Circadian rhythms of hedonic drinking behavior in mice

Accepted Manuscript Circadian rhythms of hedonic drinking behavior in mice Claire Bainier, Maria Mateo, Marie-Paule Felder-Schmittbuhl, Jorge Mendoza PII: DOI: Reference:

S0306-4522(17)30159-8 http://dx.doi.org/10.1016/j.neuroscience.2017.03.002 NSC 17646

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Neuroscience

Received Date: Revised Date: Accepted Date:

18 October 2016 15 February 2017 1 March 2017

Please cite this article as: C. Bainier, M. Mateo, M-P. Felder-Schmittbuhl, J. Mendoza, Circadian rhythms of hedonic drinking behavior in mice, Neuroscience (2017), doi: http://dx.doi.org/10.1016/j.neuroscience.2017.03.002

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Circadian rhythms of hedonic drinking behavior in mice Claire Bainier, Maria Mateo, Marie-Paule Felder-Schmittbuhl and Jorge Mendoza* Institute of Cellular and Integrative Neurosciences, CNRS UPR-3212, University of Strasbourg, Strasbourg, France

Running title: Sweet rhythms in mice

*Correspondence: Institute of Cellular and Integrative Neurosciences, CNRS UPR-3212, University of Strasbourg; 5 rue Blaise Pascal, 67084 Strasbourg, France. Tel: + (33) 0388 45 66 96; Fax: + (33) 0388 45 66 54, E-mail: [email protected]

Abbreviations AVP, Vasopressin; CP, caudate putamen; CREB, cAMP response element-binding protein; CT, circadian time; DA, dopamine; DD, constant darkness; LD, light-dark cycle; MAPK, mitogen-activated protein kinase; NAcc, nucleus accumbens; Per, Period; PK2, Prokineticin 2; Ctx, cortex; OD, optical density; ORX, orexin; SCN, suprachiasmatic nucleus; VTA, ventral tegmental area; WT, wild-type; ZT, zeitgeber time.

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Abstract In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the main circadian clock, synchronized by the light-dark cycle, which generates behavioral rhythms like feeding, drinking and activity. Notwithstanding, the main role of the SCN clock on the control of all circadian rhythms has been questioned due to the presence of clock activity in many brain areas, including those implicated in the regulation of feeding and reward. Moreover, whether circadian rhythms of particular motivated behaviors exist is unknown. Here, we evaluated the spontaneous daily and circadian behavior of consumption of a sweet caloric solution (5-10% sucrose), and the effects of sucrose intake on the expression of clock genes in the mouse brain. Mice showed a daily (in a light-dark cycle) and a circadian (in constant darkness conditions) rhythm in the intake and sucrose preference with a rise for both parameters at night (or subjective night). In addition, we observed changes in the circadian day-night expression of the clock gene Per2 in the SCN, cortex and striatum of animals ingesting sucrose compared to control mice on pure water. Finally, daily rhythms of sucrose intake and preference were abolished in Per2Brdm1 and double Per1-/- Per2Brdm1 mutant animals. These data indicate that the expression of circadian rhythms of hedonic feeding behaviors may be controlled by brain circadian clocks and Per gene expression. Keywords: circadian, motivation, sucrose, drinking, reward, Per genes

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Introduction In mammals, circadian rhythms are generated by a principal clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus (Welsh et al., 2010). To keep in synchrony with environmental changes, the endogenous activity of the SCN is adjusted by daily external signals, mainly the sunlight (day/night light changes) and other stimuli such as food or social cues (Mrosovsky, 1996; Meijer and Schwartz, 2003; Mendoza, 2007). For many years the SCN has been considered as the unique endogenous (circadian) clock in mammals. Nevertheless, in peripheral tissues (e.g., liver, heart) and in other brain regions (e.g., olfactory bulb, lateral habenula) circadian oscillators have been described (Schibler et al., 2003; Granados-Fuentes et al., 2004; Herzog, 2007; Guilding et al., 2010). These last ones may play significant roles in regulating specific behavioral rhythms (e.g., learning, olfaction, motivation) that are synchronized by the main SCN clock. Circadian clocks operate with several clock genes and proteins like Clock, Npas2, Bmal1, Per1-3, Cry1-2 and Rev-Erb, forming positive and negative loops to generate circadian oscillations (Takahashi et al., 2008). Mutations of clock genes affect circadian and noncircadian functions (Rosenwasser, 2010). Clock mutant mice show perturbations in the circadian activity of locomotion and in behavioral responses to cocaine and alcohol intake (Antoch et al., 1997; McClung et al., 2005; Ozburn et al., 2013). The addicted phenotype in these mice has been explained by an increase in the firing rate of dopaminergic (DA) neurons in the ventral tegmental area (VTA). Thus, the CLOCK protein is involved in the regulation of DAergic transmission in the brain reward circuitry (McClung et al., 2005). Conversely, chronic drug (e.g., morphine, methamphetamine, cocaine) administration up-regulates the expression of clock genes in the striatum (caudate-putamen, CP) and cortex (Ctx), and disrupts circadian rhythms of locomotor activity without affecting the expression of these genes in the SCN (Nikaido et al., 2001; Hofstetter et al., 2003; Ammon-Treiber and Hollt,

