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International audience; Using laying hens, we investigated whether position of a nest box, both within the pen and relative to other nest boxes, influenced the preference for a nest box, and how a sudden and marked change to the preferred box influenced the use of nest boxes by the hens. Groups (n = 12) of 15 Isa Warren hens were housed in pens, each with five identical nest boxes in different positions: Two single (in a corner or not) and a triplet of nest boxes (one of which in a corner). The use of nest boxes was determined by the number of eggs laid daily in each box. Three experiments, each lasting 10 days, were carried out. First, the undisturbed use of each of the nest box types was investigated, and a strong preference (P < 0.001) was found for single nest boxes in a corner, with 62% of the nest box eggs laid there. Second, each of the hen groups was moved to another pen allocated at random, and where the configuration of nest box types differed from that of their original pen. An effect of nest box type was found (P < 0.001), with 41% of all eggs laid in the single nest box in a corner, and 26% of eggs laid in the corner triplet nest box. Third, the attractiveness of the preferred nest box within each pen was reduced by removing the Astroturf mat from the nest box floor, exposing the wire netting below. This resulted in a change of nest box use (P < 0.001) from the single, corner nest box (67%; n = 6) to the corner triplet nest box (37%) and the single nest box not in a corner (35%), and from the corner triplet nest box (48%; n = 5) to the remaining triplet nest boxes (40% and 22%, respectively). The initial preference for a single nest box in a corner was probably due to a combination of isolation and view of the surroundings provided by this type of nest box. The manipulations in experiments II and III revealed that some hens were location conservative, i.e. continued laying in a corner location (or as close to that as possible), whereas others were isolation conservative, i.e. continued laying in the most isolated nest box despite it being positioned in a different area of the pen.

International audience; Sexually experienced male rats display penile erections when exposed to faeces from mammalian females in oestrus (Rampin et al., Behav Brain Res, 172:169, 2006), suggesting that specific odours indicate female receptiveness across species. However, it is unknown to what extent the sexual response observed results from an odorous conditioning acquired during sexual experience. We tested the behavioural response of male Brown Norway rats both when sexually naïve and experienced to four odours, including oestrous rat faeces and 6-methyl-5-hepten-2-one (methylheptenone; a molecule found in higher concentrations during oestrus in female rats, foxes and horses). Odour had a significant effect on the sexual response of the naïve rats, with oestrus faeces provoking significantly more erections than herb odour, and with methylheptenone and di-oestrus faeces being intermediate. This indicates that sexually naïve male rats have an unconditioned ability to detect oestrous mediated via odour. After gaining sexual experience, the response to methylheptenone, di- and oestrus faeces was significantly higher than that observed with herb odour. These results strongly suggest that methylheptenone is part of the odorous bouquet of oestrus and contributes to the olfactory determination of female receptiveness.

International audience; The central nervous system (CNS) is known to be sensitive to pollutants during its development. Uranium (U) is a heavy metal that occurs naturally in the environment as a component of the earth's crust, and populations may therefore be chronically exposed to U through drinking water and food. Previous studies have shown that the CNS is a target of U in rats exposed in adulthood. We assessed the effects of U on behavior and cholinergic system of rats exposed from birth for 10 weeks at 10 mg.L⁻¹ or 40 mg.L⁻¹. For behavioral analysis, the sleep/wake cycle (recorded by telemetry), the object recognition memory and the spatial working memory (Y-maze) were evaluated. Acetylcholine (ACh) and acetylcholinesterase (AChE) levels were evaluated in the entorhinal cortex and hippocampus. At 40 mg.L⁻¹, U exposure impaired object recognition memory (-20%), but neither spatial working memory nor the sleep/wake cycle was impaired. A significant decrease was observed in both the ACh concentration (-14%) and AChE activity (-14%) in the entorhinal cortex, but not in the hippocampus. Any significant effect on behaviour and cholinergic system was observed at 10 mg U.L⁻¹. These results demonstrate that early exposure to U during postnatal life induces a structure cerebral-dependant cholinergic response and modifies such memory process in rats. This exposure to U early in life could have potential delayed effects in adulthood.

