Jorge Mendoza
email: jmendoza@inci-cnrs.unistra.fr

Eating Rewards the Gears of the Clock

Keywords: circadian, eating, hedonic, metabolic, suprachiasmatic, clock genes

Eating behavior is regulated by metabolic and hedonic brain networks which interact each other to balance the physiological regulation of hunger and satiety. The daily balance this regulation is controlled by the central circadian clock. Importantly, metabolic and reward properties of food impact the functioning of circadian clocks, altering the oscillatory activity of the molecular clockwork and circadian rhythms. However, when feeding (metabolic or reward) is timed the whole circadian system is entrained. Furthermore, besides synchronizing the clock, the timing of both metabolic and reward eating might be crucial for health: to both improve circadian physiology as well as to treat metabolic (diabetes, obesity) and neurological diseases (mental, neurodegenerative).

Food intake is a complex behavior that involves the participation of neural substrates that function, in an interactive and balanced manner, in conjunction with peripheral physiology in various organs that indicate the energy state of the organism, in both hunger and satiety.

The classical mechanisms of the regulation of food intake involve two principal systems in the brain [START_REF] Rossi | Overlapping Brain Circuits for Homeostatic and Hedonic Feeding[END_REF]: the first, a metabolic system that regulates the amount of food necessary for the survival of the organism, and a hedonic or pleasure mechanism that regulates the quality and type of food selected and ingested [START_REF] Rossi | Overlapping Brain Circuits for Homeostatic and Hedonic Feeding[END_REF]. While the metabolic mechanisms are mainly regulated by the hypothalamus and brain stem, the hedonic processes are principally, but not uniquely, regulated by the dopamine (DA) system. Anatomically and functionally these centers in the brain are linked, and this gives a balanced regulation between the quantity and quality of what has to be eaten. In addition, food-related hormones (e.g., insulin, leptin and ghrelin) have the ability to modulate the cellular activity of brain nuclei of both systems due to the presence of specific receptors. Therefore, the physiological regulation of food intake depends on the homeostatic interaction between the brain and the periphery. When this homeostasis is lost, metabolic pathologies appear.

Since physiology is clock-controlled, food intake must be regulated in time. Therefore, centrally the circadian clock coordinates the time of metabolic and hedonic food intake, influencing the quality and quantity of food to be taken at a specific time of the day [START_REF] Mendoza | Food intake and addictive-like eating behaviors: Time to think about the circadian clock(s)[END_REF]. On the other hand, the timing of food intake can either positively (entrainment) or negatively (misalignment) affect the circadian system. Furthermore, these effects may be even more evident when particular types of diets are eaten (e.g., high fat, hypocaloric, palatable diets).

Time Eating Behavior: paced by the clock(s)

Food intake is a robust daily rhythm accompanying increased locomotor activity around the event. Mammalian diurnal species, including humans, distribute food intake across the day or light phase of the light-dark (LD) cycle imposed by the solar time [START_REF] De Castro | Circadian rhythms of the spontaneous meal pattern, macronutrient intake, and mood of humans[END_REF][START_REF] Mendoza | Setting the main circadian clock of a diurnal mammal by hypocaloric feeding[END_REF]. While nocturnal rodents, eat higher amounts of food in the early night with a second peak of feeding in the late night [START_REF] Possidente | Circadian rhythms for food and water consumption in the mouse, Mus musculus[END_REF]. Essentially, daily variations of eating patterns are synchronized to the LD cycle through the suprachiasmatic (SCN) circadian pacemaker (and their molecular clockwork; Box 1) which receives direct photic information from the retina (Box 2), and transmits this to brain structures that regulate feeding [START_REF] Mendoza | Food intake and addictive-like eating behaviors: Time to think about the circadian clock(s)[END_REF][START_REF] Patton | The suprachiasmatic nucleus[END_REF] (Figure 1).

The SCN projects to the metabolic hypothalamus via the subparaventricular zone which then distributes timing information to the paraventricular (PVN), dorsomedial (DMH) and lateral hypothalamus (LH) [START_REF] Vujovic | Projections from the subparaventricular zone define four channels of output from the circadian timing system[END_REF] (Figure 1). Cells in the LH express the neuropeptides orexins (ORX) which are involved in locomotion and feeding behaviors and show daily (synchronized to the 24h LD cycle) and circadian (self-sustained under constant light conditions) expression at both the gene and protein level. ORX release in nocturnal rodents peaks at the beginning of the night, the time at which the animals are awake and begin feeding [START_REF] Deboer | Convergence of circadian and sleep regulatory mechanisms on hypocretin-1[END_REF][START_REF] Feillet | Rev-erbalpha modulates the hypothalamic orexinergic system to influence pleasurable feeding behaviour in mice[END_REF]. Moreover, ORX have also been implicated in arousal and reward related to feeding [START_REF] Sakurai | The role of orexin in motivated behaviours[END_REF]. Thus, the SCN communicates to the LH-ORX neurons to initiate rhythms of locomotion and metabolichedonic eating.

The SCN also provides timing information to brain regions regulating the hedonic drive of eating (Figure 1). In the midbrain the DAergic ventral tegmental area (DA-VTA) projects to the nucleus accumbens (NAcc) in the ventral striatum for the regulation of motivated behaviors [START_REF] Morales | Ventral tegmental area: cellular heterogeneity, connectivity and behaviour[END_REF][START_REF] Volkow | The dopamine motive system: implications for drug and food addiction[END_REF]. In the NAcc medium spiny GABAergic neurons contain DA receptors (D1 and D2 type) which are activated when eating, and this response is still higher if a palatable diet is ingested, leading to reinforcement of this behavior. Moreover, the DA tone in VTA cells might be modulated by metabolic factors such as ghrelin and leptin to promote and inhibit, respectively, reward eating [START_REF] Volkow | The dopamine motive system: implications for drug and food addiction[END_REF][START_REF] Liu | Regulation of the mesolimbic dopamine circuit by feeding peptides[END_REF].

DA release and content in the striatum is rhythmic, and in nocturnal rats and mice higher levels occur at night, and in the day for diurnal rodents: the time of locomotion and feeding [START_REF] Hood | Endogenous dopamine regulates the rhythm of expression of the clock protein PER2 in the rat dorsal striatum via daily activation of D2 dopamine receptors[END_REF][START_REF] Ferris | Dopamine transporters govern diurnal variation in extracellular dopamine tone[END_REF][START_REF] Itzhacki | Light rescues circadian behavior and brain dopamine abnormalities in diurnal rodents exposed to a winter-like photoperiod[END_REF]. Thus, DA rhythms are likely participating in the modulation of locomotion and feeding rhythms. Anatomically, the SCN projects to the VTA indirectly via the medial preoptic nucleus (MPON) of the hypothalamus [START_REF] Luo | Circuit projection from suprachiasmatic nucleus to ventral tegmental area: a novel circadian output pathway[END_REF]; thence, VTA-DA circadian rhythms related to feeding might be induced or at least regulated by the SCN; at first, to initiate locomotion at the right time for food-seeking behaviors and secondly to modulate circadian food-reward at night [START_REF] Bainier | Circadian rhythms of hedonic drinking behavior in mice[END_REF] or day in nocturnal or diurnal species, respectively. This last point might be crucial to facilitate and promote reinforcement and conditioned learning associated to food intake.

Accordingly, the control of food intake in the brain is dependent on this balanced Metabolic-Hedonic-Circadian circuit [START_REF] Mendoza | Food intake and addictive-like eating behaviors: Time to think about the circadian clock(s)[END_REF]. However, when one of these components is out of balance food intake is altered, often leading to overconsumption and weight gain. The exposure to and intake of high caloric diets induces several changes in all these brain centers regulating feeding, including those from the circadian system.

The ad libitum intake of high caloric diets leads to alterations in circadian rhythms of behavior and on the expression of clock genes and those regulating energy balance in the hypothalamus and peripheral tissues [START_REF] Kohsaka | High-fat diet disrupts behavioral and molecular circadian rhythms in mice[END_REF]. Furthermore, the behavioral and molecular entrainment to light is affected in mice fed with a high fat diet [START_REF] Mendoza | High-fat feeding alters the clock synchronization to light[END_REF]. Taken together, these results indicate that high-fat feeding alters the functioning and light synchronization of the mammalian circadian clock.

In an experimental model in which rodents were given the choice between a high-caloric diet vs. a normocaloric diet, animals show "snacking" feeding behavior and preference for the caloric diet along the whole 24h LD cycle, disrupting thus the daily rhythm of eating and leading in rapid gain weight [START_REF] Blancas-Velazquez | Effects of a free-choice high-fat highsugar diet on brain PER2 and BMAL1 protein expression in mice[END_REF][START_REF] La Fleur | The snacking rat as model of human obesity: effects of a free-choice highfat high-sugar diet on meal patterns[END_REF]. Thus, beyond the type of diet, the loss of daily rhythms of feeding becomes an important factor in inducing obesity and metabolic diseases.