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2005; Rosenwasser et al., 2005; Morgan et al., 2006; Honma and Honma, 2009; Falcon et al., 2013). Food intake is regulated by a homeostatic and a hedonic mechanism (Saper et al., 2002). However, chronic access to highly tasty food can induce an escalating intake and alterations in brain reward circuits (Avena et al., 2008). Thus, hedonic caloric food, similar to many drugs of abuse, has strong effects in the central reward system stimulating DAergic signaling in the forebrain, which may lead to the development of addicted-like behaviors (Lenoir et al., 2007; Ahmed et al., 2013). Diets with a high sweet taste (e.g., soft drinks, fruit beverages, sucrose) are greatly rewarding and may promote an excessive consumption in an addictivelike manner (Ahmed et al., 2013; Madsen and Ahmed, 2015). Whether hedonic caloric solutions can alter the circadian system similarly to drugs of abuse like cocaine or alcohol is, however, not clear. In the present study we investigated, whether sucrose drinking behavior shows a daily and circadian pattern, as well as the effects of sucrose intake on clock gene expression in the mouse brain. Furthermore, using clock gene deficient mice, we investigated whether sucrose intake and preference are affected by a malfunctioning of the circadian clock. Material and Methods Animals and Housing For the first experiment we used young adult (6-8 weeks old) C57BL/6J male mice (Chronobiotron UPR3212). For the second, we used adult F2 homozygous Per1-/-, Per2Brdm1 and double Per1-/-, Per2Brdm1 mutant male and female mice and their wild type (WT) C57BL/6×129SvEvBrd littermates (Zheng et al., 1999; Zheng et al., 2001). Animals were housed individually in clear plastic cages in a room under a light-dark cycle (LD) 12/12h (lights on at 7:00 a.m.) with temperature (21±1°C) and humidity (55%) controlled. Food (lowfat diet 105, 12.6 kJ g-1; SAFE; distribution of metabolizable energy content as percentage: 23% protein, 65% carbohydrate and 12% fat, Augy, France) and water were available ad

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libitum during the whole experiment. The mice were under these conditions for at least three weeks before the start of sucrose exposure. All experiments were performed in accordance with the rules of the European Committee Council Directive of November 24, 1986 (86/609/EEC) and the French Department of Agriculture (licence N° 67-88 to J.M.). Sucrose intake: Two-bottle preference test We used a two-bottle choice paradigm (sucrose vs. water) with two different sucrose concentrations (5 and 10%). This procedure permits us to determine the free intake and preference for sweet solutions for long periods without handling or training of animals. First, mice were exposed to 2 days of habituation in which animals had two bottles of water to determine whether there is a difference in the intake and preference for one of the two bottles of water. Following baseline, animals had constant free access to two bottles, one of which contained water and the other a sucrose (5% or 10%) solution. Water and sucrose spillage was measured, and adjustments to intake were made. The solutions were changed regularly to prevent alteration of taste (at least three times per week). To minimize a place preference effect, bottle position was changed daily. We measured the amount (mL) of water vs. sucrose ingested, and the preference (%) for sucrose over water intake was then calculated for each animal. Experimental design Intake and preference for caloric sweet solutions under LD and DD conditions In the first experiment, male C57BL/6J mice in a 12h-12h light dark cycle were divided into three groups and exposed to a paradigm of free choice between two bottles: (Group 1, n=8), a control group with two bottles of tap water; (Group 2, n=8) a group exposed to a bottle of tap water and another bottle with 5% sucrose; and a third group (Group 3, n=5) exposed to a bottle of tap water and another bottle with 10% sucrose during 2 weeks. Sucrose concentration has been previously reported, by our group and others, to be highly preferred in