Increased activity improves broiler leg health, but also increases the heat production of the bird. This experiment investigated the effects of early open-field activity and ambient temperature on the growth and feed intake of two strains of broiler chickens. On the basis of the level of activity in an open-field test on day 3 after hatching, fast-growing Ross 208 and slow-growing i657 chickens were allocated on day 13 to one of the 48 groups. Each group included either six active or six passive birds from each strain and the groups were housed in floor-pens littered with wood chips and fitted with two heat lamps. Each group was fed ad libitum and subjected to one of the three temperature treatments: two (HH; 26°C), one (HC; 16°C to 26°C) or no (CC; 16°C) heat lamps turned on. Production and behavioural data were collected every 2 weeks until day 57. For both strains, early open-field activity had no significant effects on their subsequent behaviour or on any of the production parameters measured, and overall, the slow-growing strain was more active than the fast-growing strain. Ambient temperature had significant effects on production measures for i657 broilers, with CC chickens eating and weighing more, and with a less efficient feed conversion than HH chickens, with HC birds intermediate. A similar effect was found for Ross 208 only for feed intake from 27 to 41 days of age. Ross 208 chickens distributed themselves in the pen with a preference for cooler areas in the hottest ambient temperature treatments. In contrast, the behaviour of the slow-growing strain appeared to be relatively unaffected by the ambient temperature. In conclusion, fast-growing broilers use behavioural changes when trying to adapt to warm environments, whereas slow-growing broilers use metabolic changes to adapt to cooler ambient temperatures.

Manganese (Mn)-enhanced magnetic resonance imaging (MEMRI) is an emerging technique for visualizing neuronal pathways and mapping brain activity modulation in animal models. Spatial and intensity normalizations of MEMRI images acquired from different subjects are crucial steps as they can influence the results of groupwise analysis. However, no commonly accepted procedure has yet emerged. Here, a normalization method is proposed that performs both spatial and intensity normalizations in a single iterative process without the arbitrary choice of a reference image. Spatial and intensity normalizations benefit from this iterative process. On one hand, spatial normalization increases the accuracy of region of interest (ROI) positioning for intensity normalization. On the other hand, improving the intensity normalization of the different MEMRI images leads to a better-averaged target on which the images are spatially registered. After automatic fast brain segmentation and optimization of the normalization process, this algorithm revealed the presence of Mn up to the posterior entorhinal cortex in a tract-tracing experiment on rat olfactory pathways. Quantitative comparison of registration algorithms showed that a rigid model with anisotropic scaling is the best deformation model for intersubject registration of three-dimensional MEMRI images. Furthermore, intensity normalization errors may occur if the ROI chosen for intensity normalization intersects regions where Mn concentration differs between experimental groups. Our study suggests that cross-comparing Mn-injected animals against a Mn-free group may provide a control to avoid bias introduced by intensity normalization quality. It is essential to optimize spatial and intensity normalization as the detectability of local between-group variations in Mn concentration is directly tied to normalization quality.

International audience; The postnatal leptin surge, described particularly in rodents, has been demonstrated to be crucial for hypothalamic maturation and brain development. In the present study, the possible general effects of this hormone on maturation of numerous peripheral organs have been explored. To test this hypothesis, we used a leptin antagonist (L39A/D40A/F41A) to investigate the effects of the blockage of postnatal leptin action on neonatal growth and maturation of organs involved in metabolism regulation, reproduction and immunity. For that purpose, newborn female pups were subcutaneously injected from days 2-13 with either saline or leptin antagonist and sacrificed at weaning. Organs were submitted to histological and immunohistochemical analyses. Leptin antagonist treatment clearly impaired the maturation of pancreas, kidney, thymus and ovary. All these alterations, at the organ level, occurred without changes in the whole-body mass of the animals. Leptin antagonist treatment induced: (1) a reduction in beta cell area and a concomitant increase of a cells in Langherans islets in the pancreas, (2) a reduction in the number of glomeruli and a persistence of immature glomeruli in kidney, (3) an increase in the thymic cortical layer thickness, reflecting an unmatured stage, (4) a drastic reduction of the pool of primordial follicles, in ovaries. All these results strongly argue for a crucial role of leptin for the achievement of organ maturation, opening new perspectives in the field of leptin physiology and organ development.