Centrally, in animals exposed to the protocol of diet choice, the day-night expression of the clock proteins PER2 and BMAL1 in the mouse lateral habenula (LHb) is disrupted by the intake of the caloric diet [START_REF] Blancas-Velazquez | Effects of a free-choice high-fat highsugar diet on brain PER2 and BMAL1 protein expression in mice[END_REF]. The LHb is an epithalamic nucleus which is relevant for the control of monoamines and the regulation of palatable food intake [START_REF] Mizumori | The Lateral Habenula and Adaptive Behaviors[END_REF][START_REF] Stamatakis | Lateral Hypothalamic Area Glutamatergic Neurons and Their Projections to the Lateral Habenula Regulate Feeding and Reward[END_REF]. Interestingly, sleep deprivation, a condition that increases caloric intake and body weight gain [START_REF] Dashti | Short sleep duration and dietary intake: epidemiologic evidence, mechanisms, and health implications[END_REF], disrupts clock gene expression in the LHb [START_REF] Zhang | Sleep Deprivation Influences Circadian Gene Expression in the Lateral Habenula[END_REF], while a perturbation of the LHb clock by high caloric diets might lead to disruption of the sleep-wake cycle. This highlights a specific circadian role of the LHb in rhythmic caloric intake, likely via its control on DAergic hedonic centers [START_REF] Mendoza | Circadian neurons in the lateral habenula: Clocking motivated behaviors[END_REF].

Accordingly, it was recently reported that rhythms of DA release in the SCN are disrupted in an animal model of obesity [START_REF] Luo | Circadian peak dopaminergic activity response at the biological clock pacemaker (suprachiasmatic nucleus) area mediates the metabolic responsiveness to a highfat diet[END_REF]. Therefore, DA rhythms in the SCN and the rest of the forebrain could be necessary to maintain a healthy metabolic condition and proper food intake.

Time Eating Behavior: pacing clock(s)

Time-Restricted Eating, a good or bad clock aligner? A matter of time

Time-restricted eating (TRE) induces an increase in the locomotor activity pattern in anticipation to meal access and the synchronization of physiological rhythms and metabolic activity, indicating that feeding time is a strong synchronizer for circadian rhythms [START_REF] Patton | Circadian adaptations to meal timing: neuroendocrine mechanisms[END_REF][START_REF] Mendoza | Circadian clocks: setting time by food[END_REF] (Figure 2A). Nevertheless, the SCN (clock gene expression) remains entrained to the LD cycle, thus TRE uncouples rhythms of clock genes in peripheral [START_REF] Damiola | Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus[END_REF] (liver, lung) (Box 3) and extra-SCN [START_REF] Verwey | Food-entrainable circadian oscillators in the brain[END_REF] (hypothalamic and limbic) oscillators (Box 4) from the main SCN pacemaker.

However, the uncoupling of daily feeding rhythms from the LD cycle can lead to negative metabolic consequences. Actually, in laboratory animals, mainly mice and rats, TRE has been imposed in the light phase (when nocturnal rodents sleep) which perturbs their sleep-wake cycle. Metabolically, when mice are fed with a high-fat diet only during the light phase of the LD cycle, body weight increase is significantly higher than in animals fed at the dark phase [START_REF] Arble | Circadian timing of food intake contributes to weight gain[END_REF].

Contrary, TRE at night, when nocturnal rodents are active, leads to diverse effects in metabolism and physiology, improving circadian rhythms even in clock gene mutant mice [START_REF] Chaix | Time-Restricted Feeding Prevents Obesity and Metabolic Syndrome in Mice Lacking a Circadian Clock[END_REF][START_REF] Hatori | Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet[END_REF][START_REF] Chaix | Time-restricted feeding is a preventative and therapeutic intervention against diverse nutritional challenges[END_REF]. TRE protects against obesity induced by diet, in double global Cry1-2 mutants and in mice carrying a liver-specific deletion of Bmal1 and/or Rev-Erbα/β. More importantly, TRE at night effects against obesity, hyperinsulinemia and inflammation are observed in animals fed with a high caloric diet, even if these eat the same amount of calories as that exposed ad libitum to the diet. In both clock altered mice and/or those exposed to hypercaloric diets, TRE at night modifies gene rhythms in the liver, permitting better energy utilization and expenditure, and avoiding the development of obesity or metabolic syndrome [START_REF] Chaix | Time-Restricted Feeding Prevents Obesity and Metabolic Syndrome in Mice Lacking a Circadian Clock[END_REF][START_REF] Hatori | Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet[END_REF]. Thus, TRE might have both beneficial or negative consequences in physiology; the difference is in the time of feeding aligned with the sleep-wake cycle [START_REF] Arble | Circadian timing of food intake contributes to weight gain[END_REF] (Figure 2A). What are the signals by which TRE entrains peripheral clocks? Body temperature, hormones and peptides related to feeding are important candidate signals for the entrainment of peripheral oscillators, and even circadian clocks in extra-SCN brain sites [START_REF] Schibler | Clock-Talk: Interactions between Central and Peripheral Circadian Oscillators in Mammals[END_REF]. Recently, it was reported that oxyntomodulin (OXM) is the principal signal, induced by feeding, that entrains the circadian clock in the liver through activation of Per1 [START_REF] Landgraf | Oxyntomodulin regulates resetting of the liver circadian clock by food[END_REF]. Importantly, the effect of OXM on the liver is clock-dependent. When mice are treated with OXM at day the liver clock shifts, but not when OXM is given at night [START_REF] Landgraf | Oxyntomodulin regulates resetting of the liver circadian clock by food[END_REF]. It will be interesting to determine whether OXM can entrain other peripheral oscillators, and to identify new peripheral or central signals that specifically entrain peripheral and central oscillators by feeding time (Figure 2A).

Time-restricted eating for the master clock: a matter of time and calories

Circadian rhythms of locomotion, body temperature, hormonal release (all of them SCNoutputs), and SCN clock gene expression are phase-shifted or entrained when TRE is coupled to a caloric restriction (CR) in nocturnal and diurnal rodents. This suggests that in addition to feeding time, a caloric condition promotes resetting of the SCN circadian clock [START_REF] Mendoza | Setting the main circadian clock of a diurnal mammal by hypocaloric feeding[END_REF][START_REF] Mendoza | Feeding cues alter clock gene oscillations and photic responses in the suprachiasmatic nuclei of mice exposed to a light/dark cycle[END_REF].

CR has beneficial effects in health and lifespan through the activation of the sirtuin 1 (SIRT1) and the mitochondrial oxidative metabolism [START_REF] Libert | Metabolic and neuropsychiatric effects of calorie restriction and sirtuins[END_REF]. Interestingly, at the clock level, SIRT1 regulates the circadian rhythms of peripheral tissues and central structures, having as molecular targets BMAL1 and PER2 clock components, deacetylating both proteins [START_REF] Masri | Coupling circadian rhythms of metabolism and chromatin remodelling[END_REF].

Thus, changing the cellular metabolic state of the SCN, CR can entrain the molecular clockwork (Figure 2B).

On the other hand, SIRT1 in the VMH is relevant for the control of energy balance, mainly sensing feeding and metabolic cues. The VMH projects directly to the SCN, thus CR might induce the expression of SIRT1 in the VMH, and then, indirectly, alter the molecular timekeeping in the SCN [START_REF] Orozco-Solis | SIRT1 Relays Nutritional Inputs to the Circadian Clock Through the Sf1 Neurons of the Ventromedial Hypothalamus[END_REF]. However, as the SCN clock receives direct projections from other hypothalamic and non-hypothalamic structures related feeding, it is highly likely that entrainment of the SCN by feeding is dependent on several inputs from the whole brain.