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C57BL/6J mice at 10% concentration (Lewis et al., 2005; Feillet et al., 2015). However, it is important to note that this is a higher concentration of sucrose that, even if it was preferred by animals in the present study, is not the optimal or necessary concentration to induce a mouse to consume sucrose. To determine a daily rhythm of sucrose intake (mL) and preference (%), we measured these variables at 6h intervals on the last day of sucrose exposure: from ZT0-6, ZT6-12, ZT12-18 and ZT18-24 (zeitgeber time 0 represents lights on and ZT12 lights off). Preference was evaluated as follow: Pref. (%) = vol. sucrose intake/vol. sucrose + vol. H2O. To assess whether intake and preference of daily rhythms persist under constant light conditions, animals under a LD cycle and exposed to 10% sucrose during at least a week were placed in constant darkness (DD; dim red light 5lux) for 24 hours (n=8) to measure intake. Intake and preference to sucrose were evaluated in DD conditions at 6h intervals (CT0-6, CT6-12, CT12-18 and CT18-24) according to the lights off (ZT12) of the last day on LD as a phase reference time point, and which was referred as circadian time 12 (CT12, activity onset) in DD conditions. Mice that remained in DD conditions and exposed to 10% of sucrose were anesthetized with isoflurane and euthanized by cervical dislocation at CT6 (mid-subjective day) and CT18 (mid-subjective night). In total, these animals spent 36h and 42h in DD conditions, respectively, before sacrifice. Brains were isolated then flash-frozen by immersion in isopentane at -30°C and then stored at -80°C. An additional group of mice (designated as the control group, n=8), that received only tap water (two bottles of water), was sacrificed at the same time points (CT6 and CT18) in DD conditions. Intake and preference for caloric sweet solutions in clock mutant mice To evaluate whether daily rhythms of sucrose intake and preference are affected by a molecular disruption of the circadian clock, we used Per1-/-, Per2Brdm1, double Per1-/--Per2 Brdm1

mutant mice and their respective WT littermates (n=4 per genotype) submitted to the

same two-bottle choice protocol of sucrose intake as in the first experiment. Animals were

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first acclimatized to two bottles of water (2 days) and then exposed to one bottle of water vs. 10% sucrose for 1 week. At the last day of exposure, sucrose intake and preference were evaluated in LD conditions, at 6h intervals (ZT0-6, ZT6-12, ZT12-18 and ZT18-24), similar to the first experiment. To note, in the present study we used both male and female mutant mice. In the circadian and dopaminergic system there is an important sexual dimorphism (Mendoza et al., 2011; Michalik et al., 2015). Thus, for future experiments, it is necessary to take into account possible sex difference in the circadian and daily rhythms of sucrose intake. In situ hybridization Antisense probes were transcribed from the corresponding linearized plasmids using the appropriate polymerase in presence of [35S] UTP (1250 Ci/mmol, NEN-Dupond, Zaventem, Belgium) according to the manufacturer's protocol (MAXIscript, Ambion, USA). The in situ hybridization was performed as described elsewhere (Mendoza et al., 2010). The slides were exposed to an autoradiographic film (BioMax, Kodak, France) for 2-3 days. Quantitative analysis of the autoradiograms was performed using ImageJ (NIH software). Relative optical density (OD) was measured in both SCN (left and right hemisphere) at three different levels (rostral, medial and caudal) in one section per animal according to the mouse brain stereotaxic atlas (Paxinos and Franklin, 2004). For the rostral SCN region we took a section set at 0.3mm from bregma, and -0.46mm and -0.70mm for medial and caudal levels, respectively. For the measurements in the cortex (Ctx) we analyzed values in three sections per animal at the level of the cingulate cortex and the motor cortex (1.4mm from bregma); for the ventral striatum or nucleus accumbens (NAcc - containing both the core and the shell sub-regions of the nucleus) three sections were taken at levels between 1.34-1.18mm from bregma, and in the dorsal striatum (caudate-putamen, CP) at the level of 0.9-0.5 mm from bregma (Paxinos and Franklin, 2004). A measure from a nearby unstained area was also taken for each section.