International audience; In mammals, the olfactory sensory neurons are the only ones directly in contact with an aggressive environment. Thus, the olfactory mucosa is one of the few neuronal zones which are continuously renewed during adulthood. We have previously shown that endothelin is locally matured in the olfactory mucosa and that olfactory sensory neurons preferentially express ET(B) receptors, while ET(A) receptors are rather present in non neuronal olfactory mucosa cells. In addition to its vasoactive effect, the endothelin system is known for its pleiotropic effects including the modulation of cell population dynamics. We thus examined its potential neuroprotective effect in the olfactory mucosa using a primary culture of olfactory sensory neurons lying on non neuronal cells. While a serum deprivation led to a massive decrease of the density of olfactory sensory neurons in the primary cultures, endothelin 1 (ET-1) rescued part of the neuronal population through both ET(A) and ET(B) receptors. This effect was mainly anti-apoptotic as it reduced cleaved caspase-3 signal and nuclear condensation. Furthermore, the olfactory epithelium of ET(B)-deficient rats displayed increased apoptosis. These results strongly suggest that ET-1 acts as an anti-apoptotic factor on olfactory sensory neurons, directly through ET(B) and indirectly by limiting non neuronal cells death through ET(A).

Early survival factor deprivation in the olfactory epithelium enhances activity-dependent survival. Colloque de clôture de l'IFR 144 NeuroSud-Paris

National audience; Assurant la réception des molécules odorantes, les neurones olfactifs de la muqueuse olfactive (MO) sont en contact direct avec le milieu extérieur, exposés aux agressions permanentes des micropolluants, dont les pesticides. Ils constituent une voie d’entrée privilégiée de toxiques vers le bulbe olfactif (BO) puis le système nerveux central. Dans une étude préliminaire (ACI 2009), nous avions démontré les effets délétères d’un pesticide, la Roténone, sur le système olfactif de rat traités par injections intrapéritonéales (IP). Au cours de second travail, nous avons poursuivi cette étude sur un modèle plus proche de l’exposition réelle des animaux aux pesticides dans la nature, par inhalation répétée de roténone. Notre étude a portée sur des ratons de 0 à 4 semaines de vie postnatale, de deux souches différentes, soumis à des injections intranasales (IN) quotidiennes de roténone. Nous avons évalué les effets dose-dépendants de la roténone (1 à 100μM), pour différentes durées de traitement (1 à 3 semaines), par des mesures comportementales olfactives pendant le traitement (test de reconnaissance d’odeur maternelle), et par des approches d’immunohistochimie de la MO et du BO (marquages neurones olfactifs de la MO par β-Tubulin III et Adénylate-Cyclase III, marquage des cellules apoptotiques par Tunel, et neurones DAergiques du BO par Tyrosine Hydroxylase). Les quantifications d’immunohistochimie dans les différentes zones de MO suggèrent une apoptose accrue et une diminution de l’épaisseur du compartiment apical dendritique/ciliaire des neurones olfactifs chez les animaux traités à la roténone. Cette dégénérescence des neurones olfactifs matures de la MO s’accroit avec la durée du traitement et les concentrations de roténone. Cependant le véhicule utilisé (huile + DMSO) semble altérer les neurones, mais plutôt par une hypertrophie/inflammation de la zone dendritique/ciliaire. Cet effet étant opposé à celui de la roténone, on peut suggérer que les effets en valeur absolue de la roténone seule sont légèrement sous–évalués du fait d’un effet « antagoniste » du véhicule dans lequel la roténone est diluée. Enfin, la variabilité des effets délétères de la roténone sur les neurones semble assez importante, et différente d’une souche de rat à une autre. Les neurones dopaminergiques du BO ne semblent pas affectés par le traitement roténone IN. Pour les tests comportementaux « olfactifs », les ratons séparés de leurs mères pendant 1 à 2 heures sont placés dans un labyrinthe de choix entre deux litières, l’une de leur mère et l’autre propre. Paradoxalement, les ratons les plus fortement traités à la roténone (concentration, durée) ont montrés les meilleures performances olfactives pour se guider vers l’odeur de leur mère : taux de réussite le plus élevé, rapidité, orientation correcte. Une hypothèse déjà avancée au laboratoire dans des expériences comparables est que les neurones dont l’activité est primordiale à la survie de l’animal (odeur de la mère) ont une meilleure résistance face aux agressions. Ils auraient une survie dépendante de cette activité : plus le neurone est sollicité, activé, plus il est protégé de l’action de l’agent toxique.