Time-palatable eating: Rewarding the clock

Food intake induces the activation of the regions of the hedonic reward system. The intake of palatable diets triggers the release of DA from the VTA in the midbrain to the ventral NAcc [START_REF] Volkow | The dopamine motive system: implications for drug and food addiction[END_REF]. Moreover, DA release might also target other brain sites containing DA receptors such as the prefrontal cortex, the striatum and, importantly, the SCN [START_REF] Grippo | Direct Midbrain Dopamine Input to the Suprachiasmatic Nucleus Accelerates Circadian Entrainment[END_REF]. The VTA projects a functional pathway directly to the SCN, targeting D1-type receptors in clock cells to facilitate the re-entrainment of behavioral rhythms to a new LD cycle [START_REF] Grippo | Direct Midbrain Dopamine Input to the Suprachiasmatic Nucleus Accelerates Circadian Entrainment[END_REF]. Activation of DA receptors leads to intracellular changes in the mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB), and finally an increase in the expression of Per genes [START_REF] Akashi | Involvement of the MAP kinase cascade in resetting of the mammalian circadian clock[END_REF]. Then, DA release by feeding time can induce Per gene expression in the SCN clock via these pathways (Figure 2B). Furthermore, when feeding is highly palatable, DA release is enhanced having a major impact in the control of behavior (e.g., learning, reinforcement), including circadian behavior (synchronization) [START_REF] Volkow | The dopamine motive system: implications for drug and food addiction[END_REF][START_REF] Mendoza | A daily palatable meal without food deprivation entrains the suprachiasmatic nucleus of rats[END_REF].

In animals fed with a daily palatable diet, without food restriction, circadian rhythms of locomotor activity (Figure 2C) and clock gene expression are synchronized to the time of feeding [START_REF] Mendoza | A daily palatable meal without food deprivation entrains the suprachiasmatic nucleus of rats[END_REF][START_REF] Mendoza | Food-reward signalling in the suprachiasmatic clock[END_REF]. Moreover, in arrhythmic Per mutant mice a daily palatable snack is able to rescue and entrain behavioral rhythms [START_REF] Flores | Period-independent novel circadian oscillators revealed by timed exercise and palatable meals[END_REF]. Altogether, the effects of daily palatable meal to entrain and rescue circadian rhythms are certainly dependent, in part, on DA activity. DA release at the level of the forebrain (e.g., striatum, NAcc, SCN) is rhythmic [START_REF] Hood | Endogenous dopamine regulates the rhythm of expression of the clock protein PER2 in the rat dorsal striatum via daily activation of D2 dopamine receptors[END_REF][START_REF] Luo | Circadian peak dopaminergic activity response at the biological clock pacemaker (suprachiasmatic nucleus) area mediates the metabolic responsiveness to a highfat diet[END_REF], and synchronized by feeding (palatable or regular diet); hence, feeding time can reach SCN clock (via D1 receptors) inducing the synchronization of molecular, behavioral and physiological rhythms (Figure 2A-C). However, the neural mechanisms of palatable food entrainment become more complex when we add other systems, substrates and molecules participating in the regulation of hedonic feeding and that can affect the SCN.

Serotonin (5-HT) from the raphe nuclei is an important internal synchronizer for the SCN since the clock contains diverse types of 5-HT receptors (5-HT1A,1B, 2A, 2B and 7). 5-HT has been implicated in the non-photic synchronization of the clock via 5-HT1A and 5-HT7 receptors, the induction of both the protein kinasa A (PKA) and K + channels, and changes in Per gene expression [START_REF] Webb | Regulation of circadian rhythms in mammals by behavioral arousal[END_REF]. Intake of meals rich in carbohydrates increases hypothalamic 5-HT release, and 5-HT neurons in the raphe nuclei show c-Fos expression in animal synchronized to daily palatable meals [START_REF] Mendoza | Food-reward signalling in the suprachiasmatic clock[END_REF][START_REF] Voigt | Serotonin controlling feeding and satiety[END_REF]. Thus, 5-HT may be involved in the food-reward entrainment of the SCN clock (Figure 2B).

ORX are able to shift molecular and electrical activity of the SCN through the activation of ORX -1 and 2 receptors, changing intracellular Ca 2+ levels and leading in a suppression of Per genes [START_REF] Belle | Acute suppressive and long-term phase modulation actions of orexin on the mammalian circadian clock[END_REF]. In the entrainment of circadian rhythms and molecular clock gene expression in the SCN by daily access to a palatable feeding, ORX cells are significantly activated (c-Fos expression) [START_REF] Mendoza | Food-reward signalling in the suprachiasmatic clock[END_REF]. In addition, a molecular disruption of the clock, which leads to compulsive intake of palatable food, upregulates ORXergic activity [START_REF] Feillet | Rev-erbalpha modulates the hypothalamic orexinergic system to influence pleasurable feeding behaviour in mice[END_REF]. Thus, ORX are another internal time cue with the ability to entrain the clock when palatable feeding is timed by changing Per gene expression (Figure 2B).

Endocannabinoids are involved in the central regulation of feeding at both levels, metabolic and hedonic [START_REF] Mahler | Endocannabinoid hedonic hotspot for sensory pleasure: anandamide in nucleus accumbens shell enhances 'liking' of a sweet reward[END_REF]. 2-arachidonoylglycerol (2-AG) and N-arachidonylethanolamine (AEA), the two principal ligands of the endocannabinoids receptors (CB1 and CB2) show circadian variations in the brain, and CB1 receptors are expressed in the SCN nuclei [START_REF] Hanlon | Circadian rhythm of circulating levels of the endocannabinoid 2arachidonoylglycerol[END_REF][START_REF] Acuna-Goycolea | Cannabinoids excite circadian clock neurons[END_REF]. CB1 receptors in the SCN presynaptically regulate GABAergic transmission; thus, cannabinoids upregulate the firing rate of SCN cells and modulate the light entrainment of the clock by a presynaptic mechanism [START_REF] Acuna-Goycolea | Cannabinoids excite circadian clock neurons[END_REF]. Therefore, during palatable feeding time, CB might impact the SCN clock as an entraining signal via the modulation of the GABAergic neurotransmission and clock gene expression (Figure 2B).

Similarly, the opiod-circuitry, implicated in food-reward related behaviors, might affect the circadian system [START_REF] Difeliceantonio | Dorsolateral neostriatum contribution to incentive salience: opioid or dopamine stimulation makes one reward cue more motivationally attractive than another[END_REF]. Motivation for the intake of palatable foods is modulated by opioid (enkephalin) action in the forebrain. µ opioid receptors are contained in the forebrain including in the SCN, which is reset by selective agonists of these receptors via the ERK1/2 and GSK3β signaling pathways [START_REF] Difeliceantonio | Dorsolateral neostriatum contribution to incentive salience: opioid or dopamine stimulation makes one reward cue more motivationally attractive than another[END_REF][START_REF] Vansteensel | The opioid fentanyl affects light input, electrical activity and Per gene expression in the hamster suprachiasmatic nuclei[END_REF]. Thus, opioid signals rising after palatable feeding might be able to time code the SCN to potentiates and induce entrainment to timed food reward.

Additionally, from the periphery the metabolic-reward hormones, leptin and ghrelin, may impact the SCN to reinforce or coordinate entrainment to feeding time. In vitro, both leptin and ghrelin reset the circadian electrical and clock gene activity in the SCN [START_REF] Prosser | Leptin phase-advances the rat suprachiasmatic circadian clock in vitro[END_REF][START_REF] Yannielli | Ghrelin effects on the circadian system of mice[END_REF], likely via direct actions on leptin and ghrelin receptors, respectively [START_REF] Hakansson | Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus[END_REF][START_REF] Zigman | Expression of ghrelin receptor mRNA in the rat and the mouse brain[END_REF]; hence, in time restricted eating, high levels of ghrelin and leptin after and before feeding, respectively, are important physiological signals for the entrainment of circadian clocks bolstering the brain metabolic and hedonic drives of feeding for SCN synchronization (Figure 2B). In summary, when the clock is entrained to food with a strong palatable and reward content, several brain circuits give timing information to the SCN. The summation and temporal coordination (before, during and after feeding) of these might lead to the entrainment of the clock and the expressed behavioral and physiological rhythms.

Circadian food entrainment in humans: physiology, behavior and gene expression.

The knowledge on the effects of food entrainment on the circadian physiology of rodents has advanced profoundly, highlighting that feeding time synchronizes behavior, physiology (hormonal rhythms) and gene expression in the brain, including the SCN and peripheral tissues.

However, there is still much to be understood on the effects of meal timing on the human circadian system and health. This becomes a subject of scientific and societal interest due to the increasing epidemic of metabolic diseases worldwide such as obesity and diabetes, but also due to eating disorders (e.g., compulsive feeding, binge eating, night eating disorder). In healthy conditions, human feeding behavior follows a daily and circadian rhythm [START_REF] De Castro | Circadian rhythms of the spontaneous meal pattern, macronutrient intake, and mood of humans[END_REF]; however, this may be disrupted under particular conditions of overeating, malnutrition or by shifting the time of feeding. Accordingly, calorie intake at different times of day differentially impacts energy balance and metabolism [START_REF] Poggiogalle | Circadian regulation of glucose, lipid, and energy metabolism in humans[END_REF]. Humans, being a diurnal species, have to distribute calorie intake mainly in the early day and reduce it at night. Unfortunately, this is not the current situation in human eating behavior. Healthy subjects without a phase shift condition (e.g., shift work, jet-lag) show aberrant eating behavior with a principal caloric intake at night and less at day [START_REF] Gill | A Smartphone App Reveals Erratic Diurnal Eating Patterns in Humans that Can Be Modulated for Health Benefits[END_REF].

