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If you have ever been out to the countryside in the spring, you might have heard sheep bleating to their lambs. Sheep also bleat when they are separated from the flock or stressed in some other way. To us, all these bleats sound very similar. But do you think they also sound similar to the lambs? Or do you think the lambs know whose mother is calling and what they are saying? Scientists try to interpret the bleats of sheep by observing their behavior when they hear these sounds. They study the sound waves of recorded bleats to identify each sheep’s unique voice and even determine which emotions the sheep are feeling. They also investigate the brain to find out what is going on inside the heads of sheep when they hear and understand the sounds of other sheep. Studies show that sheep really can recognize each other’s voices and communicate vocally.

The structural connectivity of animal brains can be revealed using post-mortem diffusion-weighted magnetic resonance imaging (MRI). Despite the existence of several structural atlases of avian brains, few of them address the bird’s structural connectivity. In this study, a novel atlas of the structural connectivity is proposed for the male Japanese quail (Coturnix japonica), aiming at investigating two lines divergent on their emotionality trait: the short tonic immobility (STI) and the long tonic immobility (LTI) lines. The STI line presents a low emotionality trait, while the LTI line expresses a high emotionality trait. 21 male Japanese quail brains from both lines were scanned post-mortem for this study, using a preclinical Bruker 11.7 T MRI scanner. Diffusion-weighted MRI was performed using a 3D segmented echo planar imaging (EPI) pulsed gradient spin-echo (PGSE) sequence with a 200 $$\upmu$$μm isotropic resolution, 75 diffusion-encoding directions and a b-value fixed at 4500 s/mm2. Anatomical MRI was likewise performed using a 2D anatomical T2-weighted spin-echo (SE) sequence with a 150 $$\upmu$$μm isotropic resolution. This very first anatomical connectivity atlas of the male Japanese quail reveals 34 labeled fiber tracts and the existence of structural differences between the connectivity patterns characterizing the two lines. Thus, the link between the male Japanese quail’s connectivity and its underlying anatomical structures has reached a better understanding.

International audience; The psychoendocrine evaluation of lamb development has demonstrated that maternal deprivation and milk replacement alters health, behavior and endocrine profiles. While lambs are able to discriminate familiar and non-familiar conspecifics (mother or lamb), only lambs reared with their mother develop such clear social discrimination or preference. Lambs reared without mother display no preference for a specific lamb from its own group. Differences in exploratory and emotional behaviours between mother-reared and mother deprived lambs have also been reported. As these behavioural abilities are supported by the brain, we hypothesize that rearing with maternal deprivation and milk replacement leads to altered brain development and maturation. To test this hypothesis, we examined brain morphometric and microstructural variables extracted from in-vivo T1-weighted and diffusion-weighted magnetic resonance images acquired longitudinally (1 week, 1.5 months and 4.5 months of age) in mother-reared and mother-deprived lambs. From the morphometric variables the caudate nuclei volume was found to be smaller for mother-deprived than for mother-reared lambs. T1-weighted signal intensity and radial diffusivity were higher for mother-deprived than for mother-reared lambs in both the white and gray matters. The fractional anisotropy of the white matter was lower for mother-deprived than for mother-reared lambs. Based on these morphometric and microstructural characteristics we conclude that maternal deprivation delays and affects lamb brain growth and maturation.

La question des émotions des animaux d’élevage s’inscrit dans la volonté d’améliorer les conditions d’élevage en prenant en compte leur bien-être. La genèse des émotions repose sur la capacité cognitive des individus à percevoir, évaluer et réagir à leur environnement. La capacité de perception met en jeu différentes sensorialités et participe à la reconnaissance des éléments de l’environnement de l’animal sans nécessairement déclencher d’émotions. Les situations déclenchantes d’émotion les plus étudiées chez les animaux d’élevage sont la nouveauté, la séparation sociale ou la présence d’un être humain. De plus, les réactions émotionnelles, évaluées par les réponses comportementales, endocriniennes et neurovégétatives sont modulées par le genre, l’état physiologique ou certaines hormones comme la mélatonine. Les structures cérébrales du réseau neuronal des émotions sont impliquées dans la perception et le traitement des informations de l’environnement, et/ou dans l’expression des réponses émotionnelles. Chez les animaux d’élevage, les structures cérébrales les plus étudiées sont le système olfactif pour la perception, le cortex frontal et l’amygdale pour le traitement des informations, le noyau paraventriculaire de l’hypothalamus pour les réponses endocriniennes. D’autres travaux suggèrent le rôle potentiel de structures du tronc cérébral dans les réponses neurovégétatives et de la substance grise périaqueducale dans les réponses comportementales et plus largement dans les stratégies d’adaptation. En perspective, il est maintenant nécessaire d’étudier l’impact de l’expérience précoce sur le développement du réseau neuronal des émotions et de mieux comprendre la part de l’évaluation cognitive dans la genèse des émotions.