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National audience; The choice test is one of the most used paradigms to examine animal behaviour. Several measures are available to quantify the success in a choice test. The simplest is the binomial score of correct choice within a test (yes, no). However, it necessitates a large number of trials per animals. This is not an option when using very young animals with a need for frequent rest and feeding. Latency to make a choice can also be monitored but it requires that the test animal does make a choice. Non-choosing animals often necessitate a censoring of data, reducing the information available. We have developed a simple method to quantify direction of orientation of rat pups in a choice test (A 1 ) using scalars applied to data from a freely available tracking programme. The coordinates over time of the centre of the animal’s body and head were obtained simultaneously during video-tracking. These data allowed us to calculate two unit vectors (A 2 ), one composed by the two body markers (animal orientation vector) and one by the rear body dot to the dam faeces position (perfect orientation vector). The latter was converted into a product scalar for each video frame ranging from 1 when the pup was oriented directly towards the maternal odour, and -1 when facing the opposite direction. A representative pathway of one pup’s head position for each video frame (15 frames/s) during a test is presented in B 2 . Colour of the dots reflects orientation, ranging from white (perfect orientation towards dam’s odour source) to dark blue (opposite orientation away from dam’s odour source), i.e. 1 to -1 scalar product respectively (B 1 ). The use of scalars is likely to reduce the number of tests necessary in choice trials. Scalars could be used to determine the attraction or aversion associated with a specific stimulus placed within a test arena. Depending on the ease of marking the test animal, other species than rodents could be tested in the same way.

National audience; The welfare and health of farm animals are of critical importance for sustainable use of our environment. Paradoxically, while food quality impacts human health, modern rearing practices can be stressful for animals and affect the quality of animal production. The perception of smells and the olfactory environment have been linked to animal welfare and health (e.g. anxiety or depressive-like symptoms). There is evidence that the olfactory epithelium and bulb are responsive to stress hormones (glucocorticoids). However, the actions of glucocorticoids in these two tissues are still not fully explained. We have received seed funding (Credits Incitatifs PHASE) to evaluate those links in our model species (rat). Do glucocorticoids affect olfactory tissue and function in rat stress model? The initial step was to evaluate the potential for hormone response in the tissues of interest, by confirming the presence and localisation of the glucocorticoid receptor (GR). Immunohistochemistry showed the expression of GR is restricted to olfactory ensheathing cells (OEC), glial cells involved in processing of the olfactory signal propagation and neurone regeneration. We tested the functional significance of this observation in vitro using primary OEC cultures treated with a synthetic GR agonist (dexamethasone). The expression of various genes was affected, including ion transporters (ATP1B1), a macrophage chemoattractant (MCP1) and elements of the endothelin signalling pathways (the ET B receptor gene is repressed). We are currently exploring the significance of this latter observation by measuring endothelin response in our cells using calcium imaging. In vivo , acute administration of dexamethasone altered gene expression patterns in the OM and OB, as well as electrical activity induced by odorant exposure (measured by ElectroOlfactoGram, EOG). EOG signal recovery was delayed post-odorant stimulation in dexamethasone treated animals, this suggests olfactory neurone repolarisation might be slower. In addition to these acute stress models, we have developed a chronic unpredictable stress model in rats and we are assessing its impact on EOG and gene expression patterns. Our data shows stress hormones clearly affect olfactory response in the olfactory epithelium, at the first step of odorant detection. We currently do not know whether these changes affect smell perception in animals. To address this issue, we are in the process of developing olfactory learning tests in rats in order to study the impact of glucocorticoids on behavioural aspects of odour detection. This might allow us to determine whether stress-induced changes in olfactory function can affect animal welfare and physiology.

