D Val-Laillet
email: david.val-laillet@inra.fr

David Val

Review: Impact of food, gut-brain signals, and metabolic status on brain activity in the pig model -10 years of nutrition research using in vivo brain imaging

Keywords: Nutrition, Microbiota, Cognition, Pleasure, Behavior

The purpose of this review is to offer a panorama on 10 years of nutrition research using in vivo brain imaging in the pig model. First, we will review some work describing the brain responses to food signals including basic tastants such as sweet and bitter at both oral and visceral levels, as well as conditioned preferred and aversive flavors. Second, we will have a look at the impact of weight gain and obesity on the brain metabolism and functional responses, drawing the parallel with obese human patients. Third, we will evoke the concept of the developmental origins of health and diseases, and how the pig model can shed light on the importance of maternal nutrition during gestation and lactation for the development of the gut-brain axis and adaptation abilities of the progeny to nutritional environments. Finally, three examples of preventive or therapeutic strategies will be introduced: the use of sensory food ingredients or pre-, pro-, and post-biotics to improve metabolic and cognitive functions; the implementation of chronic vagus nerve stimulation to prevent weight gain and glucose metabolism alterations; and the development of bariatric surgery in the pig model, for the understanding of its complex mechanisms at the gutbrain level. A critical conclusion will brush the limitations of neurocognitive studies in the pig model and put in perspective the rationale and ethical concern underlying the use of pig experimentation in nutrition and neurosciences.

Introduction

The present review paper is dedicated to the exploration of brain activity via in vivo brain imaging in the pig model, in the context of nutrition and metabolic research, and to the understanding of the gut-brain axis factors modulating this brain activity. Brain activity is a rather imprecise phrasing encompassing several meanings and embracing different concepts. Throughout this review, the terms 'brain activity' will be used as an ellipsis referring to neurobiological outcomes, which can be visualized via brain-imaging techniques such as nuclear brain tomography or functional magnetic resonance imaging (fMRI). Using these brain-imaging techniques, we can investigate the cerebral blood flow, the brain glucose metabolism, or the blood-oxygen-leveldependent (BOLD) hemodynamic response, all of those being correlated with neural activity and integration processes. Brain activity or metabolism can be visualized in a resting state, or in response to specific stimulations and sensory-cognitive tasks.

Nutrition research in the conventional or miniature pig is justified by a two-way rationale: first, the (mini)pig is a farm (or pet) animal that is bred and raised to provide A c c e p t e d m a n u s c r i p t human food resources (or company); second, the pig is probably the closest animal to human beings in terms of digestive physiology [START_REF] Guilloteau | Nutritional programming of gastrointestinal tract development. Is the pig a good model for man?[END_REF], Roura et al., 2016), and it shares many other similarities with the human. Similarities include a relatively large gyrencephalic brain [START_REF] Sauleau | The pig model in brain imaging and neurosurgery[END_REF], Roura et al., 2016) associated with high cognitive abilities [START_REF] Dilger | Behavioral assessment of cognitive function using a translational neonatal piglet model[END_REF], which is why the pig is a very good preclinical research model in nutrition and neurobehavioral disorders [START_REF] Lind | The use of pigs in neuroscience: modeling brain disorders[END_REF][START_REF] Gieling | The pig as a model animal for studying cognition and neurobehavioral disorders[END_REF]. Improving health and well-being of farm (and pet) animals is not only a legal and ethical responsibility, but also a prerequisite to food quality and human health. Understanding how nutrition can modify the pig's physiology, gut microbiota, metabolism, behavior and health, within the spectrum of comparative pathology, is highly valuable to gain knowledge in human nutrition and health, and fight against food-related metabolic and behavioral disorders in both species.

The role of the microbiota-gut-brain axis in processing information and nutrients, as well as in regulating digestive processes and eating behavior, has been well documented and still attracts interest (van de [START_REF] Van De Wouw | Microbiota-Gut-Brain Axis: Modulator of Host Metabolism and Appetite[END_REF]. The brain is the conductor of almost all biological functions and behavior, including eating behavior and digestion. The way the brain responds to food/metabolic status and integrates information shapes the nutritional and emotional status of the individual, the satietyhunger alternation, the perception of pleasure, and the emergence of goal-oriented behaviors. On this gut-brain communication depends the individual's ability to adapt to its nutritional environment, by making choices and decisions that impact health and well-being. Investigating brain activity in response to food or diet is not only a fascinating fundamental research topic, but also a pathway to applied and translational research to the benefit of animal and human health.

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Different ways of tackling this topic in the pig will be presented. First, the implementation of functional and metabolic imaging in the pig model can be used to investigate the brain responses to food exteroceptive and interoceptive perception, in the context of hedonic, homeostatic, or cognitive processes. Second, investigating brain activity in swine opens the way to understanding how metabolic diseases and eating disorders are linked to diet-related neurocognitive anomalies. Many studies showed that the minipig model is a real asset to investigate obesity and metabolic syndrome (e.g. [START_REF] Johansen | The obese Gottingen as a model of the metabolic syndrome: dietary effects on obesity, insulin sensitivity, and growth hormone profile[END_REF]. This question can be explored in adult animals with a declared pathology, but also before the onset of the disease, during sensitive periods such as the perinatal life. The relatively short developmental period and lifespan of pigs, compared to humans, is consequently a unique opportunity to decipher the nutritional events/status shaping brain functions from birth to the adult age. Finally, brain imaging can be used to assess the impact of preventive or therapeutic strategies in preclinical studies. In this review, the case of functional food ingredients, neurostimulation, and bariatric surgery will be evoked.

Table 1 andFigure

Brain responses to food signals

Brain responses to basic tastes

The brain integrates a huge amount of information coming from the environment and the inside of the organism, through different communication pathways. Exposure to food and their post-ingestive consequences generates both exteroceptive (e.g. smell, taste, vision) and interoceptive (i.e. gut perceptions) signals that the brain must integrate and associate to elaborate a multisensory mental representation of food items. This mental representation includes physical and hedonic dimensions depending on the individual experience and shaping the individual expectations. One of our first objectives was consequently to map the brain responses to basic tastants, such as sweet or bitter compounds, which are known to elicit strong innate appetitive or repulsive reactions [START_REF] Steiner | Comparative expression of hedonic impact: affective reactions to taste by human infants and other primates[END_REF]. The role of gut nutrient sensing in modulating appetite and conditioning food preferences (notably via sugar gut infusions) has also been extensively described [START_REF] Sclafani | Role of gut nutrient sensing in stimulating appetite and conditioning food preferences[END_REF]. For sweet/sugar perception, our first question was to elucidate whether the detection site might condition the brain responses elicited. Glucose portal infusion (Figure 1, e) and duodenal infusion (Figure 1,d) both reduced feed intake and ingestion speed in a similar way, but induced different systemic signals and different brain responses (Table 1, [START_REF] Boubaker | Brain processing of duodenal and portal glucose sensing[END_REF], which demonstrates that gut nutrient and/or taste perception can shape brain responses related to hedonic and homeostatic regulation of food intake in the pig. The next step was to compare exteroceptive (i.e. gustatory) and interoceptive (i.e. duodenal) perceptions of sweet taste/nutrient, and more precisely to compare the impact of combined vs. dissociated oral and intestinal A c c e p t e d m a n u s c r i p t sucrose detection on the brain reward processes. Oral and/or duodenal sucrose sensing (Figure 1,c,d) induced differential cerebral blood flow changes in brain regions known to be involved in memory, reward processes and hedonic evaluation of sensory stimuli (Table 1, Clouard et al., 2014). Such information are valuable to disentangle the respective roles of oral and visceral perception of sweet/sugar in modulating food intake and motivation, and for translational research using the pig model: e.g. to assess the impact of additives or artificial sweeteners, design new food products, or improve their nutritional quality with a benefits/risks approach in terms of palatability, health, gut-brain axis, and behavior.

More recently, using fMRI, we aimed at comparing oral sweet and bitter gustatory stimulation (Table 1, [START_REF] Coquery | fMRI-Based Brain Responses to Quinine and Sucrose Gustatory Stimulation for Nutrition Research in the Minipig Model: A Proof-of-Concept Study[END_REF]. The advantage of fMRI for comparing brain responses to different food stimuli is that multiple stimulations can be performed within the same imaging session, contrary to nuclear imaging (positron emission tomography, PET, and single-photon emission computed tomography, SPECT). The fMRI modality has already been implemented and used in the pig, notably to assess brain activity after deep brain stimulation [START_REF] Knight | Nucleus accumbens deep brain stimulation results in insula and prefrontal activation: a large animal FMRI study[END_REF][START_REF] Gibson | Anterior Thalamic Deep Brain Stimulation: Functional Activation Patterns in a Large Animal Model[END_REF], but to our knowledge, the work performed by [START_REF] Coquery | fMRI-Based Brain Responses to Quinine and Sucrose Gustatory Stimulation for Nutrition Research in the Minipig Model: A Proof-of-Concept Study[END_REF] is the first to describe blood-oxygen-level dependent (BOLD) brain responses to visual (Figure 1, a) and food stimuli in the pig. We showed that quinine and sucrose stimulations (Figure 1,c) induced different brain activation patterns in gustatory, hedonic, and cognitive areas (Table 1, [START_REF] Coquery | fMRI-Based Brain Responses to Quinine and Sucrose Gustatory Stimulation for Nutrition Research in the Minipig Model: A Proof-of-Concept Study[END_REF]. This might contribute to the understanding of the effects of bitter compounds on gut physiology and behavior, for which significant effects were observed on gastric emptying, intestinal motility, gastrointestinal hormones, and food intake [START_REF] Fu | Bitter compounds delayed gastric emptying and induced intestinal smooth muscle relaxation in a pig model[END_REF], Roura et al., 2016).

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Brain responses to preferred or aversive food flavors

Previous review papers explained why and how pig models could help disentangling the complex processes underlying food perception and preferences, and how such studies might benefit biomedical research (Clouard et al., 2012b, Roura et al., 2016).

