminergic systems were affected in CORT mice treated with VEH or D11399.

Extracellular NE and 5-HT levels in the ventral hippocampus (vHPC)

Using in vivo intracerebral microdialysis, we next measured the basal extracellular NE and 5-HT levels in the vHPC of freely moving mice. One-way ANOVA revealed a significant effect of treatment factor on basal NE levels ([NE]ext) (F 2,12 =6.7; p=0.01). In particular, mice exposed to CORT-VEH (p=0.04) or CORT-D11399 (p=0.04) displayed higher [NE]ext than VEH-VEH mice. However, CORT-VEH and CORT-D11399 groups were not statistically different (p>0.05; Table 1).

In a marked contrast, one-way ANOVA for [5-HT]ext, did not unveil significant effect of treatment factor (F 2,25 =1.8; p=0.17; Table 1). However, given that 5-HT outflow measured by microdialysis reflects a balance between 5-HT uptake and release, we assumed that any increase in 5-HT release at the nerve terminals could have been counterbalanced by an active 5-HT reuptake process. We thus evaluate vHPC extracellular 5-HT levels in the presence of the SERT inhibitor escitalopram (1 µM) directly perfused through the microdialysis probes (Fig. 5A-B). One-way ANOVA indicated a significant effect of treatment factor (F 2,21 =5.1 ; p=0.016). Under this condition, CORT-VEH and CORT-D11399 mice displayed higher levels of [5-HT]ext compared to VEH-VEH mice (p=0.04 and 0.02; respectively) but no differences were detected between CORT-VEH and CORT-D11399 (p>0.05; Fig. 5B).

5-HT neuronal activity in the dorsal raphe nucleus (DRN)

Because 5-HT tone in the vHPC is highly dependent on DRN 5-HT neuronal activity, we then determined the effect of CORT given either alone or in combination with D11399 on the firing rate of this cell population. One-way ANOVA revealed a significant effect of treatment factor (F 2,85 =3.1; p=0.04). The firing rate of DRN 5-HT was significantly lower in CORT-VEH mice compared to VEH-VEH (p=0.04) and this difference was abolished in CORT-D11399 mice (p=0.38) (Fig. 6A-B). No statistical difference was detected between CORT-VEH and CORT-D11399 mice (p=0.16; Fig. 6A).

Study of the neuronal maturation in the dentate gyrus of the hippocampus in C57BL6j mice exposed to CORT

Finally, we investigated the influence of CORT  D11399 on neuronal maturation, used as an index of adult hippocampal neurogenesis. Using Kruskal-Wallis test on the number of doublecortin-positive (DCX+) cells, a significant effect of treatment factor was unveiled in the whole dentate gyrus (H 2,12 =8.24, p=0.007). CORT alone significantly decreased the number of DCX+ cells compared to VEH-VEH mice (p=0.014; Fig. 7A) but this effect was no longer statistically different when combined with D11399 (p>0.05). When evaluating the percent of DCX+ cells in the granular layer, Kruskal-Wallis test also revealed a significant effect of treatment factor (H 2,12 =6.01, p=0.04). Interestingly, a significant reduction in the percent of DCX+ cells was detected in CORT-VEH (p=0.04) mice but not in CORT-D11399 (p>0.05) (Fig. 7B-C). No statistical difference was detected between CORT-VEH and CORT-D11399 mice (p=0.054; Fig. 7B).

Discussion

In the present study, we showed that the continuous administration of D11399 in the drinking water produced anxiolytic-and antidepressant-like activities in naïve non-depressed mice. In an attempt to demonstrate the contribution of functional olfaction in such beneficial effects, we tested D11399 after its administration by oral gavage or in presence of methimazole, a drug which has been reported to promote a long-term and reversible inactivation of olfaction.

Remarkably, both procedures blocked the effects of D11399. Going one step further, we also showed that D11399 attenuated CORT-induced anxiety and depressive-like state.