Chez les mammifères, la séparation maternelle à la naissance induit un stress chronique avec pour conséquences des altérations comportementales et neurobiologiques. Ainsi, en élevage ovin, les agneaux surnuméraires sont séparés de leur mère à la naissance et nourris avec des laits artificiels, induisant de telles altérations. Des études montrent l’importance du microbiote intestinal sur le développement comportemental. Notre hypothèse est qu’une alimentation enrichie en prébiotique dès la naissance contribuerait, via une action sur le microbiote, à prévenir certains des désordres associés au stress de séparation maternelle, en particulier comportementaux.Des agnelles ont reçu le colostrum de leur mère les 2 jours suivant la naissance avant d’être séparées et nourries avec un lait commercial dépourvu (Preb-) ou enrichi avec un prébiotique (Preb+, 1% galactofructose) jusqu’au sevrage à 7 semaines. Après le sevrage, les agnelles ont toutes reçu une nourriture standard d’élevage. La maturation du microbiote fécal des agnelles a été suivie par séquençage de la région V3-V4 du gène codant l’ARNs 16S à l’âge de 3, 7 et 10 semaines. Parallèlement, leur comportement social a été comparé par une approche éthologique. La position des individus et leurs activités au sein du groupe ont été quantifiées toutes les 10 minutes pendant 3 heures par jour ainsi que les interactions affiliatives et agonistiques, et les vocalisations.Sur la période durant laquelle le galactofructose est apporté par le lait, une restructuration de la composition du microbiote révélant les propriétés bifidogènes du prébiotique est observée : la sur-implantation précoce de 3 espèces de Bifidobactéries au détriment de nombreuses communautés appartenant aux Clostridiales se traduit par une moindre richesse d’espèces dans le groupe Preb+. Elles présentent parallèlement sur le plan comportemental des indices d’association significativement plus importants et des interactions affiliatives plus fréquentes. Elles ont également tendance à manifester moins d’interactions agonistiques et à moins vocaliser. Enfin, les agnelles Preb+ consacrent davantage de temps à consommer du lait sans que ce comportement n’affecte leur croissance.Après le sevrage, une empreinte spécifique du microbiote des agnelles Preb+ est conservée et se caractérise par un meilleur équilibre de l’abondance des espèces présentes, avec notamment, une abondance augmentée des Clostridiales et une meilleure colonisation par Akkermansia muciniphila. Si les différences de comportement social s’estompent après le sevrage, en revanche les agnelles Preb+ présentent un réseau social plus structuré. Enfin, l’intégration de ces données à celles du microbiote par des analyses multidimensionnelles révèle des covariations significatives, mettant ainsi en exergue de potentielles relations entre les modulations du microbiote intestinal et le comportement social lors du développement.En conclusion, l’apport précoce de galactofructose dans le lait diminue durablement les troubles du comportement social des agnelles soumises à un allaitement artificiel et l’absence maternelle. Cette étude suggère que ce prébiotique, via ses répercussions sur la maturation du microbiote intestinal, pourrait compenser certains des effets délétères associés aux stress précoces.

Reproduction induces changes within the brain to prepare for gestation and motherhood. However, the dynamic of these central changes and their relationships with the development of maternal behavior remain poorly understood. Here, we describe a longitudinal morphometric neuroimaging study in female mice between pre-gestation and weaning, using new magnetic resonance imaging (MRI) resources comprising a high-resolution brain template, its associated tissue priors (60-μm isotropic resolution) and a corresponding mouse brain atlas (1320 regions of interest). Using these tools, we observed transient hypertrophies not only within key regions controlling gestation and maternal behavior (medial preoptic area, bed nucleus of the stria terminalis), but also in the amygdala, caudate nucleus and hippocampus. Additionally, unlike females exhibiting lower levels of maternal care, highly maternal females developed transient hypertrophies in somatosensory, entorhinal and retrosplenial cortices among other regions. Therefore, coordinated and transient brain modifications associated with maternal performance occurred during gestation and lactation.

International audience; There are many challenges to overcome in order to properly understand both the exposure to, and effects of, endocrine disruptors (EDs). This is particularly true with respect to fetal life where ED exposures are a major issue requiring toxicokinetic studies of materno-fetal exchange and identification of pathophysiological consequences. The sheep, a large, monotocous, species, is very suitable for in utero fetal catheterization allowing a modelling approach predictive of human fetal exposure. Predicting adverse effects of EDs on human health is frequently impeded by the wide interspecies differences in the regulation of endocrine functions and their effects on biological processes. Because of its similarity to humans as regards gestational and thyroid physiologies and brain ontogeny, the sheep constitutes a highly appropriate model to move one step further on thyroid disruptor hazard assessment. As a grazing animal, the sheep has also proven to be useful in the evaluation of the consequences of chronic environmental exposure to "real-life" complex mixtures at different stages of the reproductive life cycle.

International audience; AimsMedian raphe region (MRR) is an important bottom-up regulatory center for various behaviors as well as vegetative functions, but detailed descriptions and links between the two are still largely unexplored.MethodsPharmacogenetics was used to study the role of MRR in social (sociability, social interaction, resident intruder test) and emotional behavior (forced swim test) parallel with some vegetative changes (biotelemetry: core body temperature). Additionally, to validate pharmacogenetics, the effect of clozapine-N-oxide (CNO), the ligand of the artificial receptor, was studied by measuring (i) serum and brainstem concentrations of CNO and clozapine; (ii) MRR stimulation induced neurotransmitter release in hippocampus; (iii) CNO induced changes in body temperature and locomotor activity.Key findingsMRR stimulation decreased locomotion, increased friendly social behavior in the resident intruder test and enhanced depressive-like behavior. The latter was accompanied by diminished decrease in core body temperature. Thirty minutes after CNO injection clozapine was predominant in the brainstem. Nonetheless, peripheral CNO injection was able to induce glutamate release in the hippocampus. CNO had no immediate (

Automatic or interactive segmentation tools for 3D medical images have been developed to help the clinicians. Atlas-based methods are one of the most usual techniques to localized anatomical structures. They have shown to be efficient with various types of medical images and various types of organs. However, a registration step is needed to perform an atlas-based segmentation which can be very time consuming. Local atlases coupled with spatial relationships have been proposed to solve this issue. Local atlases are defined on a sub-part of the image enabling a fast registration step. The positioning of these local atlases on the whole image can be done automatically with learned spatial relationships or interactively by a user when the automatic positioning is not well performed. In this article, different classification methods possibly included in local atlases segmentation methods are compared. Human brain and sheep brain MRI images have been used as databases for the experiments. Depending on the choice of the method, segmentation quality and computation time are very different. Graph-cut or CNN segmentation methods have shown to be more suitable for interactive segmentation because of their low computation time. Multi-atlas based methods like local weighted majority voting are more suitable for automatic process.