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Specific labeling was then calculated as the difference between OD values in the area of interest and the OD unstained area. Statistical analysis Statistical analyses were performed using the Statistica® software. Differences between the experimental conditions were examined by analysis of variance (ANOVA) of independent measurements followed by a LSD post-hoc Fisher test. In addition, for time series (daily and circadian rhythms), data were analyzed using a non-linear regression (a+b*cos(2*pi*(ZTc)/24); where a=mean expression level, b=amplitude of the oscillation and c=acrophase. Differences were considered significant when p <0.05. Results Daily rhythms of consumption and preference of sucrose Under the LD cycle, control mice exposed to two bottles of water showed a significant daily rhythm of drinking behavior with a higher intake at night for the two bottles (Figure 1A, Table 1). Thus, no rhythm of preference for any bottle was observed (Figure 1B; Table 1). Animals exposed to 5% sucrose showed a significant rhythm of sucrose intake but not for water intake (Figure 1C; Table 1). Moreover, a significant daily rhythm of sucrose preference over water was observed, with a main preference at night (Figure 1D; Table 1). Similar results were observed in animals exposed to a 10% sucrose solution (Figure 1E), with a higher intake and preference for sucrose over water at night (Figure 1F; Table 1). To test whether the daily rhythm of sucrose intake persists in the absence of the LD cycle, animals exposed to a 10% sucrose solution were transferred to DD conditions. In DD conditions, the control group (two bottles of water) showed a circadian rhythm of water intake with a maximal consumption, without preference between the two bottles, at the subjective night (Figure 2A and 2B; Table 1). On the other hand, animals under DD conditions and exposed to the choice of a bottle of water vs. a bottle of 10% sucrose showed a significant

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circadian rhythm of sucrose intake with a peak at the subjective night (Figure 2C; Table 1). Moreover, preference for sucrose (10%) over water also showed a significant circadian rhythm (Figure 2D; Table 1). Clock gene expression in the brain of sucrose-exposed mice To evaluate whether night intake and preference for sucrose in mice affect the expression of clock genes in the SCN and extra-SCN oscillators, brains were collected at two different time points under DD conditions; at the mid-subjective day (circadian time 6; CT6) and midsubjective night (CT18) of animals exposed to sucrose (10%) or control animals drinking only tap water. Mice of both groups (water vs. 10% sucrose) exhibited a significant day-night difference in the expression of Per1-2 mRNA at the three levels of the SCN (rostral, medial and caudal) with higher expression at CT6 (subjective day) and lower at CT18 (subjective night) (Figure 3 and 4; Rostral, Per1, F(1,12)=21.4, p=0.0005; Per2, F(1,12)=84.9, p<0.001; Medial, Per1, F(1,12)=43.11, p=0.00002; Per2, F(1,12)=49.09, p=0.00001; Caudal, Per1, F(1,12)=7.14, p=0.02; Per2, F(1,12)=54.88, p<0.001). However, in sucrose-exposed mice, expression of Per2 in both the rostral and medial SCN was significantly lower at CT6 in comparison to the water control mice (Figure 4; Rostral, group X time interaction, F(1,12) =5.11, p=0.04; Medial, group X time interaction, F(1,12) =4.81, p=0.04). In the Ctx, the NAcc and the CP, Per1 expression was significantly higher at night (CT18) in both control and sucrose-exposed animals (Figure 3; Ctx, F(1,12)= 19.6, p<0.001; NAcc, F(1,12)= 19.2, p=0.0008; CP, F(1,12)= 16.2, p=0.002), with no differences between groups (Ctx, F(1,12)= 3.76, p=0.07; NAcc, F(1,12)= 1.83, p=0.19; CP, F(1,12)= 1.71, p=0.21). Per2 expression in the Ctx and CP, but not in NAcc, was significantly higher at CT18 in both water and sucrose exposed groups (Figure 4; Ctx, F(1,12)= 29.8, p<0.001; NAcc, F(1,12)= 1.57, p=0.23; CP, F(1,12)= 88.8, p<0.001). However, in mice exposed to 10% sucrose, Per2 expression in the Ctx and CP was higher at the subjective night (CT18) and lower at the subjective day (CT6),

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leading to an increased day-night difference with respect to controls (Figure 4; Ctx, F(1,12)= 11.2, p=0.006; CP, F(1,12)= 10.03, p=0.008). No differences between groups were observed in Per2 expression in the NAcc (Figure 4; F(1,12)= 2.96, p=0.11). Daily rhythms of sucrose intake in clock deficient mice To evaluate whether a disruption of the circadian clock may alter daily rhythms of sucrose intake and preference, we used single and double mutant mice for the Per1-2 clock genes. After a week of sucrose exposure (10%), WT mice (C57BL/6×129SvEvBrd mixed background) showed daily rhythms of sucrose intake and preference similar to WT (C57BL/6J) animals from experiment 1, with higher intake and preference at night (Figure 5; Table 2). Per1-/- mice showed a significant daily rhythm of sucrose intake (Figure 5; Table 2); however, no significant rhythm according to the