Mechanistically, our experimental observations suggest that the beneficial effects of D11399 could result from its ability to reverse CORT-induced inhibition of DRN 5-HT neuronal activity and adult-born neuron maturation at the nerve terminals namely in the dentate gyrus of the hippocampus.

The main active component of D11399 is Citrus sinensis extract with olfactory/gustatory properties. Several studies have reported that acute or chronic aerial exposure of Citrus sinensis elicits anxiolytic-and/or antidepressant-like effects in naïve non-depressed rodents (Wollfenbuttel et al., 2018). Here, we observed in naïve mice that the continuous administration of D11399 in the drinking water elicits anxiolytic-like effects in the NSF and reduces the immobility time in the FST as an index of decreased despair. These findings are interesting since it is well accepted that currently available antidepressant drugs such as the Selective Serotonin Reuptake Inhibitors (SSRIs) decrease despair while producing anxiogenic effects. Thus, one would expect that D11399 offers a wider spectrum of beneficial effects than antidepressant drugs. It is however important to mention that anxiolytic responses were not detected in the OF and EPM. Such differences could rely on the brain circuits recruited in these paradigms. Indeed, although the OF, EPM and NSF are ethological tests that investigate approach-avoidance conflict, the NSF introduces an additional component of motivation, as the food-deprived animal's drive to eat conflicts with its fear of novel open spaces [START_REF] Stedenfeld | Novelty-seeking behavior predicts vulnerability in a rodent model of depression[END_REF].

The beneficial effects of D11399, lead us to envision that this compound could interfere with the olfactory system. Indeed, although it could seem counterintuitive that the olfactory epithelium is activated following oral administration, a recent study reported than mouse was able to detect and discriminate retronasal odors [START_REF] Rebello | Direct behavioral and neurophysiological evidence for retronasal olfaction in mice[END_REF]. Such a mechanism may in turn influence stress responses and emotional state. In agreement with this hypothesis, it has been shown that surgical removal of the olfactory bulbs in rats or mice leads to a number of changes usually associated with depression [START_REF] Hendriksen | Back to the future of psychopharmacology: A perspective on animal models in drug discovery[END_REF]. In particular, bulbectomy is associated with impaired adult hippocampal neurogenesis [START_REF] Morales-Medina | The olfactory bulbectomized rat as a model of depression: the hippocampal pathway[END_REF], neurodegeneration in the lateral habenula [START_REF] Oral | How olfaction disorders can cause depression? The role of habenular degeneration[END_REF] as well as modifications of monoamonergic neuronal activities [START_REF] Masini | Dopamine overflow is increased in olfactory bulbectomized rats: an in vivo microdialysis study[END_REF][START_REF] Van Der Stelt | Permanent deficits in serotonergic functioning of olfactory bulbectomized rats: an in vivo microdialysis study[END_REF]. Although the underlying mechanisms are poorly documented, it has been proposed that such deficits result from a loss of connectivity between the olfactory bulbs and the limbic system [START_REF] Song | The olfactory bulbectomised rat as a model of depression[END_REF]. In an attempt to determine to what extent olfaction played a role in the beneficial behavioral effects of D11399, we conducted a new set of experiments using the oral gavage administration. Our results show that D11399 was no longer effective in the NSF and FST. Although these results strongly suggest a role of olfaction in D11399 activity, we cannot rule out the possibility that the olfactory epithelium was activated using this route of administration. In light of this possibility, we thus implemented another strategy aimed at testing D11399 in presence of methimazole, an antithyroid drug known to cause damage of the olfactory epithelium. As previously described [START_REF] Bergstrom | Methimazole-induced damage in the olfactory mucosa: effects on ultrastructure and glutathione levels[END_REF]. As expected, we found that methimazole induced long-lasting inhibition of olfaction. At the behavioral level, we provided evidence that methimazole by itself did not influence the spontaneous behavioral responses in the NSF and FST. Strikingly, we observed that methimazole prevented the anxiolytic-and antidepressant-like activities of D11399 in both paradigms. The latter experiments emphasize the necessity of the olfactory system in the behavioral activity of D11399 but it did not demonstrate its sufficiency. They also reinforce the idea of additional systemic effects following D11399 oral administration.