The periaqueductal gray (PAG) is a mesencephalic brain structure involved in the expression of numerous behaviours such as maternal, sexual and emotional. Histological approaches showed the PAG is composed by subdivisions with specific cell organisation, neurochemical composition and connections with the rest of the brain. The comparison of studies performed in rodents and cats as the most often examined species, suggests that PAG organisation differs between mammals. However, we should also consider the plurality of the methods used in these studies that makes difficult the comparison of the PAG organisation between species. Therefore, to study the PAG in all mammals including human, the most relevant in vivo imaging method seems to be the magnetic resonance imaging (MRI). The purpose of this review was to summarize the knowledge of the anatomical organisation of the PAG in mammals and highlights the benefits of MRI methods to extend this knowledge. Results obtained by MRI so far support the conclusions of ex vivo studies, especially to describe the subdivisions and the connections of the PAG. In these latter, diffusion-weighted MRI and functional connectivity seem the most appropriate methods. In conclusion firstly, the MRI seems to be the best judicious method to compare species and improve the comprehension of the role of the PAG. Secondly, MRI is an in vivo method aimed to manage repeated measures in the same cohort of subjects allowing to study the impact of aging and the development on the anatomical organisation of the PAG.

The periaqueductal gray (PAG) is less referred in relationship with emotions than other parts of the brain (e.g. cortex, thalamus, amygdala), most probably because of the difficulty to reach and manipulate this small and deeply lying structure. After defining how to evaluate emotions, we have reviewed the literature and summarized data of the PAG contribution to the feeling of emotions focusing on the behavioral and neurochemical considerations. In humans, emotions can be characterized by three main domains: the physiological changes, the communicative expressions, and the subjective experiences. In animals, the physiological changes can mainly be studied. Indeed, early studies have considered the PAG as an important center of the emotions-related autonomic and motoric processes. However, in vivo imaging have changed our view by highlighting the PAG as a significant player in emotions-related cognitive processes. The PAG lies on the crossroad of networks important in the regulation of emotions and therefore it should not be neglected. In vivo imaging represents a good tool for studying this structure in living organism and may reveal new information about its role beyond its importance in the neurovegetative regulation.

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International audience; The periaqueductal gray (PAG) is a mesencephalic brain structure organised in subdivisions with specific anatomical connections with the rest of the brain. These connections support the different PAG functions and especially its role in emotion. Mainly described in territorial and predatory mammals, examination of the PAG connections suggests an opposite role of the ventral and the dorsal/lateral PAG in passive and active coping style, respectively. In mammals, the organisation of PAG connections may reflect the coping style of each species. Based on this hypothesis, we investigated the anatomical connections of the PAG in sheep, a gregarious and prey species. Since emotional responses expressed by sheep are typical of active coping style, we focused our interest on the dorsal and lateral parts of the PAG. After injection of fluorogold and fluororuby, the most numerous connections occurred with the anterior cingulate gyrus, the anterior hypothalamic region, the ventromedial hypothalamic nucleus and the PAG itself. Our observations show that the sheep PAG belongs to the neuronal circuit of emotion and has specific parts as in other mammals. However, unlike other mammals, we observed very few connections between PAG and either the thalamic or the amygdalar nuclei. Interestingly, when comparing across species, the PAG connections of sheep were noticeably more like those previously described in other social species, rabbits and squirrel monkeys, than those in territorial species, rats or cats.

Remerciements : Plateforme Phénotypage-Endocrinologie, INRA, UMR PRC 0085, Centre Tours Val de Loire; International audience; Characteristics of attachment were assessed in peer- and object-reared lambs, and compared to mothered subjects by taking into consideration distress, proximity seeking, and exploration during two separation-reunion tests in both the familiar and a novel environment. Plasma cortisol and oxytocin were assayed as physiological indicators of stress and being comforted during the separation-reunion test. Rewarding properties of the familiar figures were also determined in a conditioned place preference-like paradigm. Between-group analysis revealed the existence of secure attachment with the mother, alteration of secure attachment with the peer and weaker attachment with the object. Weaker attachment was expressed by a lack of distress during separation in the home pen and no preference for the place conditioned with the familiar object. Elevated basal plasma oxytocin levels, but not cortisol, observed in maternally deprived lambs were more likely linked to the absence of a maternal figure rather than social comfort during reunion.

Transpariser un organe... et tout devient limpide !. Journées d'Animation Scientifique de la Fédération de Recherche d'Infectiologie (FéRI)

Impact of early rearing experience on brain development in sheep infant. . New animal models to understand the brain

Updated labelling and description of the gross anatomy of the sheep cortex derived from surface reconstruction of the brain. . New animal models to understand the brain

Atlas-based segmentation is a widely used method for Magnetic Resonance Imaging (MRI) segmentation. It is also a very efficient method for the automatic segmentation of brain structures. In this paper, we propose a more adaptive and interactive atlas-based method. The proposed model allows to combine several local probabilistic atlases with a topological graph. Local atlases can provide more precise information about the structure’s shape and the spatial relationships between each of these atlases are learned and stored inside a graph representation. In this way, local registrations need less computational time and image segmentation can be guided by the user in an incremental way. Pixel classification is achieved with the help of a hidden Markov random field that is able to integrate the a priori information with the intensities coming from different modalities. The proposed method was tested on the OASIS dataset, used in the MICCAI’12 challenge for multi-atlas labeling.

International audience; Farming systems can expose animals to chronic mild stress which is known to induce negative affective state. Affective state in animals, as in humans, can be assessed through behavioral cues. This study aimed to describe the effect of a chronic mild stress, known to induce a negative affective state, on sheep health through their response to vaccination. The study used 15 lambs subjected to a model of chronic mild stress for 15 weeks and 15 lambs reared under conventional farming as a control group. After 7 weeks of stressful treatment, the lambs were individually exposed to a judgment bias test to assess a putative stress-induced 'pessimism.' After 15 weeks of stressful treatment, antibody immune response was measured after an injection of a live vaccine challenge (Chlamydia abortus attenuated vaccine strain 1B). Stressed lambs displayed a pessimistic-like perception in the judgment bias test, revealing a negative affective state. Stressed and control animals showed different immunological reactions to vaccine challenge: stressed sheep had lower hemoglobin concentrations and higher platelet, granulocyte and acute-phase protein concentrations. Antibody response induced by the vaccine strain was not different between stressed and control sheep. Our results suggest that negative affective state induced by chronic stress treatment may induce a stronger inflammatory response to vaccine challenge in sheep. Improvement of animal health may be achieved through consideration of stressors that may affect the emotional and immunological state of sheep.