Similar to what has been reported in rodents, when mealtime is out of phase with the sleep wake cycle, the human circadian system is in a state of internal misalignment between central and peripheral clocks, which could lead to pathology (e.g., obesity, diabetes) [START_REF] Vetter | Circadian Biology: Uncoupling Human Body Clocks by Food Timing[END_REF]. Previous studies have reported that eating late at night increases the risk of obesity. In addition, the risk is higher still when eating earlier in the day (breakfast) is omitted [START_REF] Mchill | Later circadian timing of food intake is associated with increased body fat[END_REF].

Certain social conditions in which people are exposed to circadian disruptions such as shift work or social jet-lag lead to an increased calorie intake at night, which is significantly correlated with obesity [START_REF] Haus | Risk of obesity in male shift workers: A chronophysiological approach[END_REF][START_REF] Roenneberg | Social jetlag and obesity[END_REF]. Calorie intake at night leads to clock gene expression changes in peripheral oscillators (adipose tissue), hormonal release (leptin, insulin) and changes in plasma glucose rhythms and glucose tolerance, which altogether may induce increased appetite and food intake [START_REF] Bandin | Meal timing affects glucose tolerance, substrate oxidation and circadianrelated variables: A randomized, crossover trial[END_REF]. In shift work conditions, the daily oscillations of the hunger hormone ghrelin are shifted with a postprandial increase which might trigger (via VTA-DAergic neurons) hedonic eating at night [START_REF] Qian | Ghrelin is impacted by the endogenous circadian system and by circadian misalignment in humans[END_REF]. Contrary to this, when feeding happens at early times of day physiological responses are different. Gastrointestinal absorption is higher during the day, thus calorie intake at day improves glucose tolerance and plasma insulin levels during fasting conditions. Moreover, feelings of hunger are reduced when calorie intake is principally in the day than at night [START_REF] Poggiogalle | Circadian regulation of glucose, lipid, and energy metabolism in humans[END_REF].

Importantly, a group of subjects with prediabetes exposed to temporal restricted eating during the day, showed improvements in insulin sensitivity, β cell responsiveness, blood pressure and appetite, indicating the beneficial effects, even in a pathological condition, of timerestricted eating early at day [START_REF] Sutton | Early Time-Restricted Feeding Improves Insulin Sensitivity, Blood Pressure, and Oxidative Stress Even without Weight Loss in Men with Prediabetes[END_REF].

Timing of caloric intake affects brain DA and 5-HT metabolism in both rodents and humans [START_REF] Volkow | The dopamine motive system: implications for drug and food addiction[END_REF]. In obese subjects, caloric intake in the early morning increases the binding of the DA (DAT) and serotonin (SERT) transporters in the ventral striatum and thalamus respectively. This effect was diminished when the subjects' caloric intake was higher at night (dinner) [START_REF] Versteeg | Timing of caloric intake during weight loss differentially affects striatal dopamine transporter and thalamic serotonin transporter binding[END_REF].

A reduction of the dopamine D2 receptors availability in the striatum has been associated with body weight increase and obesity [START_REF] Volkow | Reward, dopamine and the control of food intake: implications for obesity[END_REF]. DA cell activity shows daily variations, hence, the effects of mealtime in the day on DA metabolism might be dependent on the rhythmic properties of the DAergic system. Moreover, hormonal changes (leptin and ghrelin) by timed calorie intake at the early morning could potentiate DA metabolism and contribute to reduced food intake and body weight.

Altogether, the important data on the effects of food entrainment in the human circadian system and metabolism indicate that a timed food intake aligned with the sleep-wake cycle and other hormonal rhythms improves and maintains healthy physiology. In mealtime synchronization both central systems controlling eating (metabolic and hedonic) are importantly implicated.

Conclusions and future perspectives

In the present review some of the most important, but unfortunately not all, discoveries of the effects of feeding behavior on circadian rhythms and clocks have been highlighted, as well as how food intake affects the main clock in the SCN when the caloric content and/or the hedonic value of the food change. The neural and molecular mechanisms of food entrainment of circadian clocks are not yet fully described; however, important advances indicate that the central pathways underlying food intake (metabolic and hedonic) must be involved.

However, there are still several questions regarding the mechanisms of entrainment of peripheral oscillators and the SCN clock to food timing with or without palatable diets. For example, how do reward components of palatable diets (e.g., sucrose, fat) reach the brain and then the SCN? Are these effects clock-dependent? If so, what are the optimal times to affect the SCN clock by feeding? Are clock genes implicated in SCN food entrainment?

The timing of eating has an important clinical application in human health. Thus, new proposals in research are necessary to understand the central and peripheral (circadian and non-circadian) mechanisms that underlie the effects of meal timing in physiology, and how the principal SCN clock, which was neglected as a "food-entrainable oscillator", is entrained by the time of eating.

What is the relevance of knowing these mechanisms? If we determine how feeding time synchronizes the clock in the SCN, it would be feasible to think that "feeding-time therapy" can be used in chronobiology to treat circadian and non-circadian pathologies in which the oscillatory capacity of the clock is compromised. When the SCN clock is genetically altered, this becomes highly sensitive and responsive to time feeding-metabolic-hedonic cues, even more than to light-dark cycles [START_REF] Sheward | Entrainment to feeding but not to light: circadian phenotype of VPAC2 receptor-null mice[END_REF][START_REF] Maywood | Disruption of peripheral circadian timekeeping in a mouse model of Huntington's disease and its restoration by temporally scheduled feeding[END_REF][START_REF] Abe | Daily restricted feeding resets the circadian clock in the suprachiasmatic nucleus of CS mice[END_REF]. Thus, in circadian pathological conditions (e.g., shift-work, social jet-lag, seasonal depression) in which light has been used as treatment, feeding time might replace it as a "new medication" to rescue SCN circadian rhythms. Moreover, it will not be surprising or unreasonable to think that food entrainment can be used to treat obesity, diabetes, eating disorders [START_REF] Johnston | Circadian Rhythms, Metabolism, and Chrononutrition in Rodents and Humans[END_REF], and also other mental illnesses such as depression or neurodegenerative diseases (e.g., Parkinson, Alzheimer, Huntington disease) [START_REF] Maywood | Disruption of peripheral circadian timekeeping in a mouse model of Huntington's disease and its restoration by temporally scheduled feeding[END_REF] in which a disturbed clock has to be readjusted. Further, even more interesting is the proposition of combine light and feeding therapies to set in time circadian clocks and improve health. For this, it is first necessary to understand the putative circadian and brain mechanisms shared by light and food to potentiate and facilitate the entrainment of the circadian system. driving the metabolic and hedonic pathways of eating in the hypothalamus and mesocortico limbic system respectively. Accompanying these two pathways, the suprachiasmatic (SCN) clock, and its molecular machinery (Box 1), dictates the time of both, the need and pleasure of eating. The SCN receives direct projections from retinal ganglion cells allowing the synchronization to the light-dark cycle (LD). Thus, daily hedonic and metabolic eating is time-controlled, and synchronized to the LD cycle (Box 2), giving a robust food intake at day for diurnal species (including humans) and at night for nocturnal animals (e.g., rats, mice, hamsters). The SCN sends neural and paracrine timing signals to both hedonic and metabolic centers of food intake to impose rhythms of eating. Interestingly, spite that the functional clockwork of the SCN between diurnal and diurnal species is similar, food intake is opposite during the 24h-day, suggesting that the time of eating behavior may reside somewhere in these brain feeding centers (i.e., metabolic, hedonic). and health. However, when time of feeding is misaligned to the sleep-wake cycle, metabolic, behavioral and physiological consequences emerge (e.g., body weight gain, glucose metabolism dysregulation). TRE coupled to a hypocaloric condition, or with a strong hedonic background entrains the main SCN clock. The SCN projects to the metabolic and hedonic brain feeding centers, and to peripheral oscillators via the autonomous nervous system (ANS).