Although the mechanisms of action have yet to be determined, several hypotheses can be advanced. D11399 contains D-limonene which is an aliphatic hydrocarbon classified as a cyclic monoterpene. This is also one major component in the citrus essential oils [START_REF] Dosoky | Biological activities and safety of citrus spp. Essential oils[END_REF] and we can assume that the beneficial effects of D11399 could result, at least in part, from the activation of its preferential olfactory receptors namely Olfr56 and Olfr124 [START_REF] Koyama | The effects of essential oils and terpene in relation to their routes of intake and application[END_REF]. D-limonene could also act through non-olfactory receptors expressed in the olfactory epithelium such as transient receptor potential channels (TRP) [START_REF] Lin | Olfactory neurons expressing transient receptor potential channel M5 (TRPM5) are involved in sensing semiochemicals[END_REF] that are expressed in olfactory sensory neurons. These neurons synapse with mitral cells in the olfactory bulbs projecting to multiple areas of the limbic system (eg, amygdala and hippocampus) [START_REF] Carnavan | Changes in maternal gene expression in olfactory circuits in the immediate postpartum period[END_REF][START_REF] Mainland | From molecule to mind: an integrative perspective on odor intensity[END_REF] and their activation could be a putative mechanism by which D11399 could exerts its beneficial effects on emotional state. Interestingly, Olfr56 and Olfr124 are also found in the mouse gut [START_REF] Koyama | The effects of essential oils and terpene in relation to their routes of intake and application[END_REF] and evidence suggests that their activation could affect the intestinal microflora of mice, more specifically by enhancing the relative abundance of Lactobacillus [START_REF] Wang | Effects of orange essential oil on intestinal microflora in mice[END_REF]. Given the role of microbiota in the regulation of emotional state, D11399 could also interact in the gut with these micro-organisms to contribute to its anxiolytic and antidepressant-like effects.

To confirm the therapeutic preventive effects of D11399, we decided to extend our results in relevant animal models of depression based on the subcutaneous administration of corticosterone (CORT) pellets ensuring a chronic all-day constant delivery of this stress hormone and a dose-controlled administration through drinking water. In agreement with previous data [START_REF] Demuyser | Disruption of the HPA-axis through corticosterone-release pellets induces robust depressive-like behavior and reduced BDNF levels in mice[END_REF], CORT pellets caused various endophenotypes of depression such as despair in the TST, decreased self-care in the ST and anhedonia in the SPT. We also noticed a significant decrease in the time spent and number of entries in the center of the OF compared to control animals. Our results indicate that D11399 (1.25 mg/kg/day LW dose; p.o.) reversed a part of these behavioral anomalies, strengthening its antidepressant-like profile in pathological conditions. This is consistent with previous findings reporting that Citrus sinensis exerts beneficial effects in other animal models of depression such as the chronic unpredictable mild stress (UCMS) after sniffing [START_REF] Zhang | Antidepressant-like Effect of Citrus sinensis (L.) Osbeck Essential Oil and Its Main Component Limonene on Mice[END_REF].

Remarkably, the beneficial effects of D11399 detected in CORT mice, extended beyond those detected in naïve non-stressed mice. For example, we showed that D11399 exerted anxiolytic effects in the OF while it failed to do so in naïve non-depressed mice. The latter findings are consistent with the fact that psychotropic drugs mainly act on psychopathological conditions.