COSINOR

analyses in sucrose preference

was observed (Figure 5; Table 2). Finally, single Per2Brdm1 and double Per1-/--Per2Brdm1 mutant animals did not show any significant daily rhythm of sucrose intake or preference (Figure 5; Table 2). 24h sucrose intake was not different between genotypes (WT, 17.7±1.2mL; Per1-/-, 19.1±2.1mL; Per2Brdm1, 18.5±5.8mL; Per1-/--Per2Brdm1, 15.1±3.7mL; F(3,12)= 0.23, p=0.87). Discussion In the present study we observed that intake and preference for a caloric sweet solution follow a daily and circadian rhythmic pattern in WT mice, with a main consumption and preference at the active period (night), even when animals were under constant darkness conditions (subjective night). Since this rhythmic behavior was abolished in clock gene deficient mice (Per2Brdm1 and Per1-/--Per2Brdm1 double mutants), preference and intake of sucrose must be dependent on a circadian mechanism. Moreover, day-night Per2 clock gene expression in the SCN, the CP and Ctx was affected when animals had the free choice to consume sucrose.

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Behaviors, including feeding and drinking, show a circadian component (Freund, 1970; Possidente and Birnbaum, 1979; Poirel and Larouche, 1986). In rats and mice it has been previously reported that intake of sucrose takes place mainly during the dark or activity phase (Spector and Smith, 1984; Ryabinin et al., 2003; Tonissaar et al., 2006; Feillet et al., 2015). Moreover, intake and preference in mice is strain dependent (Lewis et al., 2005), with 10% being the highest preferred concentration in C57BL6J mice. Here we report that the intake of hedonic caloric solutions also follows a daily (in LD conditions) and circadian (under DD conditions) rhythm. Whether these behavioral rhythms are dependent on the main SCN clock or another brain body clock, however, is not known (Figure 6). SCN lesions abolish circadian rhythms in drinking (Stephan and Zucker, 1972) and feeding behavior (Nagai et al., 1978; Van den Pol and Powley, 1979). However, no evidence for the effects of SCN lesions on the intake of sucrose, to our knowledge, exists yet. In the circadian system, the expression of daily rhythms in extra-SCN brain regions and in the periphery is now well documented (Herzog, 2007; Albrecht, 2012). Thus, this leads to the hypothesis that extra-SCN clocks regulate specific behaviors, or may support the circadian activity of the SCN to maintain a synchrony in the whole set of behaviors of animals (Herzog, 2007). Among the possible reward-timekeeping brain sites regulating rhythms of sucrose intake we may suggest those controlling the regulation of DA and orexin (ORX), two important transmitters for food motivated behaviors (Castro et al., 2015). DA release by the VTA to the striatum shows daily rhythms with a peak at night in rats and mice (Hampp et al., 2008; Hood et al., 2010), and firing rate of VTA neurons shows daily rhythms (Luo et al., 2008). This rise in DA release fits in time with the main intake and preference for sucrose. Diurnal rhythms of other motivated behaviors have been previously reported (Webb et al., 2015). For example, cocaine self-administration and brain self-stimulation show a daily and a circadian rhythm with a peak at night in rats (Terman and Terman, 1970, 1975; Bass et al.,

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2010). Therefore, the timekeeping mechanisms in the DAergic system may be implicated in the control of rewarded behaviors including sucrose intake (Figure 6). DA release in the forebrain (NAcc, cortex) has an important role in reinforced behaviors (Berridge, 2007), increasing locomotor activity for anticipation and seeking behaviors. DA release is stimulated by drugs of abuse such as cocaine or opioids, and by natural stimuli such as food (Di Chiara and Imperato, 1988; Hernandez and Hoebel, 1988b, a; Wise et al., 1995; Hajnal and Norgren, 2001). Here we observed changes (clock gene expression) in the principal targets for DA neurotransmission, the striatum and Ctx of animals exposed to sucrose. Per2 gene was up-regulated at night in CP and Ctx of animals drinking 10% sucrose. Similarly, we previously reported an increase of Per genes in the striatum and cortex of mice exposed to daily chocolate (Mendoza et al., 2010), and of c-Fos expression in reward-related areas in rats exposed to caloric foods (Mendoza et al., 2005; Mitra et al., 2011). Moreover, sucrose intake induces DA release in the forebrain (Bassareo et al., 2002; Rada et al., 2005; Tellez et al., 2016), and the activation of DA receptors leads to the transcription of Per genes. DA has an action on the phosphorylation of mitogen-activated protein kinase (MAPK) and the cAMP response element-binding protein (CREB) which induces an increase of Per genes expression in the brain (Yan et al., 1999; Akashi and Nishida, 2000). Similarly to the present results, cocaine or methamphetamine acute injections induce the expression of Per genes in the CP and the Ctx but not in the NAcc (Nikaido et al., 2001; Iijima et al., 2002; Falcon et al., 2013). Moreover, in vitro, the D1 receptor agonist SKF38393 stimulates the expression of Per1 and Npas2 clock genes in striatal neurons (Imbesi et al., 2009). All these studies indicate that non-natural (drugs) and natural rewards (sucrose) induce the release of DA from the VTA to the forebrain which will lead in the induction of clock genes. Per2 gene expression was also affected in the SCN of sucrose-exposed mice, significantly in the rostral and medial regions. Although the Per gene expression is mainly activated by light

