Sucrose modifies c-fos mRNA expression in the brain of rats maintained on feeding schedules

Neuroscience 192 (2011) 459 – 474

SUCROSE MODIFIES c-fos mRNA EXPRESSION IN THE BRAIN OF RATS MAINTAINED ON FEEDING SCHEDULES A. MITRA, C. LENGLOS, J. MARTIN, N. MBENDE, A. GAGNÉ AND E. TIMOFEEVA*

distinct neuronal network compared to neuronal activation produced by scheduled access to regular chow. These data provide evidence that the brain may contain different foodoscillatory systems and that food palatability may shift the neuronal activity from the medial hypothalamus to the limbic and reward-related areas even at the negative metabolic state. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Faculty of Medicine, Department of Psychiatry and Neuroscience, Université Laval, Québec (QC), G1V 4G5, Canada

Abstract—Food intake is regulated according to circadian activity, metabolic needs and the hedonic value of food. Rodents placed on a fixed feeding schedule show behavioral and physiological anticipation of mealtime referred to as food-anticipatory activity (FAA). FAA is driven by the foodentrainable oscillator (FEO), whose anatomical substrate is not yet known. Recent data have shown that restricted feeding schedules for regular chow and daily limited access to palatable food in free-feeding rats activate distinct brain regions during FAA. The combination of a deprivation regimen and scheduled access to palatable food may give rise to a more global anticipatory mechanism because the temporal cycles of energy balance would be strongly modulated by the incentive properties of palatable food; however, the neuronal response to this combined treatment is not yet known. The present study investigated how adding palatable sucrose to feeding schedules affects the pattern of brain c-fos mRNA expression during FAA (0 –3 h) and 1 h following feeding. The rats maintained on scheduled chow access increased their daily chow intake, while the rats maintained on scheduled sucrose and chow mainly increased their daily sucrose intake. Adding sucrose to scheduled feeding displaced c-fos mRNA expression from the dorsomedial and paraventricular hypothalamic nuclei and posterior lateral hypothalamus (LH) to the prefrontal cortex, lateral septum, nucleus accumbens and anterior LH. During refeeding, the rats on scheduled sucrose demonstrated higher activation of the nucleus of the solitary tract. The present results suggest that palatable sucrose combined with restricted feeding schedules activate a

Key words: food-anticipatory activity, palatable food, c-fos, neuronal activation, hypothalamus, limbic system.

Food intake is regulated according to circadian activity, metabolic needs, and the hedonic value of the food. Generally, in free-feeding conditions, rodents start to eat at the beginning of darkness. This circadian activity is driven by a central pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus (Klein et al., 1991). The SCN is regarded as the light-entrainable oscillator (LEO), whose activity is regulated according to environmental light/dark signals conveyed by the retinohypothalamic tract directly to the SCN (Hannibal and Fahrenkrug, 2005). SCN ablations eliminate or disrupt circadian activity, but do not prevent biological rhythms associated with restricted feeding schedules (RFS), indicating the presence of a foodentrainable oscillator (FEO) that may function independently of the SCN (Marchant and Mistlberger, 1997; Mistlberger, 1993; Moore and Eichler, 1972). Rodents placed on a fixed feeding schedule when daily food access is restricted to a few hours during the light period show behavioral and physiological anticipation of mealtime. This increased exploration and foraging locomotion within the hours preceding food access is referred to as food-anticipatory activity (FAA) (Stephan, 2001). Fluctuations of time-fixed caloric intake and negative metabolic state seem to be important for the development of the FAA. The caloric restriction represents a strong metabolic challenge (Pecoraro et al., 2002). Alleviation of the negative metabolic state in food-restricted rats by daily brief access to food has potential effect on the shift of circadian activity (Mendoza et al., 2008). The anatomical substrate of the FEO is not yet known. The feeding-fasting cycles have strong entraining effects on peripheral oscillators (Schibler et al., 2003; Stokkan et al., 2001; Stratmann and Schibler, 2006). However, resistance of FAA to adrenalectomy (Boulos and Terman, 1980), sub-diaphragmatic vagotomy (Comperatore and Stephan, 1990; Moreira and Krieger, 1982), capsaicininduced deafferentation (Davidson and Stephan, 1998), as well as the persistence of FAA in cirrhotic (Escobar et al., 2002) and diabetic (Davidson et al., 2002) rats cast doubt