Mechanistically, it is well known that 5-hydroxytryptamine (5-HT) but also Norepinephrine (NE) are the primary contributors to MD and that most antidepressant drugs act by enhancing monoaminergic tone in various brain areas such as the hippocampus. As mentioned earlier, the latter brain region is anatomically and functionally connected to the olfactory bulbs or the gut, both being directly or indirectly sensitive to odors. Thus, we raised the possibility that the anxiolytic/antidepressant-like effects of D11399 results from the activation of 5-HT/NE systems. Our results indicated that CORT significantly increased basal extracellular norepinephrine levels in the hippocampus. This effect of CORT is well described and contributes to activate the hypothalamic-pituitary-adrenal axis, thereby eliciting stress responses and anxiety [START_REF] Itoi | The brainstem noradrenergic systems in stress, anxiety and depression[END_REF]. However, we noticed that D11399 did not reverse CORT-induced increase in hippocampal NEext. Owing to the functional interactions between monoaminergic neurons [START_REF] Guiard | Prospect of a dopamine contribution in the next generation of antidepressant drugs: the triple reuptake inhibitors[END_REF], we then explored the influence of CORT on the serotonergic system. We did not observe any increase in basal hippocampal extracellular 5-HT levels (5-HText) in CORT mice. Because 5-HT outflow measured by microdialysis reflects a balance between 5-HT uptake and release, we assumed that any increase in 5-HT release at the nerve terminals may be counterbalanced by an active 5-HT reuptake process. If this assumption is correct, stimulating effects on basal extracellular 5-HT levels would be revealed in the presence of a SERT inhibitor. To address this possibility, we perfused the SSRI escitalopram through the dialysis probe. Under these conditions, we showed that CORT-VEH mice display higher hippocampal 5-HText than controls suggesting that CORT favored the release of 5-HT. The latter finding concurs with the fact that stress and activation of glucocorticoid receptors (GRs) increase the expression/activity of the tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme in the synthesis of neuronal 5-HT [START_REF] Donner | Two models of inescapable stress increase tph2 mRNA expression in the anxiety-related dorsomedial part of the dorsal raphe nucleus[END_REF]. Despite these experimental precautions, we did not observe any effect of D11399 on CORT-induced increase in hippocampal 5-HT release. We then studied the firing rate of 5-HT neurons in response to CORT given either alone or in combination with D11399. We showed that CORT decreased the firing of DR 5-HT neurons whereas D11399 reversed this response. To reconcile the neurochemical and electrophysiological data, it is plausible that CORT stimulated the release of 5-HT in the DR as observed in the hippocampus and that the excess of this monoamine around 5-HT cell bodies activated inhibitory somatodendritic 5-HT1A autoreceptor, which in turn, could have contributed to reduce DR 5-HT neuronal activity. On these grounds, D11399 would act by preventing the overactivation of 5-HT1A autoreceptor. Interestingly, several studies reported that administration of Citrus extracts increase hippocampal 5-HT and NE [START_REF] Komori | Potential antidepressant effects of lemon odor in rats[END_REF][START_REF] Komiya | Lemon oil vapor causes an anti-stress effect via modulating the 5-HT and DA activities in mice[END_REF] while decreasing serum levels of corticosterone [START_REF] Yi | Antidepressant-like behavioral and neurochemical effects of the citrus-associated chemical apigenin[END_REF]. Given that these studies were conducted in naïve non-depressed animals, one would expect that the chronic exposure to CORT significantly impacts brain circuits to minimize the action of D11399 on the monoaminergic systems. Finally, the nature of the active compounds, the route of administration as well as the exposure duration represent a combination of multiple factors that might explain the discrepancies between these studies and our findings.

To further investigate the mechanism of action of D11399, we finally focused our attention on adult-born immature neurons in the hippocampus used as an index of adult hippocampal neurogenesis. It is now well accepted that the enhancement of hippocampal neurogenesis is important in the mechanism of action of antidepressant drugs [START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF]. We showed that CORT mice displayed a decrease in the number of DCX-positive cells in the dentate gyrus of the hippocampus whereas D11399 did not prevent this decrease. However, during their maturation, adult-born neurons migrate from the sub-granular zone to the granular cell layer of the DG. A more detailed analysis revealed that CORT decreased the percentage of DCX+ cells in the granular layer of the DG, suggesting a maturation defect.