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in the SCN (Albrecht et al., 2001), non photic cues, including those related to metabolic and reward aspects of feeding, affect Per gene levels in the SCN (Mendoza, 2007). Daily palatable diets down-regulate Per gene expression in the SCN (Mendoza et al., 2010), and exercise or arousal cues, which have a rewarding value also reduce Per expression in the clock (Maywood et al., 1999). How these motivational cues (e.g., food, exercise) impact the SCN clock is not fully known, but this may depend on the functional relationships of the SCN with brain reward structures. The ORX system regulates feeding, reward and addiction (Castro et al., 2015), shows circadian activity (Deboer et al., 2004; Feillet et al., 2015), and projects to the SCN (Figure 6) to down-regulate the activity of the clock (Belle et al., 2014). Behaviors related to the reward effects of drugs like cocaine have been observed in the locomotor sensitization (i.e., increase of locomotion after repeated administrations of a drug) and conditional place preference (CPP) in mice (Abarca et al., 2002). Interestingly, these behaviors show daily variations and are affected in clock mutant mice. For example, Per2Brdm1, but not Per1-/-, mutant mice show an increased behavioral sensitivity and CPP to cocaine (Abarca et al., 2002). Previous studies have shown that the clock gene Per2 is an important modulator of the DAergic system (Hampp et al., 2008). Per2Brdm1 mutant mice show higher concentrations of DA at the level of the striatum due to a down-regulation effect in the monoaminoaxidase A (MAO), the enzyme that degrades catecholamines (Hampp et al., 2008). Per2Brdm1 mutant mice also voluntarily consume more alcohol than WT animals; however, food consumption and sucrose preference are not different between genotypes (Spanagel et al., 2005). Similarly, here, using the same strain of Per2Brdm1 mutant mice, we observed no differences in the amount of food (data not shown) or sucrose intake between mutants vs. WT animals. However, the rhythmic patterns of sucrose intake and preference were significantly different. In Per2Brdm1 and double Per1-/--Per2Brdm1 animals, no rhythms of sucrose intake or

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preference were found. Thus, energy intake is similar and not affected by mutations, but the rhythmic drinking behavior for sucrose is clearly altered in both Per2Brdm1and double Per1-/-Per2Brdm1 mice. Per1-/- mutant mice and WT littermates do not differ in sucrose intake, although the daily pattern preference showed a significant rhythm in WT, but not in the Per1-/mutant mice, according to the

COSINOR

analyses. Both genotypes drink sucrose during early

night, but Per1-/-mutants reduced sucrose preference at the early night in comparison to WT control animals. For the intake of alcohol or cocaine behavioral responses, whereas Per2Brdm1 mutants show behavioral alterations, Per1-/- mutants do not show robust differences to WT mice (Abarca et al., 2002; Zghoul et al., 2007). This last point may implicate a different role of Per1 vs. Per2 genes in behavior related to reward. In the present study we used both male and female mutant mice, but it will be interesting and necessary for future experiments to evaluate the possible sex difference in the circadian rhythms of motivated behaviors. In humans, variations of the Per2 gene are correlated with an increase in ethanol intake, similarly as in Per2Brdm1 mutant mice (Spanagel et al., 2005; Blomeyer et al., 2013). Under a social defeat stress Per1-/- mutant mice show a higher alcohol intake with respect to their wildtype littermates (Dong et al., 2011). In a similar manner, in young adults genetic variations of the hPer1 promoter are correlated with higher alcohol consumption and psychosocial misfortune (Dong et al., 2011). Hence, Per genes deregulation correlates with an increase or susceptibility to alcohol drinking behavior under normal or stressful situations. Therefore, in accordance with the present data, Per genes may regulate consumption of natural and nonnatural rewards and give new understanding of the neurobiological mechanisms underlying addicted behaviors. Both Per1 and Per2 genes are important in the clock mechanism and also they are induced by light. Behaviorally, Per1-/- mutants display robust circadian rhythms with a moderately shorter period (Zheng et al., 2001). However, Per2Brdm1 and double Per1-/-Per2Brdm1 mutant mice exhibit loss of circadian rhythmicity under DD conditions (Zheng et

