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Background SAL1 (salivary lipocalin) is a member of the OBP (Odorant Binding Protein) family and is involved in chemical sexual communication in pig. SAL1 and its relatives may be involved in pheromone and olfactory receptor binding and in pre-mating behaviour. The evolutionary history and the selective pressures acting on SAL1 and its orthologous genes have not yet been exhaustively described. The aim of the present work was to study the evolution of these genes, to elucidate the role of selective pressures in their evolution and the consequences for their functions. Results Here, we present the evolutionary history of SAL1 gene and its orthologous genes in mammals. We found that (1) SAL1 and its related genes arose in eutherian mammals with lineage-specific duplications in rodents, horse and cow and are lost in human, mouse lemur, bushbaby and orangutan, (2) the evolution of duplicated genes of horse, rat, mouse and guinea pig is driven by concerted evolution with extensive gene conversion events in mouse and guinea pig and by positive selection mainly acting on paralogous genes in horse and guinea pig, (3) positive selection was detected for amino acids involved in pheromone binding and amino acids putatively involved in olfactory receptor binding, (4) positive selection was also found for lineage, indicating a species-specific strategy for amino acid selection. Conclusions This work provides new insights into the evolutionary history of SAL1 and its orthologs. On one hand, some genes are subject to concerted evolution and to an increase in dosage, suggesting the need for homogeneity of sequence and function in certain species. On the other hand, positive selection plays a role in the diversification of the functions of the family and in lineage, suggesting adaptive evolution, with possible consequences for speciation and for the reinforcement of prezygotic barriers.

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International audience; With a view to alleviate the feeling of hunger in broiler breeders, different types of fibre sources were used in high-fibre diets to increase feed quantity while limiting growth to industry recommended levels. Using scatter feeding, three diets (C1: commercial control diet, 13fibre content, 80% insoluble fibre (ISF); H2: 23fibre content, 89% ISF; and L2: 23fibre content, 71% ISF) were each fed to 10 groups of 16 broiler breeder chickens. Similar growth rates were obtained on different quantities of food with all birds reaching commercial target weight at 15 weeks of age. In a hunger test, birds fed C1 ate significantly faster and showed a higher compensatory feed intake than birds on diets H2 and L2, indicating that the two high-fibre diets did reduce the level of hunger experienced by the birds. Behavioural observations carried out at 14 weeks of age showed high levels of tail pecking in birds fed C1 and almost none in birds fed L2, whereas birds fed H2 were intermediate. Stereotypic pecking at fixtures was seen twice as frequently in birds fed C1. Birds on diet L2 displayed behavioural signs indicative of discomfort, and the high water usage on this diet created problems with litter quality. Birds on diet H2 continued to show foraging behaviour throughout the day, and were more frequently engaged in dust bathing and other comfort behaviour. This experiment indicates that high-fibre diets can alleviate the feeling of hunger currently experienced by broiler breeders, and a high ratio of ISF may improve the well-being of the birds.

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International audience; Understanding how the architecture of neuronal populations contributes to brain function requires three-dimensional representations and analyses. Neuroanatomical techniques are available to locate neurons in animal brains. Repeating an experiment in different individuals yields a collection of point patterns from which common organization principles are generally difficult to extract. We recently addressed the problem of generating statistical density maps to integrate replicated point pattern data into meaningful, interpretable representations. Applications to different neuroanatomical systems illustrated the ability of our method to reveal organization rules that cannot be perceived directly on raw data. To make the method practicable for further applications, the aim of the present paper is to establish general guidelines for appropriate parameter tuning, valid result interpretation as well as efficient implementation. Accordingly, we characterize the method by analyzing the role of its main parameter, by reporting results on its statistical properties and by demonstrating its robustness, using both simulated and real neuroanatomical data.