International audience; The neuronal olfactory epithelium undergoes permanent renewal because of environmental aggression. This renewal is partly regulated by factors modulating the level of neuronal apoptosis. Among them, we had previously characterized endothelin as neuroprotective. In this study, we explored the effect of cell survival factor deprivation in the olfactory epithelium by intranasal delivery of endothelin receptors antagonists to rat pups. This treatment induced an overall increase of apoptosis in the olfactory epithelium. The responses to odorants recorded by electroolfactogram were decreased in treated animal, a result consistent with a loss of olfactory sensory neurons (OSNs). However, the treated animal performed better in an olfactory orientation test based on maternal odor compared to non-treated littermates. This improved performance could be due to activity-dependent neuronal survival of OSNs in the context of increased apoptosis level. In order to demonstrate it, we odorized pups with octanal, a known ligand for the rI7 olfactory receptor (Olr226). We quantified the number of OSN expressing rI7 by RT-qPCR and whole mount in situ hybridization. While this number was reduced by the survival factor removal treatment, this reduction was abolished by the presence of its ligand. This improved survival was optimal for low concentration of odorant and was specific for rI7-expressing OSNs. Meanwhile, the number of rI7-expressing OSNs was not affected by the odorization in non-treated littermates; showing that the activity-dependant survival of OSNs did not affect the OSN population during the 10 days of odorization in control conditions. Overall, our study shows that when apoptosis is promoted in the olfactory mucosa, the activity-dependent neuronal plasticity allows faster tuning of the olfactory sensory neuron population toward detection of environmental odorants.

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P049 Glial cells, which are supporting cells of the nervous system, are now clearly involved in nervous communications.These cells modulate odorant detection as soon as the peripheral level in the olfactorymucosa, acting on olfactory sensitive neurons. These neuronsare surrounded by glial like cells called sustentacular cells. These cells maintain both the structural and ionic integrity of the olfactory epithelium and are electrically coupled through GAP junctions. Our study focused on endothelin known to activate glial cells and to uncoupled GAP junctions in the central nervous system. Using patch clamp, we first demonstrate that endothelin also uncoupled sustentacular cells in vitro. We then confirmed in vivo by calcium imaging that endothelin limits electrical signal propagation in intact olfactory epithelial preparation, similarly as the known GAP junction uncoupling carbenoxone. Such modulation could affect olfactory response kinetics as we observed that bothendothelin and carbenoxone treatments increase EOG (Electro-OlfactoGramme) signal repolarization. Our results are thus consistent with an implication of glial cell like modulation of nervous information at the peripheral level of the nervous system.

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This work was carried out in the framework of the European project BOND (Bioelectronic Olfactory Neuron Device, http://bondproject.org/) aimed at using ORs carried by nanoscale liposomes as sensing elements of an electrochemical array, for specifically detecting target odorants from complex odorant mixtures. This project has potential applications in medical diagnosis, but also in agroalimentary or cosmetics quality control, security, and environment.