From 2011, we published the first studies using functional imaging to describe brain responses to conditioned food preferences and aversions in pigs, using 99mTc-HMPAO SPECT [START_REF] Gaultier | Flavour exposures after conditioned aversion or preference trigger different brain processes in anaesthetised pigs[END_REF][START_REF] Boubaker | Brain processing of duodenal and portal glucose sensing[END_REF], Clouard et al., 2014) and 18F-FDG PET (Clouard et al., 2012c) to map cerebral blood flow and brain glucose metabolism, respectively. We validated the use of brain imaging in anesthetized pigs and confirmed that the brain is still able to integrate and react to food sensory stimuli (e.g. flavors) according to individual positive or negative experience and associated internal states (Table 1, [START_REF] Gaultier | Flavour exposures after conditioned aversion or preference trigger different brain processes in anaesthetised pigs[END_REF], Clouard et al., 2012c). Different brain responses were identified in a neural network including frontostriatal and limbic areas, which are known in the human and rodents to be involved in the regulation of food intake and pleasure (Figure 1,b,c). The associative conditioning between post-ingestive consequences and the meal's flavors shaped further hedonic valuation and food choices, which highlighted the gut-brain mechanisms influencing eating behavior in pigs. This model might be useful to investigate the onset of food aversion further invasive medical treatments, such as radiotherapy or chemotherapy, or exacerbated food preferences mediated by hedonic reinforcers such as added sugar (Clouard et al., 2012b).

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Impact of the metabolic or emotional status on brain responses to food signals

Brain responses to food stimuli can be modulated by previous associative conditioning but also by the individual's metabolic or psychological state. In rodents, it has been shown that taste/olfactory perception capacity decreased with the visceral fat increase [START_REF] Fernandez-Garcia | An increase in visceral fat is associated with a decrease in the taste and olfactory capacity[END_REF][START_REF] Thanos | Differences in response to food stimuli in a rat model of obesity: in-vivo assessment of brain glucose metabolism[END_REF] documented significant changes in brain glucose metabolism in obese compared to lean rats exposed to appetitive food scent. Even though this question was not investigated in the pig model, we explored another relationship between the internal state and brain responses to odors. The emotional and physiological responses to stress are known to impact olfactory perception [START_REF] Hoenen | The Impact of Stress on Odor Perception[END_REF]. We recently aimed at describing the consequences of a psychosocial stress in pigs on the gut-brain axis, behavior and brain responses to olfactory food stimuli. We exposed stressed and control animals to low/high concentrations of novel food odors (Figure 1, b) and showed activations in the prepyriform, entorhinal, orbitofrontal, and anterior cingulate cortices, the dorsal striatum and hippocampus. We also showed that high-concentration olfactory stimuli induced a higher brain activity (Table 1, Coquery et al., in revision).

More interestingly, pigs with psychosocial stress had lower brain responses in the right hippocampus compared to unstressed control pigs, as well as lower magneticresonance-spectroscopy-estimated neuronal density in the hippocampus, which suggested an impact of the internal state on cognitive and limbic processes at the brain level (Table 1, Menneson et al., in press).

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Impact of weight gain and obesity on brain functions

Brain anomalies in diet-induced obese minipigs

The use of preclinical models is very important to decipher the determinants of obesity, eating disorders and metabolic diseases, but also to identify novel predictive biomarkers and innovative therapeutic strategies focused on the gut-brain axis [START_REF] Val-Laillet | Neuroimaging and neuromodulation approaches to study eating behavior, prevent and treat eating disorders and obesity[END_REF][START_REF] Obrenovich | The Co-Metabolism within the Gut-Brain Metabolic Interaction: Potential Targets for Drug Treatment and Design[END_REF]. The minipigs' limited size enables longitudinal studies at the adult age using the same imaging machines as in humans.

The first studies using the minipig as a model of obesity date back to the 80's (e.g. [START_REF] Phillips | Dietary effects on metabolism of Yucatan miniature swine selected for low and high glucose utilization[END_REF], but most of the publications appeared after the year 2000 [START_REF] Johansen | The obese Gottingen as a model of the metabolic syndrome: dietary effects on obesity, insulin sensitivity, and growth hormone profile[END_REF][START_REF] Larsen | Beta-cell function and islet morphology in normal, obese, and obese beta-cell massreduced Gottingen minipigs[END_REF][START_REF] Christoffersen | Genderassociated differences in metabolic syndrome-related parameters in Gottingen minipigs[END_REF][START_REF] Lee | Nutritional model of steatohepatitis and metabolic syndrome in the Ossabaw miniature swine[END_REF][START_REF] Madsen | Expression studies of the obesity candidate gene FTO in pig[END_REF], Val-Laillet et al., 2010a). Brain activity was described for the first time in obese minipigs in the Gottingen breed (Val-Laillet et al., 2011b), and then in the Yucatan (Ochoa et al., 2016a). The brain functional characteristics or anomalies associated with obesity and weight gain have been well described in the human, encompassing initial high-sensibility to highly palatable food stimuli in highrisk individuals (i.e. reward surfeit), but also decreased brain responses in the prefrontal cortex and striatum in obese patients (i.e. reward deficit), as well as a blunted dopaminergic system [START_REF] Burger | Variability in reward responsivity and obesity: evidence from brain imaging studies[END_REF]Stice, 2011, Blum et al., 2015).

Even though evidence suggests that chronic exposure to energy-dense palatable foods provokes a switch in the hedonic and motivational brain processes [START_REF] Burger | Variability in reward responsivity and obesity: evidence from brain imaging studies[END_REF], and that successful body weight loss can reverse some obesity-related brain anomalies [START_REF] Le | Less activation in the left dorsolateral prefrontal cortex in the reanalysis of the response to a meal in obese than in lean women and its association with successful weight loss[END_REF], there was no clear demonstration showing that these brain anomalies are induced by (or concomitant to) weight gain in obese people and not preexistent to it. Our brain imaging study in the minipig (Table 1, Val-Laillet et al., 2011b), in association with physiological data (Val-Laillet et al., 2010a, A c c e p t e d m a n u s c r i p t Val-Laillet et al., 2010c), was the first to demonstrate that diet-induced obesity could induce brain anomalies in comparison to lean subjects, including decreased basal metabolism in the prefrontal cortex and reward circuit (Figure 1, diet-induced obesity). These results suggested that some characteristics of the reward deficit phenotype observed in obese humans were probably an acquired feature of obesity, induced by regular excess food intake.

Impact of specific nutrients on brain anomalies in obese minipigs

Following the 2011 demonstration in obese minipigs, two questions arose: Are weight gain and excess calorie intake only liable for these brain anomalies described in obese subjects? And does food quality, independently from the quantity consumed, have a role in shaping brain activity in obese subjects? In two additional independent studies in obese minipigs, we decided to compare different isocaloric obesogenic diets differing in terms of lipids or carbohydrates quality, respectively. The first study compared a 5-week exposure to three obesogenic diets only differing in the nature of lipids used for their composition (i.e. fish oil / omega-3, sunflower oil / omega-6, or lard / saturated fatty acids). Omega-3 fatty acids inhibit the production of proinflammatory cytokines [START_REF] Parker | Omega-3 fatty acids and mood disorders[END_REF], and omega-3 supplementation (DHA) can increase the activity of the prefrontal cortex [START_REF] Mcnamara | Docosahexaenoic acid supplementation increases prefrontal cortex activation during sustained attention in healthy boys: a placebo-controlled, dose-ranging, functional magnetic resonance imaging study[END_REF], which is one of the brain areas that were found deactivated in obese humans and minipigs [START_REF] Le | Less activation of the left dorsolateral prefrontal cortex in response to a meal: a feature of obesity[END_REF][START_REF] Volkow | Inverse association between BMI and prefrontal metabolic activity in healthy adults[END_REF], Val-Laillet et al., 2011b). We hypothesized that omega-3 might prevent the onset of central inflammation and protect brain areas involved in food intake control. We demonstrated significant changes in terms of blood-brain barrier (BBB) permeability and basal brain glucose metabolism according to the diets (Table 1, Val-Laillet et al., 2011a and2013). Contrary to what was A c c e p t e d m a n u s c r i p t expected, we showed that a diet (overly) enriched with omega-3 did not protect the BBB nor the basal metabolism of brain areas that are dysfunctioning in the context of obesity (Figure 1, diet-induced obesity). Consistent results were obtained on intestinal permeability [START_REF] Lalles | Alterations in ileal and colonic permeability by chronic intake of high-lipid diets enriched with omega 3, omega 6 or saturated fat[END_REF]2013), suggesting global effects of the diets on the gut-brain axis. Excessive dietary omega-3 might increase the fluidity of intestinal and BBB cell membranes, therefore increasing their permeability and facilitating the invasion of pro-inflammatory compounds towards blood and brain.

Even though this hypothesis still needs validation, these results highlight the need for precise recommendations in terms of dietary omega-3, because excessive supplementation might be as deleterious as a deficiency for the gut and brain functions. The European Food Safety Authority stated that dietary recommendations for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for European adults are between 250 and 500 mg/day, and that supplemental intakes at doses up to 5 g/day do not raise safety concerns for adults in terms of cardiovascular disease risk, glucose homeostasis or immune function [START_REF] Authority | Scientific Opinion on the Tolerable Upper Intake Level of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA)[END_REF]. The omega-3 supplementation in our minipig study was 1.9 g/day in average, which corresponded to 3.2 g of omega-3 per day in a normal-weight 63-kg adult woman. This was above the recommended dose and below the tolerable upper intake level of omega-3 according to the EFSA, but no other study in our knowledge ever investigated the brain metabolism outcomes at these doses.

The second study relating nutrient quality and brain activity compared a long-term exposure to three obesogenic isocaloric diets, only differing in the nature of their carbohydrates (i.e. starch as control, compared to glucose and fructose) [START_REF] Ochoa | Effects of chronic intake of starch-, glucose-and fructose-containing diets on eating behaviour in adult minipigs[END_REF], Ochoa et al., 2016a, Ochoa et al., 2016b). Fructose, which is overly used in industrial food products, has been incriminated for its role in the onset of metabolic A c c e p t e d m a n u s c r i p t syndrome and its impact on gut-brain metabolic signals in rodents [START_REF] Dornas | Health implications of highfructose intake and current research[END_REF][START_REF] Ochoa | Dietary sugars: their detection by the gut-brain axis and their peripheral and central effects in health and diseases[END_REF][START_REF] Legeza | Fructose, Glucocorticoids and Adipose Tissue: Implications for the Metabolic Syndrome[END_REF]; but counter-arguments have been raised [START_REF] Rizkalla | Health implications of fructose consumption: A review of recent data[END_REF], and the results obtained in the minipig model feed the controversy. Obesogenic diets altered fat accretion and metabolic profiles independently of the dietary carbohydrates, but only pigs chronically fed the fructose diet developed a strong preference for it [START_REF] Ochoa | Effects of chronic intake of starch-, glucose-and fructose-containing diets on eating behaviour in adult minipigs[END_REF], Ochoa et al., 2016b).