While behavioral mechanisms of bonding between young mammals and humans have been explored, brain structures involved in the establishment of such processes are still unknown. The aim of the study was to identify brain regions activated by the presence of the caregiver. Since human positive interaction plays an important role in the bonding process, activation of specific brain structures by stroking was also examined. Twenty-four female lambs reared in groups of three were fed and stroked daily by a female caregiver between birth and 5–7 weeks of age. At 4 weeks, an isolation-reunion-separation test and a choice test revealed that lambs developed a strong bond with their caregiver. At 5–7 weeks of age, lambs were socially isolated for 90 min. They either remained isolated or met their caregiver who stroked them, or not, at regular intervals over a 90-min period. Neuronal activation was investigated at the end of the period for maximum c-Fos expression. Reunion with the caregiver appeased similarly the lambs whether stroking was provided or not. Stroking did not activate a specific brain network compared to no stroking. In both cases, brain regions associated with olfactory, visual and tactile cue processing were activated in the presence of the caregiver, suggesting a multisensory process involved. In addition, activation of the oxytocinergic system in the hypothalamic paraventricular nucleus induced by the presence of the caregiver suggests similar neuroendocrine mechanisms involved in inter-conspecific and animal-human bonding.

Les ovins sont bien connus pour développer différentes formes d’attachement social. Le déclenchement de la motivation maternelle et le développement d’un lien mutuel entre la mère et son jeune sont sous l’influence combinée de stimulations hormonales et somato-sensorielles périphériques. Ces stimulations sont fournies par la parturition chez la mère et par les premiers épisodes de tétée chez le nouveau-né. Des changements neurophysiologiques profonds sont observés dans les bulbes olfactifs principaux de la brebis, alors qu’ils sont exposés à des odeurs d’agneau nouveau-né. L’activation de l’hypothalamus consécutive à la mise bas contribue à l’expression de la motivation maternelle et à la mémorisation des odeurs de l’agneau. L’hypothalamus et l’amygdale sont également activés chez le nouveau-né suite à l’ingestion de colostrum. L’ocytocine, un neuropeptide connu pour jouer un rôle clé dans les comportements prosociaux, facilite la construction de la relation mère-jeune. Lorsque des agneaux nouveau-nés sont séparés de leur mère pour être artificiellement nourris, une pratique agricole très commune, ils sont profondément affectés. Les réponses neuroendocrinologiques suggèrent que les agneaux sont dans une situation de détresse. Cependant, si un humain prend soin d’eux, une liaison interspécifique se met en place, facilitée par des contacts sociaux positifs dans lesquels l’allaitement au biberon ou les caresses jouent un rôle clé. De tels contacts activent le système ocytocinergique, comme cela est observé dans la construction du lien mère-jeune. Ainsi, dans des conditions d’élevage artificiel, l’être humain devient une figure d’attachement saillante qui se substitue à la mère et qui peut faciliter la gestion des agneaux., The sheep is well known to develop various forms of social attachment. The onset of maternal responsiveness and the development of the mutual mother-young bond are under the combined influence of hormonal and peripheral somatosensory stimulations. These stimuli are provided by parturition in the mother and by the first suckling episodes in the neonate. Following parturition, the main olfactory bulbs of the ewe undergo profound neurophysiological changes when exposed to offspring odours while activations in the hypothalamus contribute to maternal responsiveness and memorization of lamb odours. Similarly, in the neonate the hypothalamus and the amygdala respond to colostrum ingestion. Oxytocin, a neuropeptide known to play a key role in prosocial behaviours, facilitates mother-young bonding. When new-born lambs are separated from their mother to be artificially-fed, a very common farm practice, they are profoundly affected. Neuroendocrine responses suggest that lambs are in a situation of distress. However, should a human caregiver look after them, an interspecific bond is then facilitated by positive social contacts including bottle-feeding or stroking. These contacts activate the oxytocinergic system, like in the mother-young bonding process. Thus, under artificial rearing conditions,the human becomes a salient attachment figure who may facilitate the management of lambs on farms.

La notion de bien-être chez l’animal d’élevage n’a de sens que si on lui reconnaît la capacité à ressentir des émotions. Cependant, du fait de l’absence de langage verbal, le vécu émotionnel de l’animal est difficilement mesurable, ce qui a toujours rendu son étude délicate. Les travaux récents en psychologie cognitive montrent combien l’étude des interactions entre émotions et cognition est nécessaire pour accéder aux états affectifs de l’animal et tenter ainsi d’identifier les paramètres susceptibles d’affecter son bien-être. Ce texte comporte deux volets. Le premier volet s’appuie sur les théories de l’évaluation développées chez l’Homme, lesquelles suggèrent que le déclenchement des émotions résulte de processus cognitifs d’évaluation basés sur des critères élémentaires tels que la nouveauté et la prévisibilité. Sur la base de ce cadre théorique, nous avons engagé une approche sur les animaux d’élevage visant à définir les critères d’évaluation qu’ils utilisent, et à faciliter ainsi la compréhension de leurs émotions à partir de l’étude de la combinaison des critères impliqués. Le second volet de ce texte s’appuie sur les nombreuses études menées chez l’Homme et les animaux de laboratoire, qui suggèrent que les émotions peuvent en retour influencer les capacités cognitives de l’individu en altérant ses capacités attentionnelles et mnésiques. L’approfondissement de l’étude des interactions entre émotions et cognition offre donc de nouvelles perspectives de recherche pour mieux comprendre les conditions de bien-être des animaux en élevage., Assessment of farm animal welfare requires a good understanding of the animals' affective experience, including their emotions. However, this is difficult to measure because of the absence of verbal communication. Recent studies in the field of cognitive psychology have shown that affective states can be assessed through investigation of the interactions between emotions and cognition. On the one hand, theories of the way humans appraise situations provide a conceptual framework that suggests that emotions are triggered by cognitive appraisal processes based on few elementary criteria such as novelty and predictability. In accordance with appraisal theories, we have developed an experimental approach to study the elementary criteria used by farm animals to evaluate their environment and then express a given emotional response. On the other hand, an increasing number of studies in humans and animals suggest that emotions also influence cognitive processes by modifying attention, memory and judgement. Measurement and manipulation of cognitive processes may provide new insights into how emotions and more persistent affective states in animals can be assessed. Further work on these cognitive approaches will offer new paradigms for a better understanding of the welfare of farm animals.