Interestingly, D11399 prevented this effect. This positive action concurs with our electrophysiological data of 5-HT neurons but not with the microdialysis experiments. In light of the latter data, it would be interesting to measure extracellular 5-HT levels specifically in the DG instead of the CA1 subregion of the hippocampus as described herein.

Nevertheless, our observations concur with previous data showing that chronic inhalation of Citrus sinensis restored UCMS-induced downregulation of brain-derived neurotrophic factor (BDNF) and its receptor TrkB expression in the hippocampus [START_REF] Zhang | Antidepressant-like Effect of Citrus sinensis (L.) Osbeck Essential Oil and Its Main Component Limonene on Mice[END_REF]). An indirect mechanism could explain this effect since granule cells in the DG receive, through the perforant path, massive afferents from the lateral olfactory tract [START_REF] Sosulski | Distinct representations of olfactory information in different cortical centres[END_REF][START_REF] Strauch | Orchestration of hippocampal information encoding by the piriform cortex[END_REF]. The DG appears to be sensitive to olfactory stimulation and one would expect that functional modification of this pathway might represent a substrate for the antidepressant-like properties of D11399.

A

  Citrus based sensory functional food ingredient induces antidepressant-like effects: possible involvement of an interplay between the olfactory and the serotonergic systems B. Coutens, K. Rekik, A. Harster, P. Etienne, V. Noirot, B. Frances, L. Moulédous, B. P. Guiard To cite this version: B. Coutens, K. Rekik, A. Harster, P. Etienne, V. Noirot, et al.. A Citrus based sensory functional food ingredient induces antidepressant-like effects: possible involvement of an interplay between the olfactory and the serotonergic systems. Neuroscience, 2020, 10.1016/j.neuroscience.2020.09.040. hal-02983060

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  % normal goat serum (NGS) for 2 hours. They were then incubated overnight at room temperature in polyclonal Rabbit anti-doublecortin antibody (DCX; 1/500; Synaptic Systems) in PBST containing 5% NGS. The next day, sections were rinsed in PBST and incubated for 120 minutes at room temperature in Alexa Fluor 488 Goat anti-Rabbit antibody in PBST (1/1000; Invitrogen). Sections were mounted onto slides, coverslipped using Mowiol containing Hoechst (1/10000), and stored at 4 °C. Examination of positively labeled cells was confined to the DG. Quantifications of DCX-immunoreactive (DCX+) cells were conducted on 1-in-12 series of labeled sections spaced by 360 μm spanning the rostrocaudal extent of the DG bilaterally. All immunostained sections were analyzed using a DM6000B fluorescence microscope (Leica, Germany) equipped with a motorized X-Y sensitive stage and a video camera connected to a computerized image analysis system (ExploraNova, France). The counting of labeled cells was conducted bilaterally using Mercator v.2 software (ExploraNova) that allows the measure of the DG corresponding surface. The reference volume was determined as the sum of the traced areas multiplied by the distance between sampled sections. Densities of immunopositive cells were then calculated by dividing the number of positive cells by the DG sectional volume. The absolute numbers of DCX-positive cells in the DG are expressed by multiplying the cell densities by the reference volume as