After seven months of diet, all groups presented a similar weight gain and insulin sensitivity index, and compared to starch, chronic exposure to fructose and glucose induced bilateral activations in several reward-related brain regions including prefrontal and striatal areas (Figure 1, diet-induced obesity), with no significant differences between fructose and glucose groups (Table 1, Ochoa et al., 2016a).

Consistent to the dynamic vulnerability model [START_REF] Stice | The contribution of brain reward circuits to the obesity epidemic[END_REF], it is plausible that our minipigs did not develop a blunted reward response yet, since they were food restricted for the entire experimental diet exposure and became only moderately obese. The brain metabolism differences observed here would rather be in favor of the reward surfeit theory and hypersensitization of the reward circuit, which might precede the reward deficit status, which was previously validated in the massively obese minipig model (Val-Laillet et al., 2011b).

Dynamic relationship between brain activity, body weight and diet changes

Amongst the hot questions on the relationship between excessive consumption of palatable foods and the emergence of brain dysfunctions, is the onset dynamic of these anomalies and its relationship with the body weight status. It is still unknown whether regular consumption of highly palatable foods without weight gain, or different strategies to lose weight, can also influence neural networks and related A c c e p t e d m a n u s c r i p t behavioral functions. Gautier et al. (unpublished data) aimed at following the neurocognitive processes in a cohort of adult minipigs, from a normal-weight status when exposed daily to high-fat-sugar (HFS) diet rationed not to exceed the metabolic needs, to the onset of obesity when ad libitum food intake was permitted, and finally to a third stage when animals were subjected either to a restrictive diet or bariatric surgery to make them lose weight. This is the first study describing longitudinally the impact of diet switch and/or body weight variations on metabolic and neurocognitive processes, using two in vivo brain-imaging modalities (PET and fMRI) in the same minipigs. Our preliminary and yet unpublished data indicated that brain responses to sucrose were dependent on both the metabolic/nutritional status and body weight, and that significant variations of brain activity were detected in the dorsal striatum, prefrontal cortex and insular cortex. Complete results are still pending, but we hope to identify brain-imaging markers that might predict weight gain or weight loss after nutritional interventions, in combination with metabolic markers (e.g. hormonal, transcriptomics, metabolomics, and metagenomics) to hypothesize the potential underlying microbiota-gut-brain mechanisms influencing eating behavior and disorders.

Perinatal nutrition and the gut-brain axis development

The Developmental Origins of Health and Diseases

The concept of the Developmental Origins of Health and Diseases (DOHaD), initially described by David Barker [START_REF] Barker | The malnourished baby and infant[END_REF], postulates that early nutritional environment can modify the progeny's metabolism, physiology and behavior, and influence its susceptibility to declare further pathologies. In the human, it is admitted A c c e p t e d m a n u s c r i p t that maternal obesity increases the prevalence of obesity and metabolic syndrome in the progeny [START_REF] Mcguire | Maternal obesity: consequences for children, challenges for clinicians and carers[END_REF]. The effects of high-calorie and high-lipids diets on glucose metabolism, energy balance, cardiovascular function and adiposity of the progeny have been demonstrated in rodents, pigs and non-human primates [START_REF] Albuquerque | Intake of trans fatty acid-rich hydrogenated fat during pregnancy and lactation inhibits the hypophagic effect of central insulin in the adult offspring[END_REF][START_REF] Williams | Animal models of in utero exposure to a high fat diet: a review[END_REF][START_REF] Wang | Effects of Inulin Supplementation in Low-or High-Fat Diets on Reproductive Performance of Sows and Antioxidant Defence Capacity in Sows and Offspring[END_REF], similarly to the susceptibility to declare cognitive, emotional, psychiatric, and behavioral disorders [START_REF] Bale | Early life programming and neurodevelopmental disorders[END_REF]. Maternal obesity and/or a plethoric high-fat diet during gestation and lactation also induce brain anomalies, with the possible onset of neuroinflammation, decreased spatial memory, neurogenesis and brain plasticity alterations (e.g. in the hippocampus and reward circuit). The microbiota represents a major actor of the gut-brain communication [START_REF] Bienenstock | Microbiota and the gut-brain axis[END_REF][START_REF] Carabotti | The gut-brain axis: interactions between enteric microbiota, central and enteric nervous systems[END_REF]. In mice, maternal consumption of a high-fat diet during gestation and lactation impacts the intestinal immunity and microbiota of the progeny [START_REF] Myles | Parental dietary fat intake alters offspring microbiome and immunity[END_REF], and a direct relationship exists between excessive food intake before weaning, the microbiota composition, and body adiposity accretion [START_REF] Sefcikova | Developmental changes in gut microbiota and enzyme activity predict obesity risk in rats arising from reduced nests[END_REF].

Early nutritional programming of neurocognitive functions in the minipig

The pig provides a great opportunity to investigate the consequences of nutritional programming for the gastrointestinal tract [START_REF] Guilloteau | Nutritional programming of gastrointestinal tract development. Is the pig a good model for man?[END_REF] and neurodevelopment [START_REF] Mudd | Early-Life Nutrition and Neurodevelopment: Use of the Piglet as a Translational Model[END_REF]. Recent studies focused on the impact of perinatal nutrition on brain functions in pigs, showing for instance that perinatal choline deficiency and maternal dietary choline both influence brain development in young pigs [START_REF] Mudd | Perinatal choline deficiency delays brain development and alters metabolite concentrations in the young pig[END_REF][START_REF] Mudd | Maternal Dietary Choline Status Influences Brain Gray and White Matter Development in Young Pigs[END_REF]. Though, most of the translational research on perinatal programming of obesity and associated diseases has been To contribute to fill this gap, we performed an experiment in the Yucatan minipig model and constituted two groups of sows fed either a standard or HFS diet during the last third of gestation and all the lactation period. Maternal daily food rations were calculated to prevent obesity and body weight differences between control and HFS groups (contrary to most studies in rodents). All the piglets from both groups were fed a standard diet after weaning. The HFS maternal diet increased plasma levels of triglycerides and free fatty acids in weaned piglets, but more interestingly, it significantly decreased the fecal amount of short chain fatty acids (SCFA) 100 days after birth, even though all animals were fed a standard diet after weaning [START_REF] Val-Laillet | A maternal Western diet during gestation and lactation modifies offspring's microbiota activity, A c c e p t e d m a n u s c r i p t blood lipid levels, cognitive responses, and hippocampal neurogenesis in Yucatan pigs[END_REF]. This indicates a decreased fermentation activity of the intestinal microbiota, already detected in HFS sows. An alternative hypothesis would be an increased intestinal absorption/use of SCFA, and further investigations in terms of intestinal metabolism or metagenomic sequencing are required to disentangle this question. In the holeboard test (spatial learning task), where the animals have to learn and remember the location of food rewards hidden in a complex environment, Yucatan piglets born from HFS sows had increased reference and working memory scores compared to control [START_REF] Val-Laillet | A maternal Western diet during gestation and lactation modifies offspring's microbiota activity, A c c e p t e d m a n u s c r i p t blood lipid levels, cognitive responses, and hippocampal neurogenesis in Yucatan pigs[END_REF], which corroborated previous data in conventional piglets [START_REF] Clouard | Prenatal, but not early postnatal, exposure to a Western diet improves spatial memory of pigs later in life and is paired with changes in maternal prepartum blood lipid levels[END_REF]. This might be explained by increased cognitive abilities and/or an increased motivation to perform/complete the cognitive task since it was reinforced by highly palatable food rewards (chocolatesugar-coated peanuts). Any change in the motivation to obtain these food rewards can influence the animals' memory scores. This is where the exploration of brain functions and activity is fundamental to interpret such ambivalent results.

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After the post-weaning behavioral tests, some piglets were euthanized to explore their brain plasticity and neuro-inflammation in the hippocampus and prefrontal cortex. The hippocampus is involved in spatial learning while the prefrontal cortex has a major role in hedonic valuation of food and goal-oriented behavior.

Interestingly, the piglets born from HFS mothers had a smaller hippocampal granular cell layer and a decreased neurogenesis [START_REF] Val-Laillet | A maternal Western diet during gestation and lactation modifies offspring's microbiota activity, A c c e p t e d m a n u s c r i p t blood lipid levels, cognitive responses, and hippocampal neurogenesis in Yucatan pigs[END_REF]. An increased microglial cell density was also observed in the prefrontal cortex, and the activation of microglia was increased in the hippocampus and prefrontal cortex (unpublished data). These observations rather praise in favor of decreased cognitive abilities and a possible alteration of the prefrontal processes involved in food valuation and motivation, due to neuro-inflammation processes.

Long-term impact of early nutritional programming

Some piglets were kept alive until the adult age to perform new behavioral tests, metabolic measures and brain exploration with in vivo imaging techniques. The differences observed in the young age for the holeboard test were not found again, but during the maze test (another spatial cognitive test that is not reinforced by food rewards, contrary to the holeboard test), minipigs born from HFS mothers presented more stress and reduced cognitive abilities (Table 1, [START_REF] Gautier | A maternal Western diet during gestation and lactation modifies adult offspring's cognitive and hedonic brain processes, behavior and metabolism in Yucatan minipigs[END_REF]. More interesting is the fact that these animals showed a reduced basal glucose metabolism in the prefrontal cortex and nucleus accumbens (Figure 1, maternal HFS diet), which are both parts of the brain reward circuit. We also demonstrated that these animals also presented a decreased dopamine and/or serotonin transporter availability in the striatum and hippocampus. These brain anomalies are usually observed in obese subjects, as previously demonstrated in humans (Wang et al., A c c e p t e d m a n u s c r i p t 2001, [START_REF] Le | Less activation of the left dorsolateral prefrontal cortex in response to a meal: a feature of obesity[END_REF], Volkow et al., 2008[START_REF] Volkow | Inverse association between BMI and prefrontal metabolic activity in healthy adults[END_REF] and minipigs (Val-Laillet et al., 2011b, Ochoa et al., 2016a). It is consequently striking to find them in animals that were fed a standard diet from weaning without abnormal weight gain or metabolic disease until the adult age. This suggests that HFS diet might program the brain even in the absence of weight problem and possibly modulates adaptation mechanisms to further dietary challenge. Further research is needed in this model to demonstrate that the brain and the digestive system, including gut microbiota, can influence each other with causal relationship to modulate the susceptibility to health and diseases, and notably behavioral and metabolic disorders.