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My studies are dedicated to the relationship between olfaction and nutrition in rodents. I’m interested by the plasticity of the olfactory system (mucosa and bulb) under the effect of either short- or long-term modification of nutritional cues, such a fasting or obesity using cellular, molecular and behavioral approaches. After having demonstrated the presence of various hormones involved in the control of food intake (leptin, insulin and their receptors) at several levels of the olfactory system, I’ve analyzed the variation of their expression levels in different nutritional models linked to short-term nutritional plasticity. Transcriptomic analyses have demontrated that fasting modulates the expression of several genes encoding various functions in the olfactory mucosa associated with variations in the odorant responses. Moreover, the olfactory bulb activity is also modulated by nutritional hormones and some olfactory-based behaviors are affected by a short-term manipulation of the endocrine status of animals. My perspectives are to further analyse the consequences of long-term variations of the neurodocrine status on the olfactory capabilities and to decipher the molecular mechanisms responsive of the modulation at the olfactory system periphery : identification of signaling pathways crosstalks, local translation in the apical region of the mucosa and role of the olfactory bulb in the integration of the peripheral information.; Mon programme de recherches concerne l’étude des relations olfaction-nutrition chez les rongeurs. Je m’intéresse à la plasticité du système olfactif (muqueuse et bulbe) sous l’effet de perturbations alimentaires (mise à jeun, états d’obésité) par des approches cellulaires, moléculaires, et comportementales en ciblant le rôle des hormones métaboliques. Après avoir montré l’expression de molécules cibles du contrôle de la prise alimentaire (leptine, insuline et leurs récepteurs) dans le système olfactif, j’ai mesuré leurs variations d’expression sur différents modèles alimentaires à court terme, puis entrepris de compléter l’étude en analysant les variations sur le transcriptome complet du système olfactif. Mes travaux montrent une modulation de la transcription de gènes codant pour diverses fonctions dans la muqueuse selon l’état nutritionnel qui est associée à des variations de la réponse aux odorants dès leur premier niveau de réception. Les hormones métaboliques modulent également les activités du bulbe, ce qui se traduit par une modulation de certains comportements basés sur l’olfaction selon l’état endocrinien. Le système olfactif est donc capable d’intégrer les variations aigues du statut métabolique pour moduler son fonctionnement. Tout en poursuivant ces études sur des états métaboliques installés, je me concentre aujourd’hui sur les mécanismes moléculaires responsables de la plasticité nutritionnelle à la périphérie du système olfactif selon différentes voies: dialogue des voies de signalisation dans la partie apicale de la muqueuse olfactive, rôle de la traduction locale, effet sur la plasticité du bulbe olfactif.

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The welfare and health of farm animals are of critical importance for sustainable use of our environment. Paradoxically, while food quality impacts human health, modern rearing practices can be stressful for animals and affect the quality of animal production. The perception of smells and the olfactory environment have been linked to animal welfare and health (e.g. anxiety or depressive-like symptoms). There is evidence that the olfactory epithelium and bulb are responsive to stress hormones (glucocorticoids). However, the actions of glucocorticoids in these two tissues are still not fully explained. We have received seed funding (Credits Incitatifs PHASE) to evaluate those links in our model species (rat). Do glucocorticoids affect olfactory tissue and function in rat stress model? The initial step was to evaluate the potential for hormone response in the tissues of interest, by confirming the presence and localisation of the glucocorticoid receptor (GR). Immunohistochemistry showed the expression of GR is restricted to olfactory ensheathing cells (OEC), glial cells involved in processing of the olfactory signal propagation and neurone regeneration. We tested the functional significance of this observation in vitro using primary OEC cultures treated with a synthetic GR agonist (dexamethasone). The expression of various genes was affected, including ion transporters (ATP1B1), a macrophage chemoattractant (MCP1) and elements of the endothelin signalling pathways (the ETB receptor gene is repressed). We are currently exploring the significance of this latter observation by measuring endothelin response in our cells using calcium imaging. In vivo, acute administration of dexamethasone altered gene expression patterns in the OM and OB, as well as electrical activity induced by odorant exposure (measured by ElectroOlfactoGram, EOG). EOG signal recovery was delayed post-odorant stimulation in dexamethasone treated animals, this suggests olfactory neurone repolarisation might be slower. In addition to these acute stress models, we have developed a chronic unpredictable stress model in rats and we are assessing its impact on EOG and gene expression patterns. Our data shows stress hormones clearly affect olfactory response in the olfactory epithelium, at the first step of odorant detection. We currently do not know whether these changes affect smell perception in animals. To address this issue, we are in the process of developing olfactory learning tests in rats in order to study the impact of glucocorticoids on behavioural aspects of odour detection. This might allow us to determine whether stress-induced changes in olfactory function can affect animal welfare and physiology.