Social factors hypotheses predict that social segregation between male and female adult ungulates is due to social preferences among same-sex peers and avoidance of the opposite sex, rather than aggregation due to body size dimorphism and differential needs of the sexes. It is suggested that if such social preference/avoidance exists its roots should be early in life. While juvenile ungulate males show social preference for same-sex and same-age peers a few weeks after birth, no studies have confirmed such a phenomenon in juvenile females. In this study we investigated another possible mechanism that would result in an early social preference for same-sex conspecifics among juvenile females: we hypothesized that offspring–mother attachment is stronger for daughters than for sons. Attachment was measured in lambs, Ovis aries, using three characteristics defined initially in children: proximity seeking, distress following separation and exploration in the mother's presence. Lambs were tested at 3 weeks of age in two experimental situations in which they could interact with the mother, a familiar ewe or an unfamiliar ewe. (1) During a choice test between the mother and the familiar ewe, daughters maintained closer proximity with the mother than sons did. During the test without the mother (a familiar versus an unfamiliar ewe) daughters were more distressed than sons were. (2) In an isolation–reunion–separation test, reunion with the mother increased exploratory behaviour more in daughters than in sons. Overall, our results reveal that daughters displayed stronger attachment behaviours and could be more strongly attached to their mother than sons are. Such early preference may be a key factor leading to social preference among females and social segregation between the sexes.

Remerciements :INRA, UE0332 BOURGES Domaine Expérimental Bourges-La Sapinière; Rodent studies show how prenatal stress (PS) can alter morphology in the cortico-limbic structures that support emotional and cognitive functions. PS-induced alteration is less well described in species with a gyrencephalic brain and complex earlier fetal development, and never in sheep at birth to rule out postnatal environment effects or influences of maternal behavior. This study aimed to assess the consequences of a mild chronic stress in pregnant ewes on the neurobiological development of their lambs at birth. During the last third of gestation, 7 ewes were exposed daily to various unpredictable and negative routine management-based challenges (stressed group), while 7 other ewes were housed without any additional perturbation (control group). For each group, a newborn from each litter was sacrificed at birth to collect its brain and analyze its expression levels of genes involved in neuronal dendritic morphology (Dlg4, Rac1, RhoA, Doc2b), synaptic transmission (Nr1, Grin2A, Grin2B) and glucocorticoid receptor (Nr3C1) in hippocampus (HPC), prefrontal cortex (PFC) and amygdala (AMYG). Results revealed that lambs from stressed dam (PS lambs) showed under-expression of Rac1 and Nr1 in PFC and overexpression of Dlg4 in AMYG compared to controls. To assess the morphological consequences of gene dysregulations, the dendritic morphology of pyramidal neurons was explored by Golgi–Cox staining in HPC and PFC. PS lambs had higher dendritic spine density in both structures and more stubby-type spines in the CA1 area of HPC than controls. This is the first demonstration in sheep that PS alters fetal brain, possibly reflecting functional changes in synaptic transmission to cope with adversity experienced in fetal life.

The sensitivities of galanin and melanin-concentrating hormone (MCH) neuronal systems to nutrition are poorly understood in sheep compared to rodents. The aim of this study was to describe the changes in the numbers of galanin and MCH neurones in ovariectomized ewes submitted to different nutritional levels. In the first experiment, ewes were fed ad libitum or food deprived for 24 h. In the second experiment, two groups of ewes were fed at maintenance level (group 100) or undernourished (group 40) for 167 days, after which one-half of each group was killed or refed ad libitum (group 100R and 40R) for 4 days. The MCH neuronal population located in the lateral hypothalamic area was not affected by these nutritional changes. Long-term undernutrition enhanced the number of galanin neurones located in the infundibular nucleus and the dorsal hypothalamic area (DHA), refeeding resulted in an increase of neurones in the DHA and preoptic area, but short-term starvation had no effect on any galanin subpopulations. Our data suggest that the sensitivity of MCH neuronal populations to nutrition in sheep differs from that of rodents. Various populations of galanin-containing neurones differ in sensitivity in ewes subjected to long undernutrition and refeeding but not to short starvation.

The effect of nutritional level on the immunoreactivity of corticotropin-releasing hormone (CRH) in neurones of the hypothalamic paraventricular nucleus was described in sheep, a ruminant, whose feeding strategy differs from that of monogastric species. Two groups of ewes were underfed (40%), or fed at maintenance (100%) for 167 days, after which one-half of each group was killed or ad libitum refed (at least 150% of maintenance) for 4 days before killing. The presence of CRH in the paraventricular nucleus was examined by immunohistochemistry. The number of CRH immunoreactive neurones was increased in underfed ewes, but without modification of the plasma concentration of cortisol, indicating that the rise of CRH was not released in the portal blood nor linked to the pituitary-adrenal axis. Refeeding did not modify significantly the number of CRH immunoreactive neurones in the nucleus although these neurones were increased, only in refed ewes that were previously underfed. These data differ from those for rats and mice where CRH expression is decreased or not modified by underfeeding which could reflect different effects of undernutrition on CRH immunoreactive neurones in monogastric compared to ruminants species.

Remerciements :Plateforme CIRE, Inra UMR PRC Physiologie de la Reproduction et des Comportements, Centre Tours Val de Loire; Regulation of neuroendocrine responses is often studied in animals housed indoors in individual contiguous pens. In sheep, these housing conditions are used to control the environment, facilitate biological sampling and limit social stress. However, this type of housing also prevents exploratory behaviors and could induce stereotypies, non-compliant with welfare and possibly associated with a state of stress. In this context, we investigated the impact of housing in a single-pen, with other familiar conspecifics, on emotional state by evaluating behavioral, hormonal and neuronal measures in adult ewes. We hypothesized that emotional state would be more negative in animals housed in a single-pen for one week (Pen) than in freely moving animals (Free) but less negative than in socially isolated subjects (Isol). We tested our hypothesis in ovariectomized ewes to avoid the interaction with sexual steroid variations. Our behavioral, endocrine and neuronal (Fos activation of the corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus) measures confirmed that withdrawing familiar conspecifics was sufficient to induce strong stress responses in Isol ewes, but there was no indication that Pen ewes were stressed. However, the latter showed less mastication activity than Free ewes, probably due to limited accessibility to straw. The highest plasma prolactin levels were observed in Isol and Free animals, which might result from stress and physical activity, respectively. In Free ewes, plasma dopamine was low, consistent with its inhibitory control of prolactin. However, Isol animals had both high levels of prolactin and dopamine, suggesting a dysregulated balance in socially stressed ewes. As in other species, we suggest that the regulation of prolactin by dopamine varies with stress and/or social context. Overall, this study shows that the impact of housing conditions on different neuroendocrine systems should be considered more in the future.