Impact of nutritional preventive or therapeutic strategies on brain activity

As highlighted in the previous section, data are lacking to connect the microbiota and gut physiology to neurocognitive outcomes in the pig model. Through preventive or therapeutic interventional strategies aimed at influencing the susceptibility to health and diseases, it is possible to modulate metabolism, brain and behavior. In this last section, we will review three examples of interventions for which brain activity modulation was documented in the pig model.

Impact of food functional ingredient/supplementation on brain activity

Because many evidences indicate a crucial role for the microbiota in regulating different aspects of behavior and mental health, as well as comorbidities related to increased the quality of life in patients with inflammatory bowel disease. Interestingly, they also showed changes in brain activity via fMRI, with reduced reactivity of the limbic system. Comparison data are lacking in the pig model, but the use of maternal prebiotics can impact the progeny's growth, intestinal microbiota, morphology and immunity in pigs [START_REF] Bourgot | Maternal short-chain fructooligosaccharide supplementation influences intestinal immune system maturation in piglets[END_REF][START_REF] Bourgot | Short-chain fructooligosaccharide supplementation during gestation and lactation or after weaning differentially impacts pig growth and IgA response to influenza vaccination[END_REF][START_REF] Bourgot | Maternal short-chain fructo-oligosaccharide supplementation increases intestinal cytokine secretion, goblet cell number, butyrate concentration and Lawsonia intracellularis humoral vaccine response in weaned pigs[END_REF]. The use of dietary prebiotics early in life in piglets also supported recognition memory and modulated the concentrations of SCFA in the colon, blood, and brain, as well as hippocampal serotonin [START_REF] Fleming | Dietary polydextrose and galactooligosaccharide increase exploratory behavior, improve recognition memory, and alter neurochemistry in the young pig[END_REF]. Interestingly, we recently showed that oral supplementation in butyrate might shape brain activity and plasticity [START_REF] Val-Laillet | Oral sodium butyrate impacts brain metabolism and hippocampal neurogenesis, with limited effects on gut anatomy and function in pigs[END_REF]. Butyrate is a SCFA that is naturally produced in the gut through bacterial fermentation of dietary fibers playing an essential role in gastrointestinal functions [START_REF] Guilloteau | Nutritional programming of gastrointestinal tract development. Is the pig a good model for man?[END_REF]. This microbiota metabolite (i.e. postbiotic) has a trophic role for colonocytes, modulatory properties on gut morphology and functional plasticity, and is part of the histone deacetylase (HDAC) inhibitors having positive effects on long-term memory processes [START_REF] Guilloteau | Nutritional programming of gastrointestinal tract development. Is the pig a good model for man?[END_REF], Val-Laillet et al., 2018). For that matter, some authors suggested that butyrate could prevent memory impairment and improve cognition through the upregulation of neurotrophic factors [START_REF] Moretti | Behavioral and neurochemical effects of sodium butyrate in an animal model of mania[END_REF][START_REF] Valvassori | Sodium butyrate functions as an antidepressant and improves cognition with enhanced neurotrophic expression in models of maternal deprivation and chronic mild stress[END_REF]. To tackle this question in the pig model, we compared two groups of juvenile animals that were exposed during three weeks to either oral sodium butyrate or control treatment (Table 1, [START_REF] Val-Laillet | Oral sodium butyrate impacts brain metabolism and hippocampal neurogenesis, with limited effects on gut anatomy and function in pigs[END_REF]. We demonstrated that the butyrate oral supplementation triggered basal brain glucose metabolism changes in the hippocampus and nucleus accumbens, which are involved in cognitive and hedonic processes (Figure 1,g). Interestingly, post-mortem immunohistochemistry data revealed a higher granular cell layer volume, cell proliferation and neurogenesis with A c c e p t e d m a n u s c r i p t the butyrate treatment, supporting a positive modulatory role of this postbiotic on hippocampal plasticity and neurocognitive processes [START_REF] Val-Laillet | Oral sodium butyrate impacts brain metabolism and hippocampal neurogenesis, with limited effects on gut anatomy and function in pigs[END_REF].

Apart from pre-, pro-and postbiotics, other kinds of food functional ingredients can be used to modulate behavior and brain activity. Stressful environments/events are known to alter well-being and eating behavior, sometimes leading to appetite loss and anorexia or to food cravings and hyperphagia. This has been described in humans [START_REF] Guarda | Anorexia nervosa as a motivated behavior: Relevance of anxiety, stress, fear and learning[END_REF][START_REF] Geiker | Does stress influence sleep patterns, food intake, weight gain, abdominal obesity and weight loss interventions and vice versa?[END_REF] as in pigs (Clouard et al., 2012b).

In young pigs, we demonstrated positive effects of sensory feed functional ingredients based on natural plant extracts on eating behavior, with increased feed preference and intake after a feed transition for example (Clouard et al., 2012a, Clouard andVal-Laillet, 2014). Then, we explored the brain responses to one of these feed functional ingredients and showed that the perception of the ingredient (Figure 1,b,c) in familiarized individuals induced different responses in reward and perception brain areas compared to familiar control feed flavor in naive animals (Table 1, Val-Laillet et al., 2016). In addition to potential application in animal production to improve well-being and feed intake, translational applications might be assessed in fragile human populations suffering from appetite loss, decreased motivation to eat, and/or denutrition. These food functional ingredients might also be tested in the context of degraded/stressful environments, to assess their ability to counterbalance at the brain level the effects of stressors.

Impact of chronic vagus nerve stimulation on brain activity

Chronic vagus nerve stimulation (VNS) is used in clinical practice as a therapy for refractory epilepsy and some psychiatric disorders [START_REF] Vonck | Vagal nerve stimulation--a 15-year survey of an established treatment modality in epilepsy surgery[END_REF], but it is also A c c e p t e d m a n u s c r i p t a promising potential strategy to fight against obesity and an original methodological approach to understand how the gut-brain signals transmitted via the vagus nerve can modulate brain activity, metabolism and eating behavior. The pig model was rapidly in good position amongst candidate animal models to assess VNS against obesity (for review, see Val-Laillet et al., 2010b). The vagus nerves convey much information emanating from the gut, and notably information related to gastric distension, one of the first satiation signals. We showed in young pigs that the activity of several brain areas was found significantly associated with the increase of gastric distension pressure (Figure 1, f), such as the olfactory bulb, prefrontal cortex, and amygdala [START_REF] Layec | Chronic But Not Accute Gastric Distension Activates Brain Reward Circuit[END_REF][START_REF] Sauleau | The pig model in brain imaging and neurosurgery[END_REF]. But after bilateral vagotomy, only a limited number of brain areas still responded to gastric pressure (Table 1, [START_REF] Sauleau | The pig model in brain imaging and neurosurgery[END_REF]. A pilot study also demonstrated that chronic gastric distension (via a gastric balloon, Figure 1, f) activated the nucleus accumbens [START_REF] Layec | Chronic But Not Accute Gastric Distension Activates Brain Reward Circuit[END_REF].

With VNS, the aim is to mimic the nervous gut-brain signals emitted with stomach distension. The first pilot study investigating brain responses to VNS was performed in lean juvenile pigs (Table 1, [START_REF] Biraben | Central activation after chronic vagus nerve stimulation in pigs: contribution of functional imaging[END_REF]. At that time, nuclear functional brain imaging in the pig model was emerging, and our digital pig brain atlas [START_REF] Saikali | A three-dimensional digital segmented and deformable brain atlas of the domestic pig[END_REF] was not available, which explains why identification of activated brain structures was tricky and the interpretation very limited. Two years later, we reiterated a similar experiment in obese adult minipigs and demonstrated that chronic VNS during 14 weeks decreased feed intake, prevented the animals to gain further body weight contrary to sham animals, and modified the feed preferences with a decreased consumption of high-sucrose feed and increased consumption of standard feed (Val-Laillet et al., 2010b).

A c c e p t e d m a n u s c r i p t

With time, both VNS and brain imaging procedures were improved in the pig model, and two new studies were published (Malbert et al., 2017a, Malbert et al., 2017b). A first experiment compared three chronic VNS patterns in growing pigs (S1, S2, and S3, Table 1). Only S2 and S3 activated the dorsal vagal complex (DVC) and had an impact on the prefrontal cortex, contrary to S1 (Table 1, Malbert et al., 2017b, Figure 1, h). Only S2 and S3 significantly reduced daily ingestion, the duration of eating bouts during the meal, as well as the consumption of the high-lipids diet during the three-choice feed test, where the animals had simultaneous access to control, highlipids and high-glucose feeds provided by a three-through robotic feeder (Malbert et al., 2017b). Taken together, these results demonstrate how specific nervous signals emanating from the gut can modulate the activity of the brain network involved in food intake regulation and goal-oriented behavior. This also highlights the importance of the prefrontal cortex in modulating food motivation and preferences. Another study in obese minipigs reused the same VNS procedure as that previously successfully tested (Val-Laillet et al., 2010b) to investigate the VNS impact on metabolism and brain activity (Table 1, Malbert et al., 2017a). Mean whole body insulin sensitivity and hepatic glucose uptake rate were significantly lower in non-stimulated obese minipigs compared to lean minipigs, and contrary to stimulated obese minipigs. Stimulated animals also had increased glucose metabolism in the anterior prefrontal cortex and cingulate cortex compared to non-stimulated animals (Table 1, Malbert et al., 2017a, Figure 1, h). Once again, this study demonstrated that chronically mimicking satiation signals emanating from the gut could improve the activity of the prefrontal cortex, which is known to have inhibitory inputs toward the orexigenic brain areas and to be deactivated in obese individuals.