1. The aim of this study was to evaluate how splitting the dark period (scotoperiod) affects feeding behaviour and associated intestinal measures in broilers. 2. Ross 308 broilers were reared to 37 d in groups given either a daily 8-h continuous scotoperiod (DARK 8) or an intermittent light schedule with two equally spaced 4-h scotoperiods (DARK 4þ4), which yielded the same total duration of darkness per 24 h. 3. Feeding behaviour was recorded weekly from 24-h video recordings of 24 groups each of 64 birds. Empty intestinal weights as well as their contents were measured weekly at 4 time points (n¼192). Digestive transit time was estimated on d 29 using a chromic oxide marker; production variables and the extent of foot pad dermatitis were also recorded. 4. In the 3 h prior to a scotoperiod, feeding activity increased in chickens from DARK 8 but not DARK 4þ4. This increase was reflected in a higher relative content of the crop in DARK 8 at this time. 5. Immediately following the scotoperiod, feeding activity peaked and, although the chickens in DARK 4þ4 expressed more feeding behaviour in the first 20 min after the scotoperiod, the chickens in DARK 8 had overall higher feeding activity across the day. However, DARK 4þ4 had a higher feed intake and weight gain. The occurrence and severity of foot pad dermatitis was similar between treatments. 6. In conclusion, broilers modify their feeding behaviour according to the prevailing light schedule. Eight consecutive hours of darkness reduced growth, but did not affect overall feed conversion efficiency, and did not appear to exacerbate hunger or foot pad dermatitis to any great extent.

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International audience; The mammalian olfactory mucosa (OM) is continually renewed throughout life. Owing to their position in the nasal cavity, OM cells are exposed to multiple insults, including high levels of odourants that can induce their death. OM regeneration is therefore essential to maintain olfactory function, and requires the tight control of both cell death and proliferation. Apoptosis has been implicated in OM cell death. Olfaction is one of the senses involved in food intake and depends on individual nutritional status. We have previously reported the influence of hormones related to nutritional status on odour perception and have shown that the OM is a target of insulin and leptin, two hormones known for their anti-apoptotic properties. In the present study, we investigated the potential anti-apoptotic effect of these metabolic hormones on OM cells. Both Odora cells (an olfactive cell line) and OM cells treated with etoposide, a p53 activity inducer, exhibited mitochondrial-dependent apoptosis that was inhibited by the pan-caspase inhibitor zVAD-fmk. Insulin, but not leptin, impaired this apoptotic effect. Insulin addition to the culture medium reduced p53 phosphorylation, caspase-3 and caspase-9 cleavage, and caspase-3 enzymatic activity induced by etoposide. The apoptotic wave observed in the OM after interruption of the neuronal connections between the OM and the olfactory bulb by bulbectomy was impaired by intranasal insulin treatment. These findings suggest that insulin may be involved in OM cellular dynamics, through endocrine and/or paracrine-autocrine effects of circulating or local insulin, respectively.

Chantier qualité GA; All olfactory epithelium cells, including rapidly self-renewing olfactory sensory neurons (OSN), are continuously subjected to external airborne aggressions. We hypothesized that the apical part of rat olfactory epithelia (AOE) could be the site of a local translation to be able to respond rapidly to external stimuli. We purified significant amounts of mRNAs from AOE. Sequencing of the cDNA library identified 348 mRNA species. Of these, the 220 AOE transcripts encoding proteins with known biological functions were classified in functional groups. The main functional class (40%) coded for defense, detoxification, anti-oxidant stress and innate immunity. Other classes comprised mRNAs encoding functions for neuronal metabolism and life (19%), nuclear transcription control (15%), cell survival and proliferation (13%), RNA processing and translation (12%). They did not contain any known members of the olfactory transduction pathway. The expression of a sub-set of AOE transcripts was investigated in sub-cellular AOE fractions highly enriched in ciliated dendrites and in AOE fractions after forced hemilateral OSN-specific degeneration. All the mRNAs tested were found to be: i) present in enriched ciliated dendrite preparations ii) down-regulated after OSN degeneration iii) co-purified with polysomal fractions, suggesting their commitment to local translation. We provide strong evidence that the extreme apical side of the olfactory epithelium expresses a unique transcriptome, whose function is not related to olfaction but mainly to defense and survival. The possible local translation of this transcriptome is demonstrated, in supporting cells as well as in olfactory neuron ciliated dendrites.