On the other way around the SCN is innervated by these structures and peripheral hormones which will affect the clock in food entrainment (B) At the SCN level, feeding time might affect the physiology and molecular properties of the clock through the release of diverse neurotransmitters and the activation of diverse receptors and the intercellular pathways In addition, peripheral hormones (e.g., ghrelin, leptin) synchronized by feeding time may act directly in their receptors localized in the SCN and other brain circadian clocks to potentiated food synchronization. (C) Representative actogram of the behavioral circadian rhythm (in constant darkness, DD) of wheel running activity of a mouse synchronized to daily palatable meal (chocolate) intake without food restriction (red vertical line). 5-HT, serotonin; 5-HT1A, serotonin 1A receptor; Ca 2+ , calcium; CaMKK, Calcium/calmodulin-dependent protein kinase; CB1, cannabinoid 1 receptor; CREB, cAMP-responsive element-binding protein; D1R, dopamine 1 receptor; DA, dopamine; δ-R, delta-opioid receptor E-box, enhancer box; ENDO, endocannabinoids; ERK, extracellular signal-regulated kinase; GABAA, GABAA receptor; GHSR, Growth hormone secretagogue receptor; GSK3, Glycogen synthase kinase-3; JAK, Janus Kinase; LepR, leptin receptors; MAPK, mitogen-activated protein kinase; μ-R, μu-opioid receptor; NPY, neuropeptide Y; NPY1R, neuropeptide Y1 receptor; ORX, orexin; OX-1R, orexin 1 receptor; OXM, oxyntomodulin; PKA, protein kinase A; SIRT1, sirtuin 1; STAT-3, signal transducer and activator of transcription.

Box 1. Molecular clockwork

The molecular timekeeping of the SCN is dependent on the clock genes Clock and Bmal1, two transcription factors which are able to dimerize and bind to the E-box of other genes, such as Period (Per1-3) and Cryptochrome (Cry1-2). PER-CRY, have the capacity to enter back to the nucleus to inhibit the activity of CLOCK-BMAL1, and thus their own transcription [START_REF] Takahashi | Molecular components of the circadian clock in mammals[END_REF].

The final result is the oscillatory activity (around the 24h) of these genes (excluding Clock) in the SCN, with peaks of activity during the light period for Per1-3 and Cry1-2 genes, and Bmal1 with a peak during the dark period. Another group of genes oscillating in a circadian manner and dependent on the CLOCK-BMAL1 dimer are the nuclear hormone receptors Rev-Erb (α/ β) and Ror (α, β, and γ). These genes induce the circadian rhythmicity of Bmal1: Rev-Erbα represses Bmal1 during the day while Rorβ activates it at night. Finally, clock-output or clock-controlled genes (CCG) from the SCN signal the rest of the brain and body to give the timing cue required, and keep circadian synchrony in the whole body; vasopressin (Avp) is the principal SCN output signal. Moreover, other SCN output signals are humoral or paracrine.

The peptides transforming growth factor alpha (TGFα), prokineticin 2 (PK2) and cardiotrophin-like cytokine (CLC) participate as temporal SCN signals for the circadian control of behavior [START_REF] Patton | The suprachiasmatic nucleus[END_REF]. The sophisticated clockwork enginery in the SCN has been also described in other tissues, such as the liver, heart, lung, kidney, pancreas and adipose tissue; thus, despite the initial identification of a single body pacemaker in the brain, now it is well known that several organs in the whole body contain a circadian clock indispensable for the rhythmic functions accorded to each tissue [START_REF] Albrecht | Timing to perfection: the biology of central and peripheral circadian clocks[END_REF].

Box 2. Light-synchronization of circadian clocks

The SCN is mainly synchronized to the day-night alternation [START_REF] Patton | The suprachiasmatic nucleus[END_REF]; Light is perceived in the eye by a particular layer of ganglion neurons containing the pothopigment melanopsin, also called intrinsically photosensitive retinal ganglion cells (ipRGCs). These cells project to the SCN (in the ventral neurons), through the monosynaptic retinohypothalamic tract (RHT), for entrainment to the LD cycle [START_REF] Schmidt | Melanopsin-positive intrinsically photosensitive retinal ganglion cells: from form to function[END_REF]. At cellular level light signaling promotes an increase in the influx of Ca 2+ in postsynaptic SCN cells, then an activation of protein kinases and the cAMP response element binding protein (CREB), which finally binds in the promoters of Per1-2 genes, leading to the resetting of the phase and/or entrainment of the clock.

Moreover, since ipRGCs connect with other brain structures in the hypothalamus (e.g., lateral hypothalamus, preoptic area) and epithalamus (perihabenular nucleus), light is able to modulate specific behaviors (e.g., sleep, feeding, motivation) [START_REF] Lazzerini Ospri | Mood, the Circadian System, and Melanopsin Retinal Ganglion Cells[END_REF]. Thus, the LD cycle may entrain brain circadian clocks and behavior directly and/or indirectly via the SCN. For peripheral oscillators, however, LD signals first entrain the SCN, and then the clock uses hormonal and neuronal pathways, behavioral (e.g., food intake, locomotor activity) or temperature cues to transmit the solar time information to the whole body [START_REF] Schibler | Clock-Talk: Interactions between Central and Peripheral Circadian Oscillators in Mammals[END_REF].

Box 3. Peripheral circadian oscillators

Several years have passed already from the first discovery on the expression of clock genes oscillating in the liver [START_REF] Damiola | Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus[END_REF][START_REF] Stokkan | Entrainment of the circadian clock in the liver by feeding[END_REF][START_REF] Yamazaki | Resetting central and peripheral circadian oscillators in transgenic rats[END_REF]. The liver is a peripheral circadian clock which plays an important role in physiology and metabolism. Interestingly, the first reports on the circadian activity of liver were made by serendipity by the group of Prof. Ueli Schibler. In the interest of studying the albumin gene, authors isolated the transcription factor D-element Binding Protein (DBP) with the surprising results that accumulation of Dbp in the liver is a hundred times more at night than day, and independent of the SCN and/or rhythmic hormone release [START_REF] Wuarin | Expression of the liver-enriched transcriptional activator protein DBP follows a stringent circadian rhythm[END_REF]. How important is the rhythmic gene expression in the liver? A specific Bmal1 deletion in the liver clock alters glucose metabolism inducing a decrease in the levels of circulating glucose in blood at daytime, thus, altering the endogenous rhythm of glucose production necessary for the metabolic challenges during sleep/fasting conditions [START_REF] Lamia | Physiological significance of a peripheral tissue circadian clock[END_REF].

In the pancreas, β-cells exhibit rhythmic expression of circadian genes which induces diurnal insulin secretion [START_REF] Pulimeno | Autonomous and self-sustained circadian oscillators displayed in human islet cells[END_REF][START_REF] Perelis | Pancreatic beta cell enhancers regulate rhythmic transcription of genes controlling insulin secretion[END_REF]. Considerably, clock capacity in islet pancreas cells is dependent on

Figure 1 .

 1 Figure 1. Daily and circadian rhythms of eating behavior are regulated centrally by the Metabolic-Hedonic-Circadian network. Food intake is controlled by specific brain sites

Figure 2 .

 2 Figure 2. Time eating behavior drives brain and peripheral clocks and circadian rhythms. (A) Time-restricted eating (TRE) is a strong synchronizer for extra-SCN oscillators in the brain and periphery. TRE aligned with the active phase of the organism (at day for diurnal species, and at night for nocturnal) favors synchronization and improves physiology

Acknowledgements

I would like to thank Marie-Paule Felder-Schmittbuhl, Wilf Gardner and all the members of my group (Circadian-Reward) for their constructive comments and discussions. I am gratefully acknowledged to the Agence National de la Recherche (ANR-14-CE13-0002-01 ADDiCLOCK JCJC), the consortium Danone/Foundation pour la Recherche Medicale (FRM) and the Centre National de la Recherche Scientifique (CNRS) for funding. I do apologize to all my colleagues whose relevant contributions could not be cited due to space limitations.

clock genes, since when one of the principal actors of the molecular clockwork, such as Clock or Bmal1 is lacking, the insulin secretion is altered, leading to possible development of diabetes [START_REF] Marcheva | Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes[END_REF]. Thus, the pancreatic circadian clock locally regulates insulin release in time; a circadian dysfunction of it leads to metabolic pathology. In the cardiovascular system, a circadian clock regulates daily changes in blood pressure (BP) and heart rate (HR). Clock genes and proteins (Bmal1, Per2, Rev-Erbα) and clock-output genes (Dbp) are rhythmically expressed in the heart and these rhythms persist ex vivo, indicating the presence of an autonomous circadian clock within cardiac cells [START_REF] Martino | Influence of the cardiomyocyte circadian clock on cardiac physiology and pathophysiology[END_REF]. Moreover, global and specific mutations of the BMAL1 clock gene lead in alterations in of cardiovascular functions (BP, HR), bradycardia and depressed systolic function. In humans, cardiovascular incidents (myocardial infarction) show a higher frequency in the morning (6:00-12:00 a.m.), which can be correlated to the circadian variations of cardiac functions (BP, HR, blood consistence) [START_REF] Scheer | Adverse metabolic and cardiovascular consequences of circadian misalignment[END_REF]. The clock in the cardiovascular system is synchronized by the SCN via the autonomous nervous system [START_REF] Scheer | Cardiovascular control by the suprachiasmatic nucleus: neural and neuroendocrine mechanisms in human and rat[END_REF], therefore, a misalign between the SCN and the Heart clock by circadian disruptions (jet-lag, shift work) may potentiate the risk for cardiovascular diseases [START_REF] Scheer | Adverse metabolic and cardiovascular consequences of circadian misalignment[END_REF].