A c c e p t e d m a n u s c r i p t

Impact of bariatric surgery on brain activity

To conclude this section on interventional strategies, it is interesting to evoke what is still one of the most effective therapeutic compromises against morbid obesity: the Roux-en-Y gastric bypass [START_REF] Kang | Effectiveness of bariatric surgical procedures: A systematic review and network meta-analysis of randomized controlled trials[END_REF]. A recent paper reviewed the evidences derived from neuroimaging and animal (mainly rodent) studies [START_REF] Hankir | Brain Feeding Circuits after Rouxen-Y Gastric Bypass[END_REF], explaining how a metabolic surgical procedure such as the Roux-en-Y gastric bypass (RYGB), which is drastically reorganizing gut, including peptide hormones and microbiota metabolites, is also reshaping homeostatic and hedonic brain processes. Different animal models of bariatric surgery have been described [START_REF] Rao | Animal models in bariatric surgery--a review of the surgical techniques and postsurgical physiology[END_REF] and the first demonstrations of RYGB in the pig model, in our knowledge, date back in the 90's [START_REF] Frantzides | Laparoscopic gastric bypass in a porcine model[END_REF][START_REF] Potvin | Laparoscopic Roux-en-Y gastric bypass for morbid obesity: a feasibility study in pigs[END_REF]. Since then, the pig and minipig models have been widely used to describe the functional mechanisms of RYGB. Not being exhaustive, we can refer to studies having investigated the gastrointestinal histological morphology [START_REF] Gentileschi | Histologic studies of the bypassed stomach after Roux-en-Y gastric bypass in a porcine model[END_REF], long-term weight loss in super-obese minipigs [START_REF] Birck | Laparoscopic Roux-en-Y gastric bypass in super obese Gottingen minipigs[END_REF], changes in adipose tissue distribution [START_REF] Chavez-Talavera | Rouxen-Y gastric bypass increases systemic but not portal bile acid concentrations by decreasing hepatic bile acid uptake in minipigs[END_REF], beta-cell function, biliary function, and glucose metabolism [START_REF] Lindqvist | Gastric bypass improves beta-cell function and increases beta-cell mass in a porcine model[END_REF][START_REF] Sham | Evaluating the mechanisms of improved glucose homeostasis after bariatric surgery in Ossabaw miniature swine[END_REF][START_REF] Baud | Bile Diversion in Roux-en-Y Gastric Bypass Modulates Sodium-Dependent Glucose Intestinal Uptake[END_REF][START_REF] Chavez-Talavera | Rouxen-Y gastric bypass increases systemic but not portal bile acid concentrations by decreasing hepatic bile acid uptake in minipigs[END_REF], Lindqvist et al., 2017b), as well as incretins regulation (Lindqvist et al., 2017a) for example. Despite all of this work, we found no study on the consequence of RYGB (or any other bariatric surgery) on brain activity and metabolism in pigs.

Identifying a way to reproduce the gut-brain effects of RYGB without the RYGB surgery per se would free the patients from possible deleterious consequences of invasive bariatric surgery [START_REF] Lupoli | Bariatric surgery and long-term nutritional issues[END_REF]. That is why preclinical research in a relevant model such as the pig is necessary. In a longitudinal study (unpublished A c c e p t e d m a n u s c r i p t data), we aimed at phenotyping diet-induced obese Yucatan minipigs subjected either to a RYGB (cf. [START_REF] Baud | Bile Diversion in Roux-en-Y Gastric Bypass Modulates Sodium-Dependent Glucose Intestinal Uptake[END_REF] for surgery procedure), or a pair-fed restrictive dietary treatment. Peptides and hormones measurements, gastrointestinal histological morphology and immunohistochemistry, transcriptomics, metabolomics, and metagenomics analyses were performed to provide a systemic description of the intestinal (including microbiota) and metabolic phenotype. The obese minipigs were also subjected to fMRI and PET brain imaging before and three months after the surgery (RYGB or sham) to investigate the brain responses to sucrose (Figure 1,d,f). Our preliminary results (Table 1, Bergeat et al., unpublished data) are in line with human brain imaging studies showing a modulation of the mesolimbic and frontostriatal pathway after RYGB [START_REF] Ochner | Selective reduction in neural responses to high calorie foods following gastric bypass surgery[END_REF][START_REF] Scholtz | Obese patients after gastric bypass surgery have lower brain-hedonic responses to food than after gastric banding[END_REF]. To date, the minipig is the only preclinical model for which such an analogy has been described, which opens the way to further interventional studies aiming at unraveling the complex gut-brain interactions shaping eating behavior after bariatric surgery.

Critical conclusion

This overview of 10 years of nutrition research using in vivo brain imaging in the pig illustrates how relevant this preclinical model is to disentangle the respective roles of food, gut-brain signals, and metabolic status in modulating brain activity and eating behavior. The research topics exposed in this review cover the fields of food technology, digestive physiology, sensory sciences, metabolic adaptation, nutritional programming, experimental surgery, and behavioral neurosciences. Despite the intricacy of all sensory, physiological, and metabolic signals susceptible to modulate brain activity, several interventional targets were identified in these studies. Most of A c c e p t e d m a n u s c r i p t this work remains rather descriptive and only superficially brushes mechanistic hypotheses. In the future, more applied and preclinical research investigating concrete strategies to improve health and well-being, both in animals and humans, are needed. Identifying brain activity and metabolism changes is a prerequisite to validate the existence of gut-brain processes that can be used as operational leverages. But the finality is clearly to operate actions: to identify at-risk metabolic and neurocognitive phenotypes, to improve individual adaptation abilities towards challenging diets or environments, to change individual eating habits in favor of healthy behaviors.

Coming back to the pig model, it is important to evoke its limitations/advantages and the questions arising from ethical concerns. Both these considerations should help identifying the added value of animal experimentation and provide a rationale for its reasonable use when no alternative exists, and as long as none has emerged. In terms of limitations of the pig model, the main constraints lie in the fact that all brainimaging studies presented here were performed in anesthetized animals, which moderates the analogy with human neurobehavioral studies in the same thematic. In spite of that, we identified on a recurring basis some brain activity in highly integrative neural networks that are characteristic of complex cognitive processes. Of course, brain activity during general anesthesia is weakened and different from that in the awaken state, but all these studies demonstrated that the anesthetized brain can still Although the pig is a very good model for nutritional and cognitive studies, there are dimensions of eating behavior that cannot be modeled with it, such as psychological A c c e p t e d m a n u s c r i p t and cultural influences. For example, even if a pig model of anorexia nervosa can mimic metabolic and behavioral symptoms, it has nothing to do with the real disease [START_REF] Danek | Large animals as potential models of human mental and behavioral disorders[END_REF]. Similarly, the individual history leading someone to becoming obese or food addict cannot be strictly reproduced in the animal. The emotional, motivational, and hedonic dimensions of eating can be investigated in pigs, but factors influencing them in the human are much more complex than in pigs. Cognitive control of eating is a very important leverage to change individual habits [START_REF] Val-Laillet | Neuroimaging and neuromodulation approaches to study eating behavior, prevent and treat eating disorders and obesity[END_REF], but it is specific to humans does not apply to animals. What justifies the use of the pig model though, is the opportunity to investigate questions that cannot be solved by human studies only, as well as to control specific experimental conditions in order to disentangle the respective roles of many biological factors and influences. Animal studies are still legally mandatory before testing new medicines or medical treatments in the human; a precise control of the environment and diet is possible in animal models and completely illusionary in humans; induction of a disease or disorder, or long-term interventions with possibly deleterious consequences in terms of health and well-being, cannot be justified in the human and are still tolerated in animals, when there is a clear rationale and strict ethical conduct.

All these arguments praise in favor of a translational research combining studies in relevant animal models such as the pig, as well as studies in the human, with a constant and bilateral dialog between them, to achieve better public health outcomes.

  1 summarize 10 years of collective work in nutrition research using in vivo brain imaging in the pig model at the St Gilles INRA group. Specifically, the author would like to individually acknowledge Charles-Henri Malbert for his outstanding contribution to the development of nuclear brain imaging in the pig model, as well as Nicolas Coquery and Hervé Saint-Jalmes for the implementation of pig functional magnetic resonance imaging (fMRI) on the PRISM imaging platform. In addition to published references, a few unpublished data have been included in this review to illustrate the more recent questions and perspectives currently investigated by our group.

  s c r i p t performed in rodent models, whilst the use of large animals such as pigs is scarce (Gonzalez-Bulnes and Chavatte-Palmer, 2017).

  eating and metabolic disorders (van de Wouw et al., 2017), pre-and probiotics open the way to novel biotherapeutic strategies. Pinto-Sanchez et al. (2017) recently demonstrated that a probiotic supplementation reduced depression scores and A c c e p t e d m a n u s c r i p t

  integrate and interpret positive or negative food stimuli in relation to the internal state and individual experience. They open a small window on what might happen also in the awaken animals.

  s c r i p t conference. The author also insists on the fact that all the research advances presented in this review are the result of a collective work, and therefore acknowledges all the persons involved. The complete financial disclosure is provided in the Supplementary Material S1. doi:10.5061/dryad.ch4nr. Summary of 10 years of nutrition research (2008-2018) using in vivo brain imaging in the conventional pig ([Large White x Landrace] x Piétrain) and minipig models. All these studies were performed by our INRA research group on the PRISM imaging platform of Rennes, France. The approximate age of the animals at the time of brain imaging is indicated in the second column. All references were ordered chronologically. Main imaging techniques used: 18F-fluorodeoxyglucose (18FDG) positron emission tomography (PET) to map brain glucose metabolism; 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO) single-photon computed tomography (SPECT) to map cerebral blood flow; functional magnetic resonance imaging (fMRI) based on blood-oxygenlevel dependent (BOLD).

  Gastric distension: 2; Maternal high-fat-sugar (HFS) diet: 14; Diet-induced obesity: 4, 5, 9, 16; Butyrate treatment: 11; Vagus nerve stimulation: 1, 12, 13; Roux-en-Y gastric bypass (RYGB): 16, 17. An asterisk indicates a significant modulation of the brain ROI with the type of stimulation or treatment. Absence of an asterisk for a specific ROI and stimulation/treatment corresponds either to an absence of modulation or to a missing data. The two sets of ROI represented by the frontostriatal network are highlighted (darkest blue) because they were almost systematically modulated in our studies and are part of the reward circuit described in the rodents and humans.