Le système olfactif permet aux êtres vivants de reconnaître et de discriminer un nombre quasi-illimité de molécules odorantes. La première étape de la détection odorante résulte d’une interaction entre les protéines de liaison aux odorants (OBPs) dans le mucus olfactif, les odorants et les récepteurs olfactifs (ROs) situés sur les cils des neurones sensoriels olfactifs émergeant dans l’épithélium olfactif. Cependant, la relation entre l’activation du récepteur et son état conformationnel et d’association n’est pas bien connue. Les ROs appartiennent à la famille des récepteurs couplés aux protéines G (RCPGs). L’oligomérisation est un processus important lors de la maturation et l’acheminement à la membrane plasmique des RCPGs ainsi que pour leur pharmacologie. Dans cette étude, il s'agissait d’élucider l’état d’homo-dimérisation des ROs de mammifères, et sa possible implication dans l’activation du récepteur lors de la stimulation par le ligand odorant. Pour cela, j’ai utilisé les méthodes de co-immunoprécipitation et de transfert d’énergie en résonance, FRET (Fluorescence Resonance Energy Transfer) et BRET (Bioluminescence Resonance Energy Transfer). Mes résultats montrent que le RO humain OR1740 existe sous forme de dimères en système d’expression hétérologue levure, que la dimérisation pourrait exister dès les premières étapes de la biosynthèse, et est constitutive à la membrane plasmique. De plus, la liaison de l’agoniste sur le dimère du RO induit une modulation des niveaux de BRET. La courbe en cloche obtenue pour la variation de BRET en fonction de la concentration en ligand odorant est en accord avec les précédents résultats de l'équipe INRA, selon lesquels le ligand induit un changement de conformation du dimère vers un état inactif à forte concentration en odorant. Cette démonstration de l'homo-dimérisation d'un RO confirme et valide l'une des hypothèses à la base de notre modèle décrivant les interactions tripartites entre OBPs, odorants, et ROs impliqués dans la première étape de la détection odorante. The living organisms have evolved the sense of smell to recognize and distinguish odors. The first step of odorant molecule detection involves an interaction between odorants, possibly carried through the olfactory mucus by Odorant-Binding Proteins (OBPs), and the olfactory receptors (ORs) present on the ciliae of olfactory sensory neurons emerging from the olfactory epithelium. However, the relationship between receptor activation and its association/conformational state remains largely unknown. The mammalian ORs belong to the G Protein Coupled Receptors (GPCRs) super-family. GPCR oligomerization is thought to play an important role in the maturation pathway, the trafficking to the cell surface and the functional response of the receptors. Using co-immunoprecipitation method, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) approaches, I addressed the question whether mammalian ORs exist as homo-oligomers and whether this phenomenon is involved in receptor signaling upon odorant ligand activation. My results clearly demonstrate that the OR1740, a human OR, can self-associate into dimers in a yeast heterologous expression system, that dimers may be formed early in the biosynthetic pathway, and are constitutively expressed at the plasma membrane. Moreover, an odorant agonist binding induces a modulation in BRET levels, suggesting a conformational rearrangement of the receptor related to its activation/signaling state. The bell-shaped curve observed for the variation of its functional response as a function of odorant ligand concentration corroborates previous results of the INRA team, according to which ligand-mediated conformational change can promote an inactive state of the OR dimers at high ligand concentration. This demonstration of OR homo-dimerization supports and validates one of the hypotheses at the basis of our model for tripartite OBP-odorant-OR partnership.

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Diplôme : Master

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National audience; • The animal nose is generally considered as the golden standard of chemical detector, as it is both sensitive and reliable. It is able to address a vast chemical space of hundreds of thousands of compounds. Yet, such performances would not be possible without the properties of the first sensing step in our nose: olfactory receptors. • It is thus tempting to conceive biology-inspired olfactory sensors which could take advantage of biologic noses without the constraints of either human or animal sniffing. This is what our laboratory intends to do, within the framework of a multidisciplinary, European collaborative project called "BOND", in the new, exciting, nanobiotechnology field. • Our idea is to immobilize the biological elements (olfactory receptors) in an ultra-miniaturised (possibly disposable) sensor stemming from nano-fabrication technologies, both for the supports and for electronic nano-circuits, and connected to a computer. At the crossroads of techniques in molecular biology and electronics, such a sensor would allow low-cost production for many applications. Besides, its association with artificial intelligence software, as already developed for traditional electronic noses could endow them with learning and evolutionary capabilities. • We envision the possibility of using them in such applications as medical diagnosis, environmental or occupational monitoring, and in the perfume/cosmetic industry, especially when hazardous or unknown chemicals are used or to make up for evaluation panels fatigue.