Box 4. Extra-SCN brain circadian oscillators

In the brain, extra-SCN structures have been identified as circadian oscillators with selfsustained ability and synchronized by the SCN. These are the olfactory bulb (OB) [START_REF] Granados-Fuentes | The suprachiasmatic nucleus entrains, but does not sustain, circadian rhythmicity in the olfactory bulb[END_REF] and the lateral habenula (LHb) [START_REF] Guilding | Circadian oscillators in the epithalamus[END_REF][START_REF] Salaberry | A suprachiasmatic-independent circadian clock(s) in the habenula is affected by Per gene mutations and housing light conditions in mice[END_REF]; both are good examples of self-sustained oscillators, independent of, but entrained by the SCN, and dependent on clock genes. The clock in the OB controls the circadian rhythms in odor detection, and this behavior might be relevant in nature for foraging and optimizing the quality of food sources. The LHb has been revealed as a relevant structure for the control of monoamines. Moreover, it has been implicated in the regulation of palatable and caloric food intake [START_REF] Mizumori | The Lateral Habenula and Adaptive Behaviors[END_REF][START_REF] Stamatakis | Lateral Hypothalamic Area Glutamatergic Neurons and Their Projections to the Lateral Habenula Regulate Feeding and Reward[END_REF]. In the hypothalamus, beyond the SCN, the arcuate (ARC) and dorsomedial (DMH) nuclei have circadian activity in the firing rate and clock gene expression, and these are importantly affected by feeding status in mice [START_REF] Guilding | A riot of rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus[END_REF]. However, these have been classified as slave circadian oscillators.

BRAIN


J.-B Giouse

K White

C Tromas

Nanoindentation characterization of the surface mechanical properties of a 17-4PH stainless steel substrate treated with grit blasting and coated with a Cr3C2-NiCr coating

Keywords: nanoindentation cartography, 17-4PH steel substrate, surface treatment, cermet coating, ceramic/metal interface, high velocity oxy fuel (HVOF) spraying

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INTRODUCTION

Thermal spray coatings are commonly used in many industrial applications including the automobile, petroleum or civil engineering industries. High Velocity Oxy-Fuel (HVOF) spraying is specifically useful for extreme applications like the deposition of cermet materials that requires high velocities to be deposited. Thermal spray coatings deposited by HVOF can enhance the surface properties of a material such as wear and corrosion resistance [START_REF] Pawłowski | The science and engineering of thermal spray coatings[END_REF], [START_REF] Sobolev | High velocity oxy-fuel spraying: theory, structure-property relationships and applications[END_REF].

Prior to deposition of the coating, substrate surface modification treatments, such as grit blasting may optimize the local roughness (Figure 1) to favor the mechanical interlocking of the coating [START_REF] Kumar | Influence of substrate roughness on bonding mechanism in cold spray[END_REF]- [START_REF] Fauchais | Knowledge concerning splat formation: An invited review[END_REF]. This common surface treatment sends high velocity particles (usually angular shaped alumina or silicon carbide grit) onto the substrate surface in order to cause impact damage. Reports shows opposing effect of the grit blasting on the fatigue properties. On one hand, some studies demonstrate a detrimental effect of grit blasting treatments on the fatigue lifetime, where the presence of embedded or adhered residual particles coupled with a high surface roughness can lead to important stress concentrations and a decrease of the fatigue performances [START_REF] Baleani | The Effect of Sandblasting Treatment on Endurance Properties of Titanium Alloy Hip Prostheses[END_REF], [START_REF] Multigner | Influence of the sandblasting on the subsurface microstructure of 316LVM stainless steel: Implications on the magnetic and mechanical properties[END_REF]. Cracks in the vicinity of the embedded particles were also observed in a 316 stainless steel subjected to waterjet peening [START_REF] Barriuso | Fatigue behavior of Ti6Al4V and 316 LVM blasted with ceramic particles of interest for medical devices[END_REF] which highlight the stress concentration role of such defects. In addition, the high kinetic energy associated with the process creates a complex microstructural and stress state in the subsurface, further modifying the fatigue and mechanical properties of the treated material [START_REF] Multigner | Influence of the sandblasting on the subsurface microstructure of 316LVM stainless steel: Implications on the magnetic and mechanical properties[END_REF], [START_REF] Guan | Surface nanocrystallization by sandblasting and annealing for improved mechanical and tribological properties[END_REF]- [START_REF] Tang | Production of alloyed nanocrystalline surfaces by combined punching, sandblasting and recovery treatments[END_REF]. On the other hand, it was also reported that the detrimental impact on the fatigue properties induced by the modification of the roughness and the embedded particles can sometimes be compensated and even inversed when sufficient compressive stresses are generated. Thus, several studies actually reported an increase of the fatigue properties due to the positive impact of the compressive stresses on the crack nucleation [START_REF] Cizek | Influence of plasma and cold spray deposited Ti Layers on high-cycle fatigue properties of Ti6Al4V substrates[END_REF].

Nevertheless, in the case of grit blasting used before the deposition of a coating, those defects associated with the grit blasting process have numerous effects on adhesion. First, the modification of the microstructure and stress state can modify how the splats behave when impinging the substrate. Gao and al. [START_REF] Gao | Influence of substrate hardness on deposition behavior of single porous WC-12Co particle in cold spraying[END_REF] conducted a study on the influence of hardness on the deposition of single coating splats, showing that sufficient deformation of the substrate is necessary to promote adhesion. Moreover, the presence of adhered residual grits on the substrate surface locally degrades the adhesion of the coating by modifying the wetting properties of coating particles and by blocking any interactions between the coating and the substrate.

In this paper, we propose a new methodology to characterize the impact of grit blasting on a 17-4PH steel substrate coated with a cermet Cr3C2-NiCr coating. To achieve this, the nanoindentation cartography method was used in cross section, in which the hardness data links the microstructural modifications induced by the grit blasting to the resulting local substrate mechanical properties in the vicinity of the interface. The nanoindentation measurements are coupled to atomic for microscopy (AFM) and scanning electron microscopy (SEM) observations.

II. MATERIALS AND EXPERIMENT DETAILS

a.

Materials and surface preparation

The substrate is a 17-4 precipitation hardening (17-4PH) martensitic steel. It is in the H1150 condition meaning that it underwent a heat treatment at 621°C from the solution treated condition. The surface of the steel substrate was prepared in two different ways prior the coating deposition process. One sample was grit blasted with alumina particles and another one was polished up to 1um with diamond suspension.

Following the surface treatments, both samples were coated with a cermet material made of chromium carbides Cr3C2 embedded in a NiCr metallic binder (80% Cr3C2 -20% NiCr). The coating is produced by high velocity oxy fuel (HVOF) spraying and is 250µm thick.

Samples were cut and embedded in a carbon filled resin using a hot mounting press as shown in Figure 2. Preparation of the cross section involves conventional metallographic preparation followed by chemical mechanical polishing (CMP) using non-crystallizing colloidal silica in order to achieve a minimum roughness and no work hardening of the surface. Thus, the mechanical properties measured by nanoindentation are not altered by the surface condition.

b. Analysis techniques

Microstructural observations were performed by scanning electron microscopy using a JEOL JSM-7001F. The SEM was used both in secondary (SE) and back scattered electrons (BSE).

Atomic force microscopy was carried out using a Dimension 3100 microscope from Brucker (Santa Barbara, CA) in tapping mode in order to characterize the surface resulting from the sample preparation. The AFM files were processed using the WSxM software [START_REF] Horcas | WSXM: A software for scanning probe microscopy and a tool for nanotechnology[END_REF].

c. Nanoindentation

A U-NHT nanoindenter from Anton Paar (Peseux, Switzerland) equipped with a Berkovich tip was used to perform the nanoindentation measurements. This apparatus has an active surface reference ensuring very high thermal and mechanical stabilities. The tip was carefully calibrated using the regular method, consisting of indenting a material with a known Young's modulus, in this case fused silica (E=72GPa). The hardness was determined by analyzing the elastic unloading curve using the effective indenter method [START_REF] Woirgard | An alternative method for penetration depth determination in nanoindentation measurements[END_REF], [START_REF] Oliver | Measurement of hardness and elastic modulus by instrumented indentation: Advances in understanding and refinements to methodology[END_REF].