N° Pig models Type of food-related sensory stimulation and/or dietary treatment In vivo brain imaging modalities

  At the adult age, WD progeny showed lower dopamine/serotonin transporter binding potential in hippocampus and parahippocampal cortex, as well as a trend in putamen, associated with lower basal brain activity in prefrontal cortex and nucleus accumbens compared to SD progeny Citrus odors activated prepyriform, entorhinal, orbitofrontal, and anterior cingulate cortices, dorsal striatum and hippocampus / Increased odor concentration induced higher brain activity in the reward circuit / Contrasted brain responses in stressed vs. non-stressed animals, and notably decreased hippocampal activity and neuronal density in stressed animals Figure 1. Meta-representation summarizing 10 years (2008-2018) of nutrition-related functional brain imaging data in the pig model. A) Schematic representation of a pig's brain with the main regions of interest (ROI, 1-11) explored during the studies described in Table 1. B) Schematic representation of a pig with the gut-brain axis and different levels (a-h) of sensory/nutrient stimulation or treatment. C) Synthetic table showing the brain ROI that were modulated by different types of sensory/nutrient stimulation (in green) or treatment (high-fat-sugar diet in red, possible therapies in purple). For detailed explanations, please refer to the reference numbers presented in Table 1. Visual stimulation: 15; Complex odor/flavor stimulus: 3, 7, 10, 18; Basic taste oral stimulus: 8, 15, 16; Duodenal sugar infusion: 6, 8; Portal sugar infusion: 6;

	4 9 14 Normal-weight Normal-weight Obese adult 20-	5-month exposure to standard diet 7-month exposure to a high-energy Animals that were born from	99mTc-HMPAO 18FDG PET [123I]-FP-CIT	Deactivation of the prefrontal cortex, ventral tegmental area, Compared to starch, chronic exposure to fructose and glucose	Val-Laillet et al. 2011b Ochoa et al. 2016a Gautier et al. 2018
	or obese adult month-old male adult 18-month-	(102 kcal/kg 0.75 ) or ad libitum high-fat diet enriched either with mothers fed either a standard diet	SPECT SPECT (aka	and nucleus accumbens in obese compared to lean subjects / induced bilateral brain activations in anterior and dorsolateral
	1 2 3 5 6 7 8 13 3.5-month-old 3-month-old female growing conventional pigs 3-month-old female growing conventional pigs 3.5-month-old female growing conventional pigs 2.5-year-old male Göttingen minipigs Obese adult Pitman-Moore minipigs 3-month-old female growing conventional pigs 3.5-month-old female growing conventional pigs 3.5-month-old female growing conventional pigs 18-month-old Yucatan minipigs female growing conventional pigs 17 Normal-weight and obese adult male and female Yucatan minipigs (from 1 to 2 years old) 18 Obese adult 2-year-old male and female Yucatan minipigs and female Yucatan minipigs 10 2-month-old female growing conventional pigs 11 3-month-old female growing conventional pigs 12 Normal-weight and obese adult old male and female Yucatan minipigs 15 Adult 18-month-old Yucatan minipigs 16 3.5-month-old female growing conventional pigs	Research highlights VNS triggered brain plasticity in subcortical structures of the reward circuit and activated the olfactory bulb Before vagotomy, significant brain activity associated with increase of gastric distending pressure / After vagotomy, only a limited effect of increased gastric pressure on brain activity Higher activity in corticolimbic and reward-related areas with prefered flavour / deactivation of the basal nuclei and limbic thalamic nuclei with aversive flavor m 99mTc-HMPAO SPECT 99mTc-HMPAO SPECT 99mTc-HMPAO SPECT A c c e p t e d Chronic vagus nerve stimulation (VNS) during 5 weeks Gastric distension in animals with intact vagal nerves or bilateral vagotomy Olfactogustatory stimulation during brain imaging with different flavors previously associated with positive or negative post-ingestive consequences a n u s c r i p t Western diet Prefrontal cortex and insular cortex activity negatively associated with body weight 5-week exposure to isocaloric high-energy high-fat diets enriched either with fish oil (omega 3, O3), sunflower oil (omega 6, O6), or lard (saturated fat, S) 18FDG PET / Dynamic injected CT-scan to map BBB permeability Anterior prefrontal cortex metabolism lesser for O3, intermediary for S, and higher for O6 / Decreased activity in the nucleus accumbens for O3 compared to O6 / BBB the most permeable in O3, intermediary in S, and the least permeable in O6 in cortical areas / Correlation between BBB permeability and prefrontal cortex activity Duodenal or portal glucose 20% (120 ml in 10 min) infusion after an overnight fasting 99mTc-HMPAO SPECT Both duodenal and portal glucose activated dorsolateral prefrontal cortex and primary somatosensory cortex / Duodenal glucose activated pyriform area, orbitofrontal cortex, caudate and putamen / Portal glucose activated insular cortex Olfactogustatory stimulation during brain imaging with different flavors previously associated with positive or negative post-ingestive consequences 18FDG PET Aversive or less preferred flavors deactivated prefrontal asymmetric brain responses in basal nuclei and temporal gyrus Oral (5% sucrose or artificial saliva) and/or duodenal (16% sucrose or saline, 300 ml) sucrose stimulation after an overnight fasting 99mTc-HMPAO SPECT Duodenal sucrose and combined oral/duodenal sucrose induced similar activity patterns in the putamen, ventral anterior cingulate cortex and hippocampus / Oral sucrose deactivated some brain areas in the prefrontal and insular cortices / Combined oral/duodenal sucrose activated right insular cortex and oral sucrose dissociated from caloric load induced specific activity patterns in hippocampus and parahippocampal cortex A c c e p t e d minipigs compared to sham obese and sham normal-weight 3-week exposure to water beverage with or without butyrate (110 mg/kg body weight/day) 18FDG PET Sodium butyrate compared with control treatment triggered basal brain glucose metabolism changes in nucleus accumbens and hippocampus, increased hippocampal granular cell layer volume and neurogenesis A c c e p t e d Gustatory stimulation with sucrose (5% or 16%) in the same animals at three stages: normal-weight, after 3-month of ad libitum exposure to Western diet, and 3 months after gastric bypass or restrictive diet 18FDG PET / fMRI Variations of brain activity are observed depending on the weight/metabolic status of the animals in the dorsal striatum, prefrontal cortex and insular cortex Gustatory stimulation with sucrose (5% or 16%) after an overnight fasting in obese minipigs 3 months after gastric bypass (GB) or restrictive diet (sham, pair-feeding design) fMRI Compared to sham, GB animals had higher brain activity in orbitofrontal cortex, anterior cingulate cortex, caudate nucleus and hippocampus / Compared to GB, sham animals had higher brain activity in insular cortex, somatosensory cortex and putamen A c c e p t e d cortex, activated posterior cingulate cortex, and induced m a n u s c r i p t starch, glucose or fructose -all diets were isocaloric (1 kg/day) prefrontal cortex, orbitofrontal cortex, anterior cingulate cortex, caudate and putamen Olfactogustatory stimulation with food flavor with or without functional sensory additive in animals that were habituated or not to this additive since weaning 18FDG PET Familiar feed flavors activated the prefrontal cortex / Amygdala, insular cortex, and prepyriform area were only activated in familiarized animals exposed to the feed flavor with additive / Perception of feed flavor with additive in the familiarized animals activated the dorsal striatum differently than perception of control feed flavor in naive animals 15-day chronic abdominal vagal electrical stimulation with three different procedures: a long-lasting 1000-µs pulse (S1), a series of constant-amplitude pulsons lasting 1000 µs (S2), and a series of pulsons with rising amplitude also lasting 1000 µs (S3) 18FDG PET Both S2 and S3 activated the dorsal vagal complex and increased the metabolism of different afferent cortical structures, notably the insular, prefrontal, and cingulate cortices 12-week chronic abdominal vagal electrical stimulation in obese 18FDG PET Vagal stimulation was associated with increased glucose metabolism in the cingulate and prefrontal cortices m a n u s c r i p t (SD) or a Western diet (WD) during gestation and lactation DaTscan) to map brain availability of presynaptic dopamine (and serotonin) transporter / 18FDG PET Gustatory stimulation with artificial saliva, quinine (10 mM) or sucrose (5%) after an overnight fasting fMRI Quinine and sucrose stimulation promoted different cerebral activation patterns / Insular cortex was activated with both sucrose and quinine, although some other regions were specifically activated by sucrose or quinine Gustatory stimulation with unknown odors (citrus) at low or high (10x higher) concentration in pigs with or without psychosocial stress (social isolation, environmental impoverishment, and stressful visual and auditory stimuli) fMRI / MRS (neuronal density) m a n u s c r i p t	References Biraben et al. 2008 Sauleau et al. 2009 Gaultier et al. 2011 Val-Laillet et al. 2011a, 2013 (conference proceedings) Boubaker et al. 2012 Clouard et al. 2014 Val-Laillet et al. 2018 Gautier et al. (unpublished data) Bergeat / Coquery et al. (unpublished data) Clouard et al. 2012c Val-Laillet et al. 2016 Malbert et al. 2017b Malbert et al. 2017a Coquery et al. 2018 Coquery et al. (in revision) / Menneson et al. (in revision)
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Acknowledgments

The author would like to acknowledge the scientific committee of the 14 th International Symposium of Digestive Physiology of Pigs, and especially Eugeni Roura, for having invited him to write this review and present his work at the

Declaration of interest

The author declares no conflict of interest.

Ethics committee

The experiments performed by our group and presented in this review paper were conducted in accordance with the current ethical standards of the European

Software and data repository resources

Most of the data presented in this review paper were not deposited in an official repository, except brain imaging data from two articles, which are available from the DRYAD public repository (http://datadryad.org/). For [START_REF] Val-Laillet | Familiarity to a Feed Additive Modulates Its Effects on Brain Responses in Reward and Memory Regions in the Pig Model[END_REF], please refer to doi.org/10.5061/dryad.ck3g3. For Ochoa et al. (2016a), please refer to


Dali Ma
email: dali.ma@ens-lyon.fr

François Leulier
email: francois.leulier@ens-lyon.fr

A new transkingdom dimension to NO signaling

Bacterial-derived metabolites profoundly influence the host's cellular and organismal physiology. Seth et al. reports that via interspecies S-nitrosylation, microbiota-derived nitric oxide directly alters the host's Argonaute family protein activity, and consequently impinges on the overall post-transcriptional gene silencing program through the microRNA (miRNA) machinery.

Text

At the end of his life, Alfred Nobel considered it the "irony of fate" that for angina treatment, the doctors handed him a prescription of nitroglycerine, the very explosive compound that brought Nobel his vast fortune. Fast forward a hundred years, Nobel might delight in another irony that part of his extraordinary largess would be awarded to three scientists for their "discoveries concerning nitric oxide as a signaling molecule in the cardiovascular system". We now know that nitroglycerine releases nitric oxide (NO) into to the blood stream, and this diatomic, freely diffusible gas acts as a potent vasodilator, a neurotransmitter, and a versatile signalling molecule that governs many fundamental cellular processes that are universal and essential for life.