The hypothalamus plays an important role in the control of food intake in different species, but there is little relevant information for ruminants like sheep. In order to study the putative role of several hypothalamic nuclei in food intake in sheep, Fos expression, a marker of cellular activity, was compared by immunohistochemistry between fed and unfed ewes. The expression of Fos protein was stimulated in the supraoptic nucleus of fed ewes, whereas it was increased in the paraventricular nucleus of unfed animals. In the latter nucleus, Fos immunoreactivity was mainly localized close to the third ventricle, an area corresponding to the parvocellular system of the nucleus, but never in the magnocellular system. In the paraventricular nucleus, the number of corticotrophin releasing factor-immunoreactive neurons and the number of Fos/corticotrophin releasing factor double-labelled neurons were not affected by feeding or lack of feeding. The number of Fos-immunoreactive neurons was higher in the lateral septum, the infundibular, the ventromedial and in the dorsomedial nuclei of unfed ewes than in those of fed ewes. Our results show for the first time that the dorsomedial and ventromedial nuclei are involved in the control of feeding in sheep as in rodents. The supraoptic nucleus of sheep is activated by the same conditions as in rodents but, conversely, the paraventricular nucleus is activated in unfed sheep, whereas in rodents and primates, this nucleus is activated by satiety as well as by fasting. In sheep, unlike in rodents, corticotrophin releasing factor did not appear to be involved in short-term regulation of food intake.

International audience; Although the physiological role of galanin has been demonstrated in several endocrine regulations in sheep, the anatomical characteristics of this neuronal system has never been studied. The distribution of galanin-containing neurones was described by immunohistochemistry using galanin antiserum in the diencephalon of adult awes, both ovariectomized or treated with colchicine.Galanin-immunoreactivity was found throughout the diencephalon. In the ovariectomized ewes, galanin-immunoreactive neurones were mainly observed in the medial preoptic area and the infundibular nucleus. The highest density of immunoreactive fibres was found in the external layer of the median eminence. Numerous galanin-immunoreactive fibres were also observed in the preoptic area, the mediobasal hypothalamus, the periphery of the supraoptic and the paraventricular nuclei. With colchicine treatment, the number of labelled neurones increased, and additional galanin-immunoreactive perikarya were observed in the bed nucleus of the stria terminalis, the lateral septum, the supraoptic. the paraventricular and the periventricular nuclei and the paraventricular nucleus of the thalamus. In the caudal part of the diencephalon. the density of labelled neurones was lower in both groups of animals than in other species studied. Regardless of treatment, labelling was not seen in the suprachiasmatic nucleus and only rarely in the ventromedial nucleus.These results describe, for the first time. the distribution of galanin-immunoreactive neurones in the sheep diencephalon. Compared to other species studied, distribution in the sheep diencephalon has several distinct differences. In ovariectomized animals, the medial preoptic area presents more labelled neurones in sheep than in monkeys, whereas in the supraoptic nucleus the density of labelled neurones is lower in sheep than in humans or opossums. After colchicine treatment only very few differences were observed between sheep and rats, but in contrast to other species, the suprachiasmatic nucleus of the sheep does not contain labelled neurones.

Remerciements : INRA, UMR PRC Plate-forme CIRE 37380 Nouzilly; Sheep are gregarious mammals with complex social interactions. As such, they are very sensitive to social isolation and constitute a relevant animal model to study specifically the biological consequences of social stress. We examined previously the behavioral and endocrine responses in ewes isolated socially in the familiar conspecific withdrawal model (FCW) and showed that stressful responses increased and maintenance behaviors decreased, confirming that social isolation is a strong stressor in sheep. Melatonin synchronizes seasonal and circadian rhythms; and several studies reported its implication in cognitive processes as emotion. Here we investigated its role in the modulation of social stressful responses. Firstly, we studied ewes in the FCW model during the day (characterized by low melatonin levels) and the night (characterized by high melatonin levels). We found lower stressful responses (significant lower levels of cortisol plasma, number of foot pawings, of circling attempts) during the night as compared to the day. To investigate whether these effects were due to melatonin or to darkness, we submitted ewes to FCW during the night with lights on, a condition that suppresses melatonin secretion. Ewes infused with melatonin under these conditions showed decreased stressful responses (significant lower levels cortisol plasma, number of vocalizations, time spent with the head out of the cage) as compared to ewes infused with saline. These findings demonstrate that melatonin diminishes the endocrine and behavioral impact of social isolation in ewes and support the idea that melatonin has a calming effect in socially stressful situations.

Concernant l'affiliation Inra "Physiologie de la reproduction et des comportements" : le codique 6175 indiqué sur la publication est erroné. Le bon codique est 0085.; Chronic stress can be described as a long-term negative affective state induced by an accumulation of negative emotional experiences that alters an individual's interactions with the environment. In humans, chronic stress induces both persistent judgment biases and learning deficits. We investigated whether similar cognitive biases could occur in sheep. Twenty-four lambs were subjected to a chronic stress treatment for 9 weeks (exposure to various unpredictable, uncontrollable and aversive events regularly encountered in ordinary agricultural practices) and 24 other lambs were used as a control group. Before treatment all the lambs had been trained to approach or avoid a food bucket depending on its location. After treatment, the lambs were individually exposed to two tests: (i) a food bucket approach/avoidance test, by testing the response of lambs to ambiguous locations of the bucket, i.e. a judgment bias test, (ii) a learning test consisting of training on six consecutive days to distinguish between two visual stimuli differing in their color and shape (acquisition phase) and re-exposure to the test conditions 2 days after the end of the acquisition phase (recall phase). In the judgment bias test, treated lambs took longer to approach the ambiguous locations of the bucket. During the learning test, treated lambs had lower numbers of correct choices than control lambs in the acquisition and recall phase. Hence sheep experiencing repeated aversive events demonstrated pessimistic-like judgment biases and learning deficits. These may be useful measures to assess long-term emotional states in animals.