In order to characterize the local mechanical response, the nanoindentation cartography method has been applied. This method consists in performing a regular matrix of idents in order to reconstruct a hardness map [START_REF] Ulm | Statistical Indentation Techniques for Hydrated Nanocomposites: Concrete, Bone, and Shale[END_REF]- [START_REF] Tromas | Hardness cartography to increase the nanoindentation resolution in heterogeneous materials: Application to a Ni-based single-crystal superalloy[END_REF]. The nanoindentation cartography method allows the investigation of the local mechanical properties in heterogeneous materials and the correlation of the hardness variations with the chemical or microstructural heterogeneities of the material. For the cartography, the array was designed with a maximum indentation depth of 100nm and a distance between adjacent imprints of 3µm. These parameters were selected to minimize the step size of the array, and thus increase the resolution of the cartography, while ruling out the influence of the surface morphology. In the case of a Berkovich indenter, the diameter of an imprint is about 7 times its depth and it is common to use a minimum distance of 5 times the diameter, i.e. about 35 times the maximum indentation depth between two successive indentations.

A color chart has been associated with the hardness values, and a bicubic interpolation has been applied to build the map from the discrete dataset. Such an interpolation method was chosen as it leads to smoother images with fewer interpolation artifacts than either the nearest neighbor or the bilinear interpolations [START_REF] Tromas | Hardness cartography to increase the nanoindentation resolution in heterogeneous materials: Application to a Ni-based single-crystal superalloy[END_REF].

III. RESULTS AND DISCUSSION

a. Microstructure analysis with the SEM. The average diameter of the particles is 20µm. Fully embedded particles were also observed, as shown later. When trapped, the particles modify the wetting properties of the coating particles during the deposition and impede the interaction between the coating and the substrate resulting locally in poorly bonded regions [START_REF] Soltz | The Effect of Substrate Contaminates on the Life of Epoxy Coatings Submerged in Sea Water[END_REF], [START_REF] Momber | Blast cleaning technology[END_REF]. Embedded alumina particles originate from important deformation of the substrate overlaying the particles during the process. It is usually associated with a complex surrounding stress state that can lead ultimately to failure [START_REF] Baleani | The Effect of Sandblasting Treatment on Endurance Properties of Titanium Alloy Hip Prostheses[END_REF], [START_REF] Multigner | Superficial severe plastic deformation of 316 LVM stainless steel through grit blasting: Effects on its microstructure and subsurface mechanical properties[END_REF], [START_REF] Price | Effect of cold spray deposition of a titanium coating on fatigue behavior of a titanium alloy[END_REF]. The SEM image in Figure 4b reveals that the process also affects the microstructure of the substrate. Two microstructurally dissimilar regions can be distinguished inside the substrate. The region just beneath the interface is associated with an ultrafine-grained microstructure and is about 10-20µm. The rest of the substrate is characterized by a grain size gradient up to the point where the process does not affect the microstructure anymore and the common lath martensitic structure is observed.

b. Cartography

Figure 6 shows the hardness cartography determined from an array of 765 indents. The step size between each indentation is 3µm. For a better analysis, the color scale has been adjusted to the substrate hardness values (the coating hardness values have been saturated

and appear in grey contrast). The substrate presents a hardness gradient ranging from 4700MPa at 100µm away from the interface up to 5800MPa at the interface. The Young's Modulus was also measured and remains constant throughout the substrate thickness at 219 ± 12GPa. A similar value of 225GPa using the same method was found [START_REF] Gross | Bimetallic MIM Medical Components -ASM International[END_REF], thereby validating the hardness nanoindentation measurements. Moreover, the use of displacement control ensured that all indentations have the same size so that artefacts associated with any possible tip calibration uncertainties or with indentation size effect are averted.

A strong increase of the hardness is observed with a gradient of about 11MPa/µm and a hardness reaching a maximum of 5800MPa close to the interface. This hardness increase can have several consequences on the adhesion of the coating. First, it is likely to promote brittleness and modify the surface fatigue properties of the substrate. Furthermore, it can affect the coating build-up during the early stage of deposition. Indeed, research conducted by Gao et al. [START_REF] Gao | Influence of substrate hardness on deposition behavior of single porous WC-12Co particle in cold spraying[END_REF] revealed that a higher hardness of the substrate is associated with more particles rebounding from the substrate surface during the first stage of deposition. The non-grit blasted sample exhibits no evidence of hardness gradient in the substrate.

Comparison with cartography of the grit blasted sample shown Figure 6 suggests that the grit blasting modifies the mechanical properties and increases the hardness. It also suggests that the coating deposition, despite high kinetic energy involved, does not induce any visible changes of the mechanical properties of the substrate. Yet, one would have expected some hardening due to the impact of the carbides contained in the coating. Thus, the contribution of the NiCr metallic phase in absorbing the deformation is probably non-negligible.

The impact of the grit blasting process on the microstructure was investigated by imaging the microstructure of the substrate at different distance from the interface. Figure 8b was taken in the bulk where the substrate has not been affected by the grit blasting process. The microstructure is a regular lath microstructure characteristic of martensitic steels [START_REF] Jr | Microstructure and cleavage in lath martensitic steels[END_REF] . Figure 8a was taken in the substrate, a few microns away of the interface, where the substrate is highly affected by the grit blasting process. The lath microstructure disappeared, replaced by a finer-grained structure. The comparison of the microstructure shown in Figure 8a and b suggest that the high values of hardness measured at the interface is related to grain refinement induced by the grit blasting process. Several papers have reported a change of microstructure when blasting high velocity particles on a metallic surface. The mechanisms of refinement are similar to the ones observed in severe plastic deformation processes such as high-energy shot peening or surface mechanical attrition treatment [START_REF] Mao | Surface nanocrystallization by mechanical punching process for improving microstructure and properties of Cu-30Ni alloy[END_REF]- [START_REF] Wu | Microstructure and evolution of mechanically-induced ultrafine grain in surface layer of AL-alloy subjected to USSP[END_REF]. The plastic strains induced by the impacting particles are accommodated by a change of microstructure through dislocation motion and grain subdivision [START_REF] Lu | Nanostructured surface layer on metallic materials induced by surface mechanical attrition treatment[END_REF]. A gradient of microstructure and mechanical properties is usually observed in the impacted material because of the gradient of strain and strain rate induced.

c. Indentation size effect

The nanoindentation cartography has been performed at a low penetration depth (100nm) in order to reduce the matrix step size and thus to increase the spatial resolution. However, one may wonder if the hardness values depend on the penetration depth (ISE). For this reason, the effect of the penetration depth on the hardness measurement was investigated.

Indentation size effect (ISE) describes the scale dependence observed in various materials for which the hardness increases with decreasing the indentation size. In order to characterize a potential ISE in the substrate, fifteen rows of indents were made containing 15 indentations each for a total of 225 indentations. Each row corresponds to a different maximum penetration depth ranging between 80 and 700nm. The indentations were made in the bulk where the microstructure and hardness are considered homogeneous. The results of the ISE characterization are presented Figure 9. Each data point is an average of 15 hardness values.

The hardness remains constant throughout the range, which indicates that there is no scaledependence in the hardness values. The uncertainties at small penetration depths remain acceptable, which further validates the indentation procedure shown in Figure 3a.

d. Embedded alumina particle

Grit blasting leaves residual particles on the substrate surface, which can adhere to the substrate or completely embed into the substrate surface. are ranging from 6.2 to 7.3GPa. Outside this region, the hardness ranges between 5.1 and 5.9GPa.

The SEM and AFM observations suggest that the vicinity of embedded alumina particles is severely altered and the mechanisms of refinement described earlier take place to a greater extent. It is commonly thought that the coating/substrate interface is the preferential path for failure. However, the presence of cracks around the embedded particles (red arrows in Figure 10a) indicates that the particles act as stress raisers and are preferential sites for crack nucleation. Additionally, we can also observe that those cracks propagate in the substrate, in the layer of affected microstructure, which suggests that the coating/substrate interface is not necessarily where the failure will occur. It is confirmed by the nanohardness cartography results, which indicate that the area surrounding the particles has higher hardness, which is likely to promote brittleness and thus crack propagation. Figure 11 further illustrates the crack propagation in the substrate at different locations of the substrate/coating interface. In all optical images, a crack has nucleated from an alumina particle and has propagated through the substrate.