In the current issue of Cell, a study of Seth et al. uncovers a new dimension to NO signaling, in that microbial-derived NO can also alter host biology through direct Snitrosylation, namely the covalent attachment of a -.NO group to the thiol moiety of a conserved cysteine residue within a protein (Figure 1). First described in 1992 [START_REF] Stamler | S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds[END_REF], S-nitrosylation is now recognized as a major post-translational modification mechanism and a principle NO signaling transduction modality. In the past few years, the reported number of S-nitrosylated proteins rapidly increased from 3,000 to 7,000 in literature. Dysregulation in the S-nitrosylation is implicated in a broad range of human pathologies [START_REF] Hess | Regulation by S-nitrosylation of protein posttranslational modification[END_REF].

Virtually all living organisms produce their own intercellular NO during the conversion of L-arginine to L-citulline, via the organisms' intrinsic nitric oxide synthase. C.elegans, a bacteriavore, however, is an exception. Lacking nitric oxide synthase in its genome, C.elegans relies exclusively on the NO derived from their closely associated microbes for relevant physiological and cellular functions. NO produced by B.subtilis, a natural bacteria food substrate and a constituent member of C.elegans microbiota, can prolong worm life [START_REF] Gusarov | Bacterial nitric oxide extends the lifespan of C. elegans[END_REF]. These observations raise the hypothesis that in worms, all NOdependent processes, including S-nitrosylation, must critically depend on bacteria provision.

Seth et al. first surveyed the S-nitrosylation profile of the proteomes derived from developing worms, and detected a clear S-nitrosylation signal in over 900 host proteins in animals grown on wild-type bacteria. However, in worms fed on bacteria mutant for NO production (Δnos in B. subtilis or ΔnarG in E. coli), the signal strength is dampened by 60%. This provides a first glimpse into how extensively a host's proteome can be modified by a single bacteria-derived metabolite. Among the 900 candidates. Seth et al. zeroed in on one candidate, the Argonaute (Ago)-related protein (ALG-1), a homologue of the human Ago2, a core component of the RNA-induced silencing complex (RISC).

Ago family proteins, along with the miRNA-RISC complex are highly conserved across the eukaryotic kingdoms. They are dedicated to orchestrate gene silencing programs via the microRNA (miRNA) machinery. Once docked onto RISC, the miRNAs guide the RISC to find their cognate mRNA. Upon the formation of miRNA and target mRNA complex, Ago2 proteins interacts with a conserved GW family protein, such as GW182, which further recruits downstream effectors to ultimately mediate posttranscriptional translation repression and mRNA decay [START_REF] Treiber | Regulation of microRNA biogenesis and its crosstalk with other cellular pathways[END_REF]. Seth et al observed that ALG-1 is S-nitrosylated in worms fed with wild-type bacteria, the signal is again decreased by 60% simply by feeding worms with ΔnarG E. coli orΔnos B. subtilis, confirming that bacteria-derived NO plays a key role to S-nitrosylate Ago.

To search for the specific S-nitrosylated cysteine residue, Seth et al employed HEK293 cells, whose endogenous Ago2 is basally S-ntirosylated, and can be further augmented by exogenous addition NO. Among the 22 cysteine residues in Ago2, Seth et al. determined that the primary S-nitrosylation site is Cys691, corresponding to Cys855 in ALG-1. Replacing this particular cysteine residue with a serine dampens the nitrosylation signal in both HEK293T cells and worms.

Depending on the cellular context, Ago2 protein undergoes extensive post-translational modifications that regulate its function and turnover, which in turn alters the activities and dynamics of its associated miRNAs [START_REF] Jee | Alteration of miRNA activity via context-specific modifications of Argonaute proteins[END_REF]. Proxyl-4 hydroxylation and ubiquitination affect Ago protein stability. Phosphorylation of Ago2 at serine S387 by the human protooncogene Akt3 can strengthen the binding between Ago2 and GW182 [START_REF] Horman | Akt-mediated phosphorylation of argonaute 2 downregulates cleavage and upregulates translational repression of MicroRNA targets[END_REF]. Interestingly, AGO2 and GW182 are reciprocally coimmunoprecipiated in cultured cells and in worms, but their binding is significantly weakened by exogenous NO treatment, or by the C691S mutation. Furthermore, in developing C. elegans that have consumed Δnos B.bacillis, greater quantity of AIN-1 remains bound to ALG-1. Therefore, S-nitrosylation at the conserved cysteine residue disrupts the protein complex formation between Ago proteins and GW182, a prerequisite for post-transcriptional mRNA silencing. Overall, S-nitrosylation on Argonaute protein via microbial provision of NO can serve as a molecular signal to relieve translational repression on a plethora of host target genes.

What are the cellular consequences of interfering with Ago2-GW182 binding by Snitrosylation? In C. elegans, ALG-1 specifically binds the 3'end of let-7 miRNA, one of first "founder" microRNAs discovered in worms [START_REF] Reinhart | The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[END_REF]. The Ago-let7 interaction is also highly evolutionarily conserved across the eukaryotic kingdom. Seth et al. utilized the well-characterized Hmga2 luciferase assay to assess let-7 activity, while modifying the S-nitrosylation status of Ago-2. In the presence of NO, S-nitroslyation on Ago2 effectively de-represses the luciferase activity. Notably, the C691S Ago2 mutant also de-represses the luciferase activity, but such derepression is refractory to NO treatment. This observation confirms that S-nitrosylation on C691S interferes Ago2 and GW182 interaction to relieve translational inhibition, but the cysteine residue itself is necessary for such binding for silencing mRNA target. Consistent with this observation, Cys691 resides in the vicinity of the GW182 interaction site of Ago2, away from the miRNA-mRNA complex binding motif. Therefore, the aberrant reporter activity mediated by the C691S mutation is unlikely to be attributed to the potential interruption of Ago-miRNA binding.

The let-7 miRNA is conserved from worms to humans. Mutations in let-7 itself, or in the let-7 target sequence of mRNAs can lead to developmental defects and poor disease prognosis [START_REF] Bussing | let-7 microRNAs in development, stem cells and cancer[END_REF]. Since S-nitrosylation on Ago potentially interferes with let7-mediated gene silencing, Seth et al searched for conspicuous developmental or physiological consequences at the organismal level. let-7 mutant worms die of vulva rupture during development, due to the failure to repress a single target: lin41, which results in vulva morphogenesis defect [START_REF] Ecsedi | The let-7 microRNA directs vulval development through a single target[END_REF]. In a lin-7 temperature sensitive mutant (n2853), consuming wild-type B. subtilis leads to ectopic increase of lin-41 mRNA and 35% animal lethality at restrictive temperature, but feeding the worms with Δnos B. subtilis can decrease lin-41 expression level and increase worm viability by 2-fold. Furthermore, worms bearing the C885S mutation in ALG-1 are lethal, but such lethality is rescued in the n2853 mutant. However, as the S-nitrosylation site is abolished in the C855S mutant, the vulva bursting phenotype can no longer be rescued by ingesting Δnos bacteria. These observations provide strong evidence that microbiota-mediated Snitrosylation on worm AGO protein can alter vulva bursting phenotype and have profound developmental consequences.

It is estimated that up to 10% circulating mammalian serum metabolites can be attributed to bacterial origin (Wikoff et al., 2009), and trans-kingdom communications between bacteria and their eukaryotic associates have long been observed from yeast to man. For example, lactate produced by S.gallinurum robustly induces the heritable expression of the [gar+] prion in Saccharomyces cerevisiae, and so that the yeast overcomes glucose repression [START_REF] Garcia | A common bacterial metabolite elicits prion-based bypass of glucose repression[END_REF]; short-chain fatty acids (SCFAs), such as acetate and propionate derived from microbial digestion of dietary fibers can activate signal transduction events downstream of a defined set of orphan G-protein coupledreceptors [START_REF] Tan | The role of short-chain fatty acids in health and disease[END_REF]. However, the molecular mechanisms explaining how these microbial-derived molecules can alter the host's biology remain largely unexplored. By narrowing down the chemical action of a particular microbial compound to a specific Snitrosylation event, on a specific cysteine residue within a host protein, Seth et al. unravels the mechanism of such interspecies communication with unprecedented precision in the host-microbe interaction field. This study unequivocally demonstrates that the resident microbiota can directly control host protein function through a small metabolite, and such modification on a single residue can profoundly alter the entire transcriptional and translational landscape, along with the developmental trajectory of a host. Apart from Argonaute, Seth et al. found >900 S-nitrosylated proteins in worms fed exclusively on wildtype bacteria. They account for almost 5% of the worm genome and comprise a treasure trove of metabolic and redox enzymes, stress-sensors, proteasome subunits and other important regulators. Future studies should be devoted to understand how microbiota-mediated S-nitrosylation impact the function of these proteins and the biological processes that implicate them. In retrospect, can the developmental defects and physiological deficits observed in different germ-free animal models be attributed to microbial S-nitrosylation? If so, how and to what extent? Moreover, in the eukaryotic animals that generate their own intracellular NO, what proportion of S-nitrosylation in the overall proteome can be traced to microbial origin? Can microbial-derived NO impose regulatory control on the host's own NO synthesis pathway? Answers to these questions will enhance our understanding how mechanistically our microbial partners shapes different aspects of our biology, and the pathologies associated with such interspecies exchange of metabolites.

Wikoff, W.R., Anfora, A.T., Liu, J., Schultz, P.G., Lesley, S.A., Peters, E.C., and Siuzdak, G. (2009). Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proc Natl Acad Sci U S A 106, 3698-3703.

Figure 1. Interspecies S-nitrosylation of host Ago protein affects worm development

As reported by Seth et al, the aerobic E.Coli and the anaerobic B. Subtilis are closely associated with their C.elegans host. Nitric oxide (NO) derived from these bacteria directly S-nitrosylates the worm Ago homologue on a conserved cysteine residue. Snitrosylation weakens the Ago-GW182 interaction, and consequently relieves the let-7 microRNA mediated inhibition on lin-41. Such S-nitrosylation event thus impacts vulva morphogenesis and worm development. By inference, other yet unknown host cellular and physiological events can also be affected by microbial S-nitrosylation (colored boxes).