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Babies with intra-uterine growth restriction (IUGR) are at increased risk for experiencing negative neonatal outcomes due to their general developmental delay. The present study aimed to investigate the effects of a short postnatal leptin supply on the growth, structure, and functionality of several organs at weaning. IUGR piglets were injected from day 0 to day 5 with either 0.5 mg/kg/d leptin (IUGRLep) or saline (IUGRSal) and euthanized at day 21. Their organs were collected, weighed, and sampled for histological, biochemical, and immunohistochemical analyses. Leptin induced an increase in body weight and the relative weights of the liver, spleen, pancreas, kidneys, and small intestine without any changes in triglycerides, glucose and cholesterol levels. Notable structural and functional changes occurred in the ovaries, pancreas, and secondary lymphoid organs. The ovaries of IUGRLep piglets contained less oogonia but more oocytes enclosed in primordial and growing follicles than the ovaries of IUGRSal piglets, and FOXO3A staining grade was higher in the germ cells of IUGRLep piglets. Within the exocrine parenchyma of the pancreas, IUGRLep piglets presented a high rate of apoptotic cells associated with a higher trypsin activity. In the spleen and the Peyer's patches, B lymphocyte follicles were much larger in IUGRLep piglets than in IUGRSal piglets. Moreover, IUGRLep piglets showed numerous CD79(+) cells in well-differentiated follicle structures, suggesting a more mature immune system. This study highlights a new role for leptin in general developmental processes and may provide new insight into IUGR pathology.

International audience; Hetero- and homo-oligomerization of G protein-coupled receptors (GPCRs) has been addressed in the past years using various approaches such as co-immunoprecipitation, fluorescence resonance energy transfer and bioluminescence resonance energy transfer (BRET). Here, we report the methodological details from a previously published study to investigate the relationships between oligomerization and activation states of olfactory receptors (ORs). This methodology combines heterologous expression of ORs in Saccharomyces cerevisiae and BRET assays on membrane fractions, in particular, upon odorant stimulation. We have demonstrated that ORs constitutively homodimerize at the plasma membrane and that high odorant concentrations promote a conformational change of the dimer, which becomes inactive. We proposed a model in which one odorant molecule binding the dimer would induce activation, while two odorant molecules, each binding one protomer of the dimer, would blunt signaling.

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International audience; Natural vesicles produced from genetically engineered cells with tailored membrane receptor composition are promising building blocks for biosensing devices [1]. This is particularly true for the case of G - protein coupled receptors (GPC Rs) present in many sensing processes in cells, whose functionality crucially depends on their lipid environment. However, the controlled production of natural vesicles containing GPCRs and their GPCRs expression level are among the outstanding challenges in the road map to realize practical biomolecular devices based on GPCRs. Largely expressed in the human body, GPCRs are implicated in a wide range of disease pathways [2], including cancer [3,4], cardiomyopathy, and schizophrenia. Around 50% of the drugs on the market regulate GPCR function, 30% of which directly target GPCRs [5]. Herein we present the production and characterization of membrane nanovesicles (nanosomes) of about 100 nm diameter from Saccaromyces Cerevisiae carrying the heterologously expre ssed olfactory receptor c - myc - OR1740 [6], a member of the family of GPCRs and representative of important candidates for the development of bioelectronic noses [7]. The nanosomes size in solution was characterized by Dynamic Light Scattering (DLS), Nanopar ticle Tracking Analysis (NTA), and Cryo - Electron Microscopy. In addition, we describe a universal direct novel c - myc - GPCR quantification without the necessity of a previous protein purification step. The quantification was carried out by a highly promising strategy based on a competitive ELISA format assay using c - myc tag - bioconjugates as model conjugates that mimics the behavior of the c - myc olfactory receptor on nanovesicles. A custom made antibody (anti - c - myc) was produced giving assay detectability in t he picomolar range and allowing the quantification of the c - myc - OR proteins. The number of olfactory receptors (OR17 - 40) per nanovesicle obtained was (30 +/- 6) OR/NS. Evidences of the OR17 - 40 functionality have been obtained by Biacore (SPR) measurements. Present results constitute an important step in the practical realization of biosensor devices based on natural nanovesicles integrating G - protein coupled membrane receptors.