This study tested whether lines of Japanese quails divergently selected for a fear response, the tonic immobility, might constitute a reliable bird model for studying anxiety. Previous studies demonstrated that the selection modifies the general underlying emotionality of the birds rather than exerting its effect only on tonic immobility. The behavioural effects of intraperitoneal injections of diazepam, an anxiolytic drug, were assessed in two lines of quail selected either for their short (STI) or long (LTI) duration of tonic immobility. Effects of diazepam were examined in two tests used for measuring emotionality in birds, the open field and the tonic immobility tests. After being placed in the centre of the open field, birds with a high emotionality (LTI quails) stayed longer in the centre of the apparatus than STI quail. Diazepam had anxiolytic effect in LTI birds as it increased the time spent in the outer area. This effect of diazepam appears to be selective because the drug has no effect on other behaviours such as distress calls or escape attempts. The drug has also no effect on the tonic immobility response in any of the two lines. These findings reveal an “anxiogenic” trait of LTI birds in the open field test that can be modulated by the administration of an anxiolytic drug. Therefore quails selected for LTI and STI represent a valuable model to study the mechanisms underlying anxiety in birds.

Paradigms used so far to study the effects of social isolation in sheep confound the effects of social isolation with those of other stressors (e.g. new environment) and showed contradictory effects after multiple social isolations. We propose here to characterize and examine the repeatable effects of social isolation induced by the familiar conspecific withdrawal (FCW). This latter test consists of socially isolating the ewe by the removal of group mates from the room test for 3 h. Behavioral and endocrine responses of adult ovariectomized-estradiol implanted ewes were compared 90 min before and 90 min after FCW, which was applied three times every fourteen days. We observed that each FCW induced significant increases in plasma cortisol level, in the number of vocalizations, foot pawing, circling attempts and a significant decrease in time spent lying down. An increase in plasma cortisol levels and decrease in duration of maintenance behaviors were significantly lower after the third FCW than after the first one. These differences could be explained by higher plasma cortisol levels and lower duration of maintenance behaviors before the third FCW than before the first FCW suggesting an anticipation of the social isolation period. These data indicate that social isolation is sufficient to induce distress with stable stressful responses after multiple exposures to familiar conspecific withdrawal.

This chapter describes how large animal models can be used to improve our knowledge of neuroscience and brain disorders. Various animal models have been used in Magnetic Resonance Imaging (MRI). Rodents and non-human primates are the most commonly used, but they present a number of drawbacks; for example, the rodent brain is smaller than that of humans and thus a higher spatial resolution is required. In addition, there are significant differences between human and rodent brain morphology: for example the rodent brain is smooth, whereas that of the human is gyrencephalic. By contrast, the brain of large mammals such as the pig, sheep or goat is gyrencephalic and has greater similarities with the human brain (Lind et al. 2007). The Gottingen minipig is increasingly used in experimental neuroscience, to investigate brain disorders and is a suitable alternative model to non-human primates for economic, ethical and genetical homogeneity reasons.

International audience; The vagal motor system is involved in the regulation of cardiorespiratory and gastrointestinal functions. Vagal motor neurons are localized near or adjacent to catecholaminergic neurons, but their co-localisation seems species dependent, present in the cat but absent in the rabbit. In pig, a species commonly used as an experimental model in humans brain disorders (sudden infant death syndrome, hypoxia), the relationship is poorly understood. We aimed at describing the distribution of vagal motor neurons and tyrosine hydroxylase-immunoreactive (-ir) neurons by using a double staining method in combination with retrograde tracing of vagal efferent neurons. After fluorogold impregnation of the central part of the sectioned left cervical vagal trunk, two main vagal motor neuronal populations were located in the dorsal motor nucleus of the vagus nerve (DMX) and in the area of the nucleus ambiguus (Amb). Like in the human, the DMX was composed of different subpopulations of neurons with the same morphological characteristics. Immunohistochemistry of catecholaminergic synthesizing enzymes differentiated two main sites containing vagal motor populations: the dorsomedial and the ventrolateral medulla. TH-ir was rarely seen in vagal motor neurons of the DMX, but TH-ir neurons were present around the two main vagal motor neuronal populations that contained TH-ir fibres. The anatomical organisation of the vagal motor and the catecholaminergic neuronal systems are similar to those described in humans and suggest that the involvement of the catecholamines in the control of the vagal motor system may be similar in pigs and in humans. (C) 2009 Elsevier B.V. All rights reserved.

International audience; The cortical (CoA) and the medial (MeA) nuclei of the amygdala are involved in the processing of olfactory information relevant to social recognition in the ewe. To better understand the neural pathways responsible for these effects, the connections of both CoA and MeA with the telencephalic and diencephalic regions were studied by injecting an anterograde (Biotin-Dextran-Amine, BDA) or a retrograde (Fluorogold, FG) neuronal tracer into either the CoA or the MeA. Concerning the primary olfactory structures, the CoA receives inputs from both the main olfactory bulb and the accessory olfactory bulb (AOB), while the MeA is innervated by cells only from the AOB. Among the other olfactory structures, only the entorhinal cortex and the tenia tecta are connected with both the CoA and the MeA. With respect to the other secondary olfactory structures, the connections with the CoA and the MeA show segregating neuronal routes. The CoA is connected with the accessory olfactory nucleus, the piriform, the endopiriform and the orbitofrontal cortices while the MeA exhibited connections with the nucleus of the lateral olfactory tract, the perirhinal and the insular cortices. Concerning the diencephalic structures, only the MeA receives projections from the PVN and the MBH. On the other hand, we showed that the BNST is the major site of connection with both the CoA and the MeA. Reciprocal projections were observed between the CoA and the MeA and between both nuclei and the basal or the lateral nuclei of the amygdala with the exception of the CoA which does not send inputs to the lateral nucleus. These data are discussed in relation with olfactory learning in the context of sexual and maternal behavior in sheep. (C) 2008 Elsevier B.V. All rights reserved.