The results of the nanoindentation cartography and the microstructural analysis highlight the adverse effect of the grit blasting process. Those observations are similar to what was observed in other studies [START_REF] Cizek | Influence of plasma and cold spray deposited Ti Layers on high-cycle fatigue properties of Ti6Al4V substrates[END_REF], [START_REF] Price | Effect of cold spray deposition of a titanium coating on fatigue behavior of a titanium alloy[END_REF], [START_REF] Ghelichi | Microstructure and fatigue behavior of cold spray coated Al5052[END_REF], which validates the use of our method for the characterization of coated materials. However, our methodology alone does not allow concluding on the overall impact of the grit blasting on the adhesion. Despite the obvious detrimental effects of the grit blasting on the adhesion (cracks and brittle substrate subsurface), other factors need to be accounted for. Indeed, Cizek et al. [START_REF] Cizek | Influence of plasma and cold spray deposited Ti Layers on high-cycle fatigue properties of Ti6Al4V substrates[END_REF] reported an increase of the fatigue properties (cantilever-beam cyclic bend loading) despite the presence of embedded residual grits. This was explained by the compressive stresses in the substrate induced by the surface preparation treatment which compensate the stress concentration induced by the increase of roughness and the embedded particles.

IV. CONCLUSION

The impact of grit blasting on a 17-4PH steel substrate coated with a cermet Cr3C2-NiCr coating was investigated using an original method based on nanoindentation cartography coupled to microscopic observations. Despite the beneficial effect of grit blasting on adhesion through roughness modification, it was shown that the grit blasting process affects the microstructure and mechanical properties of the substrate subsurface (an average increase of more than 20% in hardness at the interface compared to the bulk values was measured). The impact of the coating deposition process on the substrate was shown to be close to zero. The potential adverse effects of the grit blasting treatment were confirmed by a local investigation of an embedded residual alumina particle. The SEM and AFM imaging, coupled to the hardness map reconstruction from nanoindentation measurements, revealed an ultra-finegrained microstructure associated with a further, and very local, increase of hardness (40% higher than the bulk values). Cracks nucleating from the edge of the particle were also observed suggesting that failure could preferentially occur in the layer affected by the grit blasting and not at the coating substrate interface.

Obviously, other factors could be more critical in understanding the failure of such systems.

Failure in the coating or at the interface is more likely to occur. However, in a process of improving the performance of the coating, the local investigation of the impact of the substrate surface preparation is critical. Moreover, in light of the results presented in this paper, the nanoindentation cartography method couple to high resolution imaging techniques could enable to reinterpret the adhesion problems of thermal spray coating deposited on grit blasted substrate.

Figure 1 .

 1 Figure 1. Simplified steps occurring during a grit blasting process. The particles are blasted onto the substrate surface to increase the roughness and favor the mechanical interlocking of the coating particles. Some alumina particles remains embedded in the substrate after the process.

Figure 2 .

 2 Figure 2. Preparation steps of the samples. Each sample is cut from a bar and mounted into a conductive carbon resin. (a) Schematic of the coated bar as received by the manufacturer. The coating is in red and the substrate in grey. (b) Schematic representation of the samples as mounted in the resin. Two samples were used and faced each other to favor a uniform polishing. (c) Photograph of the samples mounted in a conductive carbon filled resin.

Figure 3a illustrates the

  Figure 3a illustrates the loading sequence used during the nanoindentation tests in this work. The loading is performed in depth control to ensure that each indentation have the same maximum penetration depth, without regard to hardness. The unloading is performed in load control at 400 N/min. Figure 3b presents a typical nanoindentation curve obtained in cross section in the 17-4PH steel using the loading profile shown Figure 3a.

Figure 3 .

 3 Figure 3. (a) Indentation profile. The loading is depth controlled to assure a constant penetration of all the indentations while the unloading is load controlled (b) Typical nanoindentation curve obtained in cross section in the 17-4PH steel using the loading profile shown Figure 3a.

Figure 4

 4 Figure 4 summarizes the results of the microstructural characterization of the interface. An optical micrograph of the interface, shown in Figure 4a, identifies the presence of two residual alumina particles from the grit blasting. Figure 4b is a zoomed image of the interface taken

Figure 4 .

 4 Figure 4. (a) Optical microscopy image of the coating/substrate interface showing adhered and embedded alumina particles. (b) SEM image of the interface in cross section revealing a change of microstructure of the substrate when approaching the interface. Atomic force microscopy was used to inspect the surface after metallographic and chemical mechanical polishing (CMP) preparation and the results are shown Figure 5. Figure 5b is the 3D representation of the picture shown in 5a. The line represents the position of the height profile shown in 5c, which shows that the coating is standing 150nm above the substrate, resulting from different polishing rate during sample preparation. The slope of the profile extends over 12 m corresponding to a slope of approximately 0.01°. A Route Mean Square (RMS) roughness of about 4nm was achieved in the 17-4 PH substrate. Both the slope and the roughness are sufficient for the nanoindentation measurements.

Figure 5 .

 5 Figure 5. Data obtained from atomic force microscopy. (a) Topography image of the surface at the interface after CMP with colloidal silica. The blue line represents the position of the profile shown in Figure 5c. (b) 3D representation of the topography image shown in (a). The difference in depth between the substrate and the coating is clearly observable. The reader is referred to the profile shown in (c) for Z scale. (c) Height profile taken at the interface. Both the substrate and coating are flat. The slope corresponds to the near interface region of the substrate affected by the grit blasting and resulting from differential polishing rate during CMP.

Figure 6 .

 6 Figure 6. Nanohardness cartography built from a 17*45 nanoindentation array at the interface between the coating and the substrate. The hardness map is superimposed to the optical microscopy image of the indented area. The substrate surface was grit blasted with alumina particles prior to the deposition of the coating. The coating is on the left side and was voluntarily saturated to increase the contrast for the representation of hardness in the substrate.

Figure 7

 7 Figure 7 shows the hardness cartography of the non-grit blasted substrate determined from 450 indents. The nanohardness remains constant throughout the substrate thickness at approximately 4700MPa.

Figure 7 .

 7 Figure 7. Nanohardness cartography built from a 15*30 nanoindentation array at the interface between the coating and the substrate. The hardness map is superimposed to the optical microscopy image of

Figure 8 .

 8 Figure 8. Electronic microscopy images revealing the microstructure of the substrate taken a) away of the interface b) at the interface. The microstructure at the interface is made of very small grains associated with deformation patterns while the microstructure in the bulk shows a regular lath microstructure.

Figure 9 .

 9 Figure 9. Nanohardness as a function of the penetration depth for ISE investigation. Each data point is the average of 15 hardness values obtained from indentations made at the same depth for a total of 225 indentations. The errors bars correspond to the standard deviation calculated from those 15 values. The hardness is constant throughout the entire range, which means no evidence of scale dependence in the hardness measurements is observed.

Figure 10

 10 illustrates the steps in performing and analyzing the cartography from a 12*15 indentations array around an alumina particle. The SEM image (Figure10a) of the indented area taken using back scattered electrons, using a step size of 2µm provided increased resolution of this ultra-fine-grained microstructure. The yellow dash line separates this microstructure from the rest of the material.The diameter of the affected region is about 2-3µm. Small cracks nucleating from the particle (see red arrows on Figure10a) and propagating into the substrate are clearly observable. An AFM image of the particle is shown Figure10b. It shows deformation patterns on the surface, which follows the ultra-fine-grained microstructure observed in Figure10a. In addition, the high resolution AFM resolves surface features for correlation with the hardness cartography shown in Figure10c. A nearest neighbor interpolation method was used to reconstruct the hardness map. Figure10csuggests that the hardness is notably higher in the vicinity of the particle. However, the hardness variations apply over very short distances, which make it difficult to extract clear information from the hardness map. To address this issue, one could think of increasing the resolution of the cartography by decreasing the step size of the array but further decreasing the step size would induce interactions between neighboring indentations. One solution is then to look at the individual hardness values as shown in Figure10c. The hardness values of the ultra-fine-grained region (delimited by the yellow dashed line)

Figure 10 .

 10 Figure 10. Nanoindentation array on an alumina particle. (a) SEM image showing the microstructure around the particle. The yellow line delimits an ultra-fine-grained structure in the vicinity of the particle. (b) Same area imaged by AFM (3D top view) that shows the deformation patterns of the surface around the particle. (c) Nanohardness map around the alumina particle. Individual hardness values are also indicated. The hardness values of the ultra-fine-grained structure are 40% higher than the bulk values.

Figure 11 .

 11 Figure 11. (a) -(d) Optical images showing different regions of the interface where cracks have nucleated from embedded alumina particles. The red arrows indicate the likely initiation site of the crack from the particle.

AKNOWLEDGEMENT

This work was partially funded by the French Government program Investissements d'Avenir (LABEX INTERACTIFS, reference ANR-11-LABX-0017-01). We thank Anne-Marie Archambault for assistance with preparing the samples, and Dominique Eidy for helping on the SEM observations.