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International audience; Olfaction influences food choice and intake. Conversely, the nutritional and metabolic status modifies odour detection. In an effort to understand the olfactory-metabolic crosstalk, we have localized orexigenic and anorexigenic hormones and their receptors in the olfactory epithelium and bulb of rats, and looked for their effect on the olfactory signal. 1) Concerning the orexigenic hormones: - in fasted rats, transcription of orexins (A & B) and their receptors (1 & 2) increased in the olfactory epithelium and, in the bulb, in mitral, periglomerular and granular cells. An orexin antagonist decreased locomotor activity of fasted animals, their time spent sniffing and bulb cells c-Fos response. Orexins also enhanced the amplitude of the olfactory epithelium electroolfactogram. - similarly, NPY enhanced the electroolfactogram amplitude (30%) in fasted rats, mainly through Y1 receptors and less through Y5. 2) Concerning the anorexigenic hormones: - in the olfactory epithelium, the transcription of leptin and its receptor (long and short forms) were enhanced by fasting. In fasted rats, leptin decreased locomotor activity, time spent sniffing and bulb cells c-Fos response, as well as electroolfactogram amplitude. - fasting induced an increase in the number of receptors in the olfactory epithelium, together with an increase in local insulin production. Interestingly, insulin decreased the electroolfactogram amplitude of the olfactory epithelium by 30%. These results are consistent with an enhanced interest, especially for food odours, in fasted animals, in contrast to decreased odour stimulation in satiated ones. Endly, we analyzed the olfactory epithelium transcriptome of fasted vs satiated rats. Fasting decreased transcription of immunity-related genes, perhaps in connection with stress, while it increased some transcripts related to cell-dynamics or signal transduction. Thus transcriptomic data were largely correlated with enhanced olfactory activity upon fasting

Soutenance : 21 juin 2010 Diplôme : Master

Nagnan-Le Meillour : agent mis à disposition par l'unité INRA 1197. ; Native porcine odorant-binding protein (OBP) bears eleven sites of phosphorylation, which are not always occupied in the molecular population, suggesting that different isoforms could co-exist in animal tissues. As phosphorylation is a dynamic process resulting in temporary conformational changes that regulate the function of target proteins, we investigated the possibility that OBP isoforms could display different binding affinities to biologically relevant ligands. The availability of recombinant proteins is of particular interest for the study of protein/ligand structure-function relationships, but prokaryotic expression systems do not perform eukaryotic post-translational modifications. To investigate the role of phosphorylation in the binding capacities of OBP isoforms, we produced recombinant porcine OBP in two eukaryotic systems, the yeast, Pichia pastoris, and the mammalian CHO cell line. Isoforms were separated by anion exchange HPLC, and their phosphorylation sites were mapped by MALDI-TOF mass spectrometry and compared to those of the native protein. Binding experiments with ligands of biological relevance in the pig, Sus scrofa, were performed by fluorescence spectroscopy on two isoforms of recombinant OBP expressed in the yeast. The two isoforms, differing only by their phosphorylation pattern, displayed different binding properties, suggesting that binding specificity is driven by phosphorylation.

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In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear “compartments”. Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types., Author Summary Several reports suggest functional relationships within the spatial organization of the nucleus, gene regulation and cell differentiation. However, it still remains difficult to extract common rules, mostly because i) most data have been gathered on limited sets of nuclear elements and in nuclei outside their normal physiological environment, and ii) few three-dimensional (3D) quantitative measures have been performed. Thus, we questioned whether common nuclear organization principles exist in the animal and plant kingdoms. For that purpose, we investigated the 3D distribution of centromeres/chromocenters in five populations of animal and plant nuclei: rabbit embryos at 8-cell and blastocyst stages, rabbit mammary gland epithelial cells and Arabidopsis thaliana plantlets. We set up adapted procedures to segment confocal images and developed a new analytical methodology based on distances between positions within the nucleus and centromeres/chromocenters. We showed that in all systems, despite large differences in chromosome number (44 in rabbit; 10 in A. thaliana) and genome size (rabbit estimated size 2.77 Gbp; A. thaliana 125 Mbp), centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation. This suggests that, whatever their specific features, conserved rules govern the spatial distribution of genomes in nuclei of differentiated cells.

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International audience; The perception of thousands of odours by about 380 human olfactory receptors (ORs) results from a combinatorial coding, in which one OR recognizes multiple odorants and odorants are recognized by different combinations of ORs. We used data of 95 odorant molecules tested on two human ORs, OR1G1 (class II) and OR52D1 (class I), to perform a 3D molecular modelling study of ligands using Catalyst/HypoGen software. The sorting-out procedure previously used for OR1G1 ligands was transposed to OR52D1 ligands. The 3D-QSAR models obtained for OR1G1 and OR52D1 ligands essentially differ by distance between their features. This result suggests a path to decipher the odotopes of OR1G1 and OR52D1 agonists in order to investigate the role of ORs in olfactory coding.