-with the use of spikes enriched in Pu 239 -low uncertainties for Pu amount determination : 0.17-0.28% [START_REF] Handbook | Analytical Techniques for the Detection of Plutonium[END_REF][START_REF]International target values 2010 measurement uncertainties in safeguarding nuclear materials[END_REF] • Redox Titrimetry :

-based on potentiometric method (Waterbury&Metz [START_REF] Waterbury | [END_REF] or Mac Donald&Savage [4]) -level of uncertainty: 0.21-0.28% [START_REF]International target values 2010 measurement uncertainties in safeguarding nuclear materials[END_REF] • Coulometry -based on controlled potential coulometry [START_REF] Scott | International conference on the Peaceful Uses of Atomic Energy[END_REF][START_REF] Shults | [END_REF][START_REF] Holland | proceedings of the 3rd ESARDA Symposium[END_REF] -lowest level of uncertainty: 0.10-0.14% [START_REF]International target values 2010 measurement uncertainties in safeguarding nuclear materials[END_REF][START_REF] Holland | proceedings of the 3rd ESARDA Symposium[END_REF][START_REF] Ruas | [END_REF][START_REF]Controlled-potential coulometry assay of plutonium[END_REF] 5 Controlled potential coulometry of Pu -critical values: En ≥ 2, significant bias; En < 2, non significant bias.

 no bias observed for the series of results (5 replicates)

EQRAIN Pu Standard characterization

• Fidelity analysis:

- First approach: application of the law of the propagation of variances

Outlines• 3 [ 1 ]

 31 CETAMA: who are we? • Metrology of plutonium: what for? • Highly accurate determination of Pu content: what are the most appropriate techniques? • Controlled potential coulometry of Pu: what performance can we expect? • Implementation of coulometry: what are the difficulties? • Uncertainty evaluations: which best approach? • Perspectives of coulometry: what challenge for this technique? Presentation of CETAMA • Commission for establishment of analytical methods • CEA, Nuclear division, located in Marcoule (south of France) • Commission created in 1961 for diffusion of good practice in analytical science within the nuclear field • Main activities: -Organization of working groups for exchanging information on analytical techniques and methods (15 working groups, 380 members in Europe) -Fabrication, certification and marketing of Certified Reference Materials (CRM) -Organization and interpretation of interlaboratory comparisons -Writing of methods, draft standards and reference documents [1] • Laboratory of Metrology of Nuclear Materials in Atalante facility (Marcoule) -fabrication of CRM -development of analytical tools dedicated to actinides metrology Validation of method for ICP-AES; from sample to results, EDP sciences, (2017). Metrology of Plutonium • Control of nuclear fissile materials • Knowledge of quantity and grade of Pu matter • Guarantee of fissile material accountancy -for safety in nuclear facility as regards criticality -to prevent diversion of matter (proliferation risk) • Need of reference material -to ensure validation of analytical methods and calibration of equipment -to perform reliable an accurate plutonium content determination 4 IAEA headquarters in Vienna MP2 plutonium metal Best analytical tools for determining Pu mass content with high accuracy • ID-TIMS (Isotope Dilution-Thermoionization Mass Spectroscopy):

7 • 2 e 1 • 2 • 8 [ 1 ] 2 •

 7212812 the measurement of quantity of electricity during an electrochemical reaction -on the reversible reaction involving Pu(IV)/Pu(III) redox pair: • Faraday Law: -relates the quantity of electricity Q to the amount of Pu, m Pu , -within a range of potentials with no competing reactions • Primary measurement: -calibration against physical standards of high level of accuracy  time (s) and electric current (A) -absolute method 6 F: Faraday constant, F=96485.34 C/mol M: molar mass of Pu, M=239.076 g/mol n: exchanged electron = 1 Protocol of measurement • Phase 1: preliminary reduction to the Pu 3+ oxidation state • Phase 2: oxidation of Pu 3+ to Pu 4+ Calculation of the fraction of plutonium electrolysed, f [1] : -f = correction factor <1:  depends on E°', E 1 and E 2  calculated from the Nersnt Equation describing equilibrium after phase 1 and Pu 4+ = fraction of Pu 4+ not reduced at first stage e Pu 3+ = fraction Pu 3+ not oxidized at second stage [1] Holland, M. K., Weiss, J. R., and Pietri, Anal. Chem., 50, 236, (1978). E°'=formal potential of Pu 4+ /Pu 3+ redox pair E°' ± 230 mV Implementation of coulometry • Step 0: Dilution of sample (optional) -with dilution factor DF=m 2 /m Step 1: weighing of sample and fuming to dryness -addition of H 2 O 2 to reduce Pu(VI) to Pu(IV) [1] -addition of H 2 SO 4 to stabilize Pu(IV) in the form of crystals of Pu(SO 4 ) Step 2: blank measurement Q b -HNO 3 0.9M + sulfamic acid (to prevent nitrous acid interference) -same sequence as coulometric titration E 1 (reduction), E 2 (oxidation) -measurement of total charge Q 1 , sum of the charge attributed to double layer capacitive current Q c and to reversible reactions in the electrolyte Q f (residual current i r1 ) -Expression of net blank charge Q b : ASTM C 1108-99, "Standard Test Method for Plutonium by Controlled-Potential Coulometry", ASTM international, West Conshohocken, PA, published February 1999. Coulometric titration of Pu • Step 3 : Pu measurement -dissolution of Pu(SO 4 ) 2 with blank -reduction/oxidation sequence: E 1 , E 2 -measurement of Q 2 during oxidation step, residual current i r2 and duration t Expression of results : -net charge Qs of Pu titration :-effective charge Q after correction of the blank



  mathematical expression of Pu concentration in g/kg:  hypothesis: uncertainties on M Pu , F and f neglected. Second approach: Monte Carlo Method (MCM)  calculation based on propagation of probability distribution  pertinent when [1]: • the measurand mathematical model is complicated (non linear expressions) • the uncertainties of the input are not of the same order of magnitude  appropriate to estimate uncertainties for f and m Pu or [Pu];  sampling of a great number N of values of input quantities at random and according to their probability distribution (N10 6 ) (JMP®13 SAS institute Inc.) 13 [1] M. Crozet, C. Rivier, Journal of Radioanalytical and Nuclear Chemistry 302, 103-115 coulometry = accurate method for dertimining Pu amount in solution  performance of Coulometry at LAMMAN in accordance with ITV (u(r)=0.1% to be compared with u r ([Pu])=0.045-0.047%) Conclusion and perspectives • Coulometry -robust technique for determining Pu amount -with high accuracy -well adapted for metrology laboratories working on actinides • Challenge for our lab -coulometric titration of Pu in presence of uranium -coulometric titration of uranium -upgrading of our equipment

s [Pu] (%)

	• Trueness analysis:
	LF04	6.2932	6.2952	+0.03	0.064
	LF 05	6.2832	6.2785	-0.08
	LF 06	6.3168	6.3191	+0.04
	LF 07	6.3011	6.2952	-0.09
	LF 08	6.2983	6.2936	-0.07

-expression of normalized deviation E n : 11 X mean , concentration mean value of experimental replicates; X ref , concentration reference value; u (exp), standard uncertainty on experimental concentration; u (ref), standard uncertainty on the reference concentration.


Damien Féron

Valérie L'hostis

Stéphane Sarrade

P T Anastas

J C Warner

Green workshop "The perspectives on sustainable building and products -challenges and opportunities. Anticorrosion in sustainable building industry" EUROCORR 2018 Wednesday 12, 2018 -KRAKOW, POLAND

Illustration -Corrosion inhibitors (Directive REACH)

Chromium is a surprisingly common ingredient in anticorrosion coatings such as the paint pretreatment being sprayed on the airplane fuselage, but chromium ions are a serious health threat when inhaled or ingested Solutions:

-Large developments for new and « green » inhibitors silane coatings for aerospace (?)

| PAGE 8 ''Green Workshop'', Eurocorr 2018

  of corrosion is 3-4% of the world's Gross Domestic Product One quarter of the steel annual production is destroyed by corrosion Source: World Corrosion Organization (granted NGO by the United Nations) Illustrations from the web Atmospheric corrosion in a chemical plant Corrosion cracking of the fuselage of airplane (April 28, 1988 -1 fatal over 95) Pitting corrosion of an oil tank Mississippi Bridge, Minneapolis, August 1, 2007, 13 fatal structural weakness caused by corrosion ERIKA (1999) "result of structural weakness caused by corrosion" 10. Design for innocuous degradation 11. Pollution prevention 12. Safer for accident prevention Principle 1… Prevention: it is better to prevent waste/pollution than to treat and clean up after it has been done Illustration -Pollution linked to the copper roofs In many cities, copper roofs are used since centuries, but with the recent requirements regarding the polluted waters, raining waters on these roofs are becoming too concentrated in metallic cations … Solutions : -More resistant alloys -Pre-oxidized copper surfaces C. Leygraf & al., Atmospheric corrosion, ECS series, 2000 | PAGE 5 ''Green Workshop'', Eurocorr 2018 Principle 2… Atoms economy: economy of "raw materials" Illustration -extension of exploitation time of industrial equipements Initial nuclear power plants were planned for 30 years, new ones are planned for 60 years of operation and the exploitation of some old ones have been extended to 60 years or more, one reason being a very good corrosion resistance of the alloys. Atoms economy: 7 000 tonnes for the confinement building, 550 tonnes for the steam generator, 330 à 510 tonnes for the vessel, …. Beznau nuclear power plant (1969, 365 Mwe, initially planned for 40 years, the oldest nuclear power plant in operation today | PAGE 6 ''Green Workshop'', Eurocorr 2018 Principle 3… Less hazardous conditions: working and living in safer conditions with less hazardous chemicals Illustration -the control of corrosion is needed for safe operation of plants and civil structures Actions:-In France 302 events are linked to corrosion and are analyzed on a governmental web site with the objective to inform and prevent https://www.aria.developpement-durable.gouv.fr/wp-content/files_mf/SY_corrosion_JFM_FR_20052014.Chemical products should be designed to affect their design function while minimizing their toxicity

Principle 5…

Benin solvents: The use of auxiliary substances (e.g., solvents) should be made unnecessary wherever possible and, innocuous when used.

Illustration -protective coatings for anticorrosive purposes

Principle 6…

Design for energy efficiency

Illustration -Efficiency of electricity production

Thermal electricity generation process is limited by the Carnot efficiency = (T source -T sink ) / T source . High temperatures of T source lead to a better efficiency, but corrosion is thermally activated.

Challenges:

-Thermally resistant alloys -High temperature corrosion

Principle 7…

Use of renewable feedstocks: A raw material or feedstock should be renewable rather than depleting whenever technically and economically practicable.

Illustration -galvanizing

• Galvanization is the process of coating iron and steel with zinc is widely used in applications where corrosion resistance is needed.

Principle 10…

Design for innocuous degradation: design a product including for its final degradation.

Illustration -recycling metals and alloys

Evolution

Illustration -corrosion monitoring

Metallic cations are pollutants for natural environments Corrosion Monitoring is a process that evaluates and monitors equipment components, structures, process units, and facilities for signs of corrosion.