Elodie Chaillou and Dominique Grouselle contributed equally to this work.; International audience; As in other species, exogenous administration of ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptors can stimulates feeding behaviour and GH secretion in the sheep. However, the importance of endogenous ghrelin for these two functions as well as its central or peripheral origin remained to be established. In the present study, cerebrospinal fluid (CSF) ghrelin concentrations were measured in five anoestrous ewes and found to be more than 1000-fold lower than circulating plasma levels, in keeping with the even lower concentration in hypothalamic compared to abomasum tissue extracts. Cluster analysis indicated that CSF ghrelin levels were markedly pulsatile, with a greater number of peaks than plasma ghrelin. Pulsatility parameters were closer for GH and CSF ghrelin than between GH and plasma ghrelin. Plasma ghrelin and GH levels were significantly correlated in three out of five ewes but CSF ghrelin and GH in one ewe only. Half of the CSF ghrelin episodes were preceded by a ghrelin peak in plasma with a 22-min delay. Cross-correlations between plasma GH and plasma or CSF ghrelin did not reach significance but a trend towards cross-correlation was observed from 20 to 0 min between plasma and CSF ghrelin. At 09.00 h, when food was returned to ewes, voluntary food intake did not elicit a consistent change in plasma or CSF ghrelin levels. By contrast, a peripheral ghrelin injection (1 mg, i.v.) immediately stimulated feeding behaviour and GH secretion. These effects were concomitant with a more than ten-fold increase in plasma ghrelin levels, whereas CSF ghrelin values only doubled 40-50 min after the injection. This suggests that peripherally-injected ghrelin crosses the blood-brain barrier, but only in low amount and with relatively slow kinetics compared to its effects on GH release and food intake. Taken together, the results obtained in the present study support the notion that, in the ovariectomised-oestradiol implanted sheep model, peripheral ghrelin injection rapidly induces GH secretion, and feeding behaviour, probably by acting on growth hormone secretagogue receptor subtype 1 located in brain regions in which the blood-brain barrier is not complete (e.g. the arcuate nucleus).

Circulating leptin is regulated by food intake in the long, medium and short term; however, little is known about putative remnant effects of these successive regulations at any given time. To clarify this, two experiments were conducted in adult sheep, during which body condition parameters and plasma leptin were measured. During experiment 1, twenty ewes with normal body condition were either well fed (101 % of maintenance energy requirements (MER)) or underfed (41 % MER) for 166 d, then rapidly re-fed (at a mean of 208 % MER) for 3 d. Leptinaemia decreased after 14 d of underfeeding, remained depressed until day 166 and did not increase after 3 d re-feeding, whereas it was increased (+153 %;P P P P

The identification and role of neuropeptides in the control of food intake and energy balance have been extensively studied in rodents, and for more than ten years, similar studies have been performed in sheep. As a photoperiodic ruminant, sheep are an interesting alternative animal model to rodents. In this review, we summarize the results obtained in sheep concerning the distribution of peptide-containing neurones in the hypothalamus and their central role in the control of food intake and energy balance, and compared them with relevant data from rodents. Even if the general organization and the role of hypothalamic neuropeptides are similar in sheep and rodents, numerous differences have been observed between these two species. In sheep, the magnocellular neurones of the paraventricular and supraoptic nuclei are characterized by the low density and the lack of galanin- and neuropeptide-Y-containing neurones, respectively. The sheep pituitary stalk presents neurones containing neuropeptides such as neuropeptide-Y or beta-endorphin, which are also found in the deep part of the infundibular nucleus. In this structure, several neuronal populations, including galanin, agouti-gene related peptide, somatostatin, are sensitive to energy balance variations, undernutrition or overfeeding, which may specifically modify neuropeptide levels in discrete neuronal subgroups. This feature is well illustrated by the number of neuropeptide- Y labelled neurones, that increases in the lateral part of the infundibular nucleus of undernourished ewes and decreases in the ventral part of overfed ewes. Conversely, after 24 hours of food deprivation, the number of neuropeptide-Y-immunolabelled neurones is unchanged in the sheep infundibular nucleus, whereas increased levels of this neuropeptide are described, in rats, by radioimmuno-assay. In conclusion, our review shows that peptide-containing neurone systems, involved in the regulation of food intake and energy balance in sheep, are generally similar to those observed in other species, but they present specific differences according to the physiological characteristics of the animal model.

Conversely to rodents, the involvement of hypothalamic neuropeptide Y (NPY) neurons in the control of nutrition is poorly understood in ruminants such as sheep. Therefore, the aim of this work was to describe the NPY neurons of the diencephalon in ewes submitted to different diets. In colchicine-treated animals, large populations of NPY-immunoreactive (-ir) neurons were observed in a ventral and a lateral subpopulation of the infundibular nucleus (IN), in the median eminence, the pituitary stalk, and the dorsomedian and dorsocaudal nuclei. No labeled perikaryon was observed in the magnocellular neurons of the hypothalamus, although numerous labeled fibers were noted in the neural part of the pituitary. The pattern of distribution of NPY-ir neurons in the sheep hypothalamus is similar in many ways to those of rodents, but it presents also many specific characteristics that have not been previously described. In ewes that were fasted for 24 hours, or fed ad libitum, the number of NPY-ir neurons was the same whatever the hypothalamic structures. In underfed ewes (40% of maintenance for 24 weeks), the lateral subpopulation of the IN presented a higher number of NPY-ir neurons than observed in the 100% fed ewes. Conversely, in the ventral subpopulation, the animals refed ad libitum (at least 150% of maintenance for 4 days) presented a lower number of NPY-ir neurons than the other groups. The other NPY neuronal populations of the hypothalamus were not significantly modified by the dietary treatments. For the first time, we demonstrated the presence of two functionally distinct subpopulations of NPY neurons in the sheep IN. The variations of labeled neurons were correlated with plasma nonesterified fatty acid levels but not with leptinemia.

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