  Altogether our results highlight an anxiolytic/antidepressant-like activity of the sensory functional food ingredient D11399 (1.25 mg/kg/day LW dose; p.o.) in adult male mice and this could result from the activation of a brain circuit arising from the olfactory system and dynamically influence the activity of serotonergic neurons in the dorsal raphe. Although multiple source of evidence indicate that 5-HT neurons send dense innervation in the olfactory system to regulate olfaction(Steinfeld et al., 2015), the present study raises the possibility that the reciprocal functional interactions may also occur to regulate emotional state. Deciphering such a circuit might pave the way to identify new therapeutic targets to relieve depressive symptoms. This work received financial support from Phode. Bruno Guiard and Lionel Mouledous currently receive investigator-initiated research support from Phode. Bruno Guiard also served as a consultant in the areas of target identification and validation and new compound development for Lundbeck and Servier in 2011-12 and Theranexus since 2016. Authors' contribution CNRS: B.C., K.R. and A.S.H. performed the surgical procedures and conducted the behavioral, neurochemical, electrophysiological and immunohistochemical experiments. They also performed data analyses. B.F., L.M., B.P.G. supervised the study, interpreted the results and wrote the manuscript.Phode: P.E. and V.N. provided the D11399 and its vehicle (D12250). They also participated in the discussion regarding the design of the study and assisted the academic authors in the proofreading of the manuscript.

Figures legend Figure 1 :

 legend1 Figures legend

Figure 2 :

 2 Figure 2: Attenuation of D11399-induced antidepressant-like effects after oral gavage

Figure 3 :

 3 Figure 3: Attenuation of D11399-induced antidepressant-like effects after the inactivation of olfaction by methimazole in non-stressed mice. Four groups of mice were

Figure 4 :

 4 Figure 4: D11399 reverses CORT-induced anxiety and depressive-like phenotype.

Figure 5 :

 5 Figure 5: D11399 does not modify CORT-induced increase in 5-HT release in the ventral hippocampus.

Figure 6 :

 6 Figure 6: D11399 reverses CORT-induced decrease in dorsal raphe 5-HT neuronal

Figure 7 :

 7 Figure 7: D11399 reverses CORT-induced decrease in hippocampal neuronal

Table 1 :

 1 D11399 does not modify CORT-induced increase in basal hippocampal extracellular norepinephrine and serotonin levels. Basal extracellular levels of NE and 5-HT in the hippocampus of VEH-VEH (control), CORT-VEH and CORT-D11399 mice. *p<0.05: significantly different from VEH-VEH mice.

Figure S2 :

 S2 Figure S2: Validation of methimazole-induced loss of olfaction. Naïve mice were placed for 3 min in a new cage containing a blotting paper soaked in female urine. Data are mean ± SEM of time spent (A) and latency (B) to sniff the piece of paper 2, 5 or 10 days after the injection of saline (NaCl 0.9%) or methimazole (100 mg/kg; i.p.) (n=5-10 mice per group). *p<0.05, **p<0.01 and ***p<0.001: significantly different from NaCl injected mice.

This study received financial support from Phodé

ACKNOWLEDGMENTS

We thank Nathalie Legendre for the coordination of the research project. The authors also greatly acknowledge the

Conflict of interest statement

In order to test whether scent of D11399 had reward properties, a drinking preference test was performed. Thirty-two CD1 mice were housed 4 per cage for one week with free access to one bottle of D11399 and one bottle of water. In order to avoid place preference, the bottles were changed every day by swapping the position. Consumptions of water and D11399 were recorded every day. Results were represented by the ratio: D11399 consumption/ Water consumption. Ratios greater than 1 indicated a preference for the D11399.

Figure S1. Drinking preference test (D11399 vs. water).

Mice had the choice (two bottles) to consume D11399 or water for one week. Drinking consumption was recorded once a day and results were represented as mean ± SEM of D11399/water consumption ratio. Equal consumption of the two bottles (i.e., no preference) corresponds to a ratio =1 represented by the dotted line. Preference for D113900 correspond to a ratio >1. *p<0.05: significantly different from theorical value fixed to 1 using a Wilcoxon test (n=8 cages of 4 mice).

Supplemental Figure 2

Experimental procedure (Olfaction test) Olfaction was assessed by evaluating the capacity of mice to detect female urine. 50 µl of freshly collected urine were dropped on a small piece of blotting paper. The paper was then buried 1cm beneath the surface in a random position in a new cage containing about 3 cm of clean bedding. The latency to find the paper was recorded, as well as the time spent sniffing the paper over a total period of 3 minutes.