We present a procedure that (i) automates the homology modeling of mammalian olfactory receptors (ORs) based on the six three-dimensional (3D) structures of G protein-coupled receptors (GPCRs) available so far and (ii) performs the docking of odorants on these models, using the concept of colony energy to score the complexes. ORs exhibit low-sequence similarities with other GPCR and current alignment methods often fail to provide a reliable alignment. Here, we use a fold recognition technique to obtain a robust initial alignment. We then apply our procedure to a human OR that we have previously functionally characterized. The analysis of the resulting in silico complexes, supported by receptor mutagenesis and functional assays in a heterologous expression system, suggests that antagonists dock in the upper part of the binding pocket whereas agonists dock in the narrow lower part. We propose that the potency of agonists in activating receptors depends on their ability to establish tight interactions with the floor of the binding pocket. We developed a web site that allows the user to upload a GPCR sequence, choose a ligand in a library and obtain the 3D structure of the free receptor and ligand-receptor complex (http://genome.jouy.inra.fr/GPCRautomodel).

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International audience; Recently published work and emerging research efforts have suggested that the olfactory system is intimately linked with the endocrine systems that regulate or modify energy balance. Although much attention has been focused on the parallels between taste transduction and neuroendocrine controls of digestion due to the novel discovery of taste receptors and molecular components shared by the tongue and gut, the equivalent body of knowledge that has accumulated for the olfactory system, has largely been overlooked. During regular cycles of food intake or disorders of endocrine function, olfaction is modulated in response to changing levels of various molecules, such as ghrelin, orexins, neuropeptide Y, insulin, leptin, and cholecystokinin. In view of the worldwide health concern regarding the rising incidence of diabetes, obesity, and related metabolic disorders, we present a comprehensive review that addresses the current knowledge of hormonal modulation of olfactory perception and how disruption of hormonal signaling in the olfactory system can affect energy homeostasis.

International audience; An overall view of olfactory structures activated by biologically relevant odors in conscious rats has not been available up to now. Manganese (Mn)-enhanced MRI (MEMRI) could be an appropriate method to provide such a view because it is based on the detection of Mn, a persistent intra-cellular functional contrast agent. Whereas MEMRI has been used for anatomical labeling of olfactory pathways, functional imaging analyses have not yet been performed beyond the olfactory bulb. Here, we have used MEMRI for the functional imaging of rat central olfactory structures and for comparing activation maps obtained with odors conveying different biological messages. The odors used were those of male fox feces, that triggers an innate fear reaction, and of chocolate-flavored cereals, that is linked to a food reward. They were used to stimulate conscious rats previously treated by intranasal instillation of Mn at a dose optimized to ensure reproducible contrast in MR images of whole brain at 4.7 T, while sparing olfactory perception. MEMRI activation maps showed Mn enhancement throughout the primary olfactory cortex. Mn enhancement elicited by fox odor differed from that elicited by deodorized air (used as a control), whereas Mn enhancement elicited by chocolate odor did not. By providing an overall image of brain structures activated in conscious rats by odorous stimulation, these results demonstrate the interest of MEMRI for functional studies of olfaction in small laboratory animals. Our results also suggest that the activation of deep brain olfactory structures depends on the biological significance of the odorous message

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International audience; Located between the environment and the brain, the olfactory epithelium (OE) is a unique neuroepithelium subjected to several external aggressions for which original defense strategies are essential for long-term tissue integrity. Taking advantage of a simple biochemical fractionation of the tissue, we performed a fine examination of OE apical region in terms of subcellular machineries and molecular components. We provide several lines of evidence supporting the transport and local translation of the subset of mRNA previously identified in olfactory dendrites (Persuy et al. 2011). We discuss a possible link between local translation and neuronal activities in these self-renewing neurons, whose function is essential for animal survival and as a protective barrier for the brain.

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