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al., 1999; Zheng et al., 2001). In light resetting, whereas Per1-/- mutants do not show phase advances in behavior when they receive a light pulse at the late night, Per2Brdm1 animals are not able to delay the phase of rhythms in behavior when light pulse is at the early night (Albrecht et al., 2001). Therefore, both genes are differentially implicated in the control of rhythmic behaviors, including not only general locomotor activity but also motivated behaviors such as sucrose intake. Conclusion The present study demonstrates that sucrose intake and preference are rhythmic in mice, with higher intake at night (activity phase) in correlation with the higher expression of clock genes in the CP and Ctx. Moreover, this work provides evidence that these rhythms persist in DD conditions, and that animals lacking clock genes show altered rhythms of sucrose intake and preference. To summarize, the present data give new insights into the circadian regulation of palatable-feeding behaviors that can be useful for the understanding of the brain timekeeping mechanism implicated in palatable food intake, overconsumption and addiction. Funding and Disclosure Funding sources of the present study were provided by the Agence National de la Recherche (ANR-14-CE13-0002-01 ADDiCLOCK JCJC to JM), Foundation pour la Recherche Medicale/Danone consortium (JM) and the Centre National de la Recherche Scientifique (JM). Authors declare no conflict of interest. Acknowledgements We thank deeply Prof. Urs Albrecht (University of Fribourg) for providing the Per1-/- and Per2Brdm1 mutant mice, and Dr. Tando Maduna for comments and suggestions in the manuscript.

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Figure legends Figure 1. Daily rhythms of drinking behavior in mice. (A) Daily rhythms of water intake in the control group exposed to two bottles of tap water under a LD 12h-12h cycle. Animals drank water mainly at night, but no differences in the preference (B) between the two bottles were found. (C) In animals exposed to a bottle of tap water and a bottle of 5% sucrose, a daily rhythm of intake for sucrose, but not water, was found. (D) Moreover, sucrose preference was significantly higher at night. (E-F) Similar results were observed in the group of animals exposed to 10% of sucrose. Symbols represent the mean ± SEM. Suc, sucrose; H2O, water. Figure 2. Circadian rhythms of drinking behavior in mice. (A) Circadian rhythms of water intake in the control group exposed to two bottles of tap water in constant darkness conditions. Animals drank water mainly at the subjective night but, similar to LD conditions, no preference (B) for any of the two bottles was found. (C-D) In animals exposed to a bottle of tap water and a bottle of 10% sucrose, under DD conditions, a significant circadian rhythm of intake and preference for sucrose was found. Symbols represent the mean ± SEM. Suc, sucrose; H2O, water. Figure 3. Brain Per1 gene expression in mice. Subjective day-night expression of Per1 gene in the SCN, NAcc, CP and Ctx of animals drinking tap water or sucrose (10%) under DD conditions. (Top) Representative autoradiographic images of Per1 mRNA expression across forebrain of control and sucrose-exposed animals sampled at the circadian (CT) time 6 and 18 (mid-subjective day and night respectively). (Bottom) Circadian expression of the Per1 mRNA in different levels of the SCN (rostral, medial and caudal), nucleus accumbens (NAcc, both shell and core), caudate-putamen (CP) and cortex (Ctx, motor and cingulated cortex) from control animals (drinking tap water, black symbols) and mice drinking 10% sucrose (pink symbols). Symbols represent the mean ± SEM.