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National audience; This study addresses the functional mapping of deep brain regions activated by odours in rats. For this purpose, manganese-enhanced MRI (MEMRI) was chosen. This method uses manganese (Mn) as an exogenous contrast agent (1). Manganese, a calcium analogue, is recruited by activated neurons and then slowly eliminated. This allows stimulations to be performed on conscious animals and images to be subsequently acquired under anaesthesia. Image contrast depends on intra-neuronal Mn concentration, and so reflects neuronal activity throughout the stimulation period. However, a major drawback of this technique is the toxic effect of manganese, which may alter locomotor behaviour and/or olfactory perception. First, we defined the maximal manganese dose that provided a good contrast in fMRI studies while simultaneously preserving normal behaviour in animals when Mn was injected into nostrils (2). Also, Mn remanence prevents control and activation state images being obtained in a single-subject experiment. Images have to be acquired through multisubject studies and spatial variation of Mn concentration assessed by groupwise image comparison. We have developed an original image processing sequence comprising (i) a semi-supervised brain segmentation method based on fast adjustment of an average three-dimensional brain model obtained from 20 manual segmentations and (ii) an iterative algorithm normalizing images in both spatial and intensity dimensions, independently of an a priori image target. Preliminary results obtained from large cohorts of rats indicate that the association of an optimal dose preserving olfactory perception, of an image processing chain for segmentation and normalization and a voxelwise statistical test for comparing means, will highlight deep brain regions involved in odour processing.

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Laboratoire d'accueil : UMR 8576 Unité de Glycobiologie Structurale et Fonctionnelle de l'Université de Lille 1 Diplôme : Dr. d'Université

Udgivelsesdato: December Feather pecking (FP) remains a major welfare and economic problem in laying hens. FP has been found to be related to other behavioural characteristics, such as fearfulness. There are indications that fearful birds are more likely to develop FP. Furthermore, FP can lead to increased fearfulness in the victims. To investigate further the relationship between FP and fearfulness, feather damage and behavioural fear responses were recorded in three White Leghorn lines of laying hens: a line selected for high FP (HFP line), a line selected for low FP (LFP line) and an unselected control line (10th generation of selection). We used 64 birds per line housed in 16 four-bird cages (cage was the experimental unit). At 25 weeks of age, birds were subjected to a tonic immobility (TI) test and a combined human approach (HA) and novel object (NO) test, and plumage condition was recorded. Line differences in fear responses between the HFP and LFP lines were not found, neither in the TI-test, nor in the HA or NO test. As expected, birds from the HFP line had considerably more feather damage than birds from the LFP line and birds from the unselected control line were intermediate. Cages that withdrew from the NO 30 s after placement had more feather damage on the back compared with cages that did not show a withdrawal response. These results suggest that although relationships were found between feather damage and fear response at cage level, lines divergently selected on feather pecking behaviour do not differ in their fear responses. Divergent selection on feather pecking may have altered pecking motivation rather than fearfulness.

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Background: The caudal brainstem plays an important role in short-term satiation and in the control of meal termination. Meal-related stimuli sensed by the gastrointestinal (GI) tract are transmitted to the area postrema (AP) via the bloodstream, or to the nucleus tractus solitarii (NTS) via the vagus nerve. Little is known about the encoding of macronutrient-specific signals in the caudal brainstem. We hypothesized that sucrose and casein peptone activate spatially distinct subpopulations of NTS neurons and thus characterized the latter using statistical three-dimensional modeling. [br/]Methodology/Principal Findings: Using immunolabeling of the proto-oncogene Fos as a marker of neuronal activity, in combination with a statistical three-dimensional modeling approach, we have shown that NTS neurons activated by sucrose or peptone gavage occupy distinct, although partially overlapping, positions. Specifically, when compared to their homologues in peptone-treated mice, three-dimensional models calculated from neuronal density maps following sucrose gavage showed that Fos-positive neurons occupy a more lateral position at the rostral end of the NTS, and a more dorsal position at the caudal end. [br/]Conclusion/Significance: To our knowledge, this is the first time that subpopulations of NTS neurons have be distinguished according to the spatial organization of their functional response. Such neuronal activity patterns may be of particular relevance to understanding the mechanisms that support the central encoding of signals related to the presence of macronutrients in the GI tract during digestion. Finally, this finding also illustrates the usefulness of statistical threedimensional modeling to functional neuroanatomical studies.

International audience; The present work focuses on the development of an immunosensing surface to build a portable olfactory system for the detection of complex mixture of odorants. Homogeneous cell derived vesicles expressing the olfactory receptors were produced and immobilized with efficiency onto a gold substrate through an optimized surface functionalization method.

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