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Figure 4. Brain Per2 gene expression in mice. Subjective day-night expression of Per2 gene in the SCN, NAcc, CP and Ctx of animals drinking tap water or sucrose (10%) under DD conditions. (Top) Representative autoradiographic images of Per2 mRNA expression across forebrain of control and sucrose-exposed animals sampled at the CT6 and CT18. (Bottom) Subjective day-night Per2 expression in the SCN (anterior and median) and CP was significantly down regulated at the subjective day time point (CT6), and up-regulated at the subjective night (CT18) in the Ctx. Asterisks indicate significant differences between groups (H2O vs. sucrose; p<0.05 Post hoc LSD test). Symbols represent mean ± SEM. Figure 5. Daily rhythms of drinking behavior in clock mutant mice. Rhythmic behavior of sucrose consumption and preference (closed symbols) and water intake (open symbols) in WT, Per1-/-, Per2Brdm1 and double Per1-/-- Per2Brdm1 mutant mice. WT and Per1-/- mutant animals show significant daily rhythms in sucrose intake. WT animals, but not Per1-/- mutant, showed a significant rhythm of sucrose preference. Both Per2Brdm1 and double Per1-/-Per2Brdm1 do not show rhythms in sucrose intake and preference. Symbols represent mean ± SEM. Figure 6. Circuit model for circadian regulation of sucrose intake. The SCN synchronizes forebrain oscillators (via clock output genes, vasopressin, AVP; prokineticin 2, PK2) which show circadian rhythms of Per genes expression in opposite phase to the SCN. Sucrose intake shows daily and circadian rhythms (with a higher intake and preference at night) that might depend directly or indirectly in the forebrain and SCN circadian activity, respectively, and the Per genes expression. Sucrose intake has feedback effects (as a non-photic zeitgeber) on the clock gene expression in both forebrain structures and the SCN, probably trough neurotransmission pathways implicated in reward (e.g., dopamine, orexin, opioids).

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Table 1. Summary of statistics for the COSINOR analyses of drinking behavior in mice in a 12/12h light-dark cycle (LD) or constant darkness conditions (DD), and exposed to two Mean

Amplitude

Acrophase (ZT/CT)

p

LD H2O Intake (Left) mL H2O Intake (Right) mL Preference (Left vs.Right) %

2.29 1.73 56.46

0.65 0.46 0.45

19.58 20.89 19.08

0.001 0.001 0.98

H2O Intake, mL Sucrose intake (5%) mL Preference (Suc vs.H2O) %

1.54 4.29 69.56

0.35 2.43 7.54

22.52 18.94 17.39

0.11 0.001 0.04

H2O Intake, mL Sucrose intake (10%) mL Preference (Suc vs.H2O) %

1.21 2.73 64.30

0.21 1.97 11.17

18.25 16.55 17.55

0.15 0.0004 0.03

DD H2O intake (Left) mL H2O intake (Right) mL Preference (Lvs.R) %

2.18 2.48 48.24

2.92 3.47 5.79

15.58 15.62 20.20

0.0003 0.0004 0.50

H2O intake, mL Sucrose intake (10%) mL Preference (Suc vs.H2O) %

2.05 5.39 66.74

1.56 5.36 10.45

15.44 16.45 18.17

0.001 <0.0001 0.02

bottles of water (control), or 5 or 10% of sucrose (Suc.) and a bottle of water (H2O). Acrophases are in ZT for LD experiment and CT for experiments in DD.

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Table 2. Summary of statistics for the COSINOR analysis of drinking behavior in WT and Per mutant mice in a 12/12 h light-dark cycle (LD) and exposed to either 10% of sucrose or a Genotype

Mean

Amplitude

Acrophase (ZT)

p

WT Water intake (mL) Sucrose intake (mL) Sucrose preference (%)

1.58 4.44 71.27

0.24 2.71 7.34

17.70 15.52 15.07

0.20 0.0004 0.02

Per1-/Water intake (mL) Sucrose intake (mL) Sucrose preference (%)

2.55 4.79 64.04

0.17 1.65 5.26

16.14 13.98 13.22

0.73 0.01 0.26

Per2Brdm1 Water intake (mL) Sucrose intake (mL) Sucrose preference (%)

2.88 4.62 56.53

0.28 0.39 1.83

17.29 17.31 15.11

0.15 0.93 0.94

Per1-/-Per2Brdm1 Water intake (mL) Sucrose intake (mL) Sucrose preference (%)

1.58 3.78 68.76

0.43 0.72 1.77

17.06 14.93 15.95

0.05 0.55 0.91

bottle of water.

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Highlights -Sucrose intake and preference show a daily and a circadian rhythm in mice. -The Per2 gene expression in the brain is affected in sucrose-exposed animals. -Per2 mutations lead to a loss of daily rhythms of sucrose intake and preference. -Circadian rhythms of hedonic feeding are modulated by a brain timekeeping mechanism.

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