*Corresponding author. Tel: ⫹1-418-656-8711 ext. 3749; fax: ⫹1-418656-4942. E-mail address: [email protected] (E. Timofeeva). Abbreviations: AcbCoL, lateral part of the nucleus accumbens core; AcbCoM, medial part of the nucleus accumbens core; AcbSh, nucleus accumbens shell; ANOVA, analysis of variance; AP, area postrema; DMHc, compact part of the dorsomedial hypothalamic nucleus; DMHv, ventral part of the dorsomedial hypothalamic nucleus; FAA, foodanticipatory activity; FEO, food-entrainable oscillator; HPA, hypothalamic-pituitary adrenal axis; IL, infralimbic cortex; LEO, light-entrainable oscillator; LHapf, anterior part of the perifornical lateral hypothalamic area; LHcpf, caudal part of the perifornical lateral hypothalamic area; LSmv, medioventral part of the lateral septum; NTSc, caudal part of the nucleus of the solitary tract; NTSm, medial part of the nucleus of the solitary tract; NTSr, rostral part of the nucleus of the solitary tract; OD, optical density; OFC, orbitofrontal cortex; PFCg, prefrontal cingulate cortex; Pir, piriform cortex; PVHm, magnocellular part of the paraventricular hypothalamic nucleus; PVHp, parvocellular part of the paraventricular hypothalamic nucleus; PVTa, anterior part of the paraventricular thalamic nucleus; RFS, restricted feeding schedules; SCN, suprachiasmatic nucleus; SHi, septohippocampal nucleus; SON, supraoptic nucleus; VO2, volume of oxygen consumption; ZT, Zeitgeber time.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.06.033

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on the peripheral location of the FEO. In the central nervous system, several ablations of a single brain structure were not effective (Davidson et al., 2001a,b; Honma et al., 1987; Mistlberger and Mumby, 1992; Mistlberger and Rusak, 1988) or were inconsistent (Gooley et al., 2006; Landry et al., 2007a,b; Nakahara et al., 2004) for preventing food entrainment. Analyses of neuronal activation during FAA, assessed by detection of the induction of expression of immediate early gene c-fos, have suggested that the FEO may be represented by a distributed neuronal network (Poulin and Timofeeva, 2008). This inference is supported by neural tissue culturing showing that the brain contains multiple, damped circadian oscillators outside the SCN and that phasing of these oscillators to one another may play a critical role in coordination of behavioral and metabolic activity (Abe et al., 2002). The hedonic value of food may also entrain the FAA. To engender anticipatory activity in ad-libitum-fed rats, palatable food might be nutrient-rich and might be consumed in sufficient amounts (Mistlberger and Rusak, 1987; Stephan and Davidson, 1998; Waddington Lamont et al., 2007). FAA induced by daily access to 5 g of chocolate in ad-libitum-fed rats (Mendoza et al., 2005b; Mistlberger and Rusak, 1987) was accompanied by an increase in Fos protein expression in the limbic system nuclei, but not in the hypothalamus (Mendoza et al., 2005b). Conversely, the rats entrained on the chow-restricted schedules showed a significant increase in the induction of c-fos mRNA and Fos protein expression in the hypothalamic regions in addition to some extra-hypothalamic sites (Mendoza et al., 2005b; Poulin and Timofeeva, 2008). Therefore, feeding–fasting cycles and scheduled access to palatable food can entrain the FAA by affecting the distinctive central neuronal network. The stronger behavioral effects of the combination of a negative metabolic state and scheduled access to palatable food (Pecoraro et al., 2002) raise the possibility that these combined treatments may affect a larger or distinct neuronal network compared to that seen in rats maintained on chow-restricted schedules or on the time-fixed feeding of palatable food. To test these hypotheses, a highly palatable sucrose solution was added to the chow-restricted schedules, and the time course of c-fos mRNA expression was detected during food anticipation and following feeding in rats maintained for 3 weeks on scheduled access to chow or on combined access to sucrose and chow. Detecting c-fos mRNA expression allowed rapid simultaneous screening of neuronal activation throughout the entire brain. The present study has shown that rats maintained on scheduled access to chow increased their daily chow intake, while the rats maintained on scheduled access to sucrose and chow mainly increased their daily sucrose intake. During food anticipation, the sucrose schedules displaced the neuronal activity from the tuberal dorsomedial, lateral, and paraventricular hypothalamus to the prefrontal cortex, ventral striatum, and the anterior lateral hypothalamus. These results suggest that palatable sucrose combined with restricted feeding schedules activates a distinct neuronal

network compared to neuronal activation produced by scheduled access to regular chow.

EXPERIMENTAL PROCEDURES Animals and housing conditions Male Wistar rats (n⫽76), aged 6 weeks, were purchased from the Canadian Breeding Laboratories (St.-Constant, QC, Canada). Animals were housed individually in plastic cages lined with wood shavings and maintained on a 12:12-h dark–light cycle [light-on between 06h00 (Zeitgeber time 0 —ZT0) and 18h00 (ZT12)], with ambient temperature of 23⫾1 °C, free access to tap water, and the standard laboratory rat diet (Rat/Mouse/Hamster chow; 1000 Formula; 12.9 kJ/g; Agway Prolab), unless otherwise specified. Rats were acclimated to environmental conditions for 13 days before starting the experimental procedure. Therefore, at the beginning of entrainment, the rats were at their late adolescent-early adult age. The present experiments involved young rats to avoid age-related decline in FAA (Tanaka et al., 2000). All rats were cared for and handled according to the Canadian Guide for the Care and Use of Laboratory Animals, and the present protocol was approved by our institutional animal care committee.

Feeding schedules During 3 weeks, four groups of rats (60 rats in total) were fed regular chow ad libitum (AL groups). The rats maintained on feeding schedules had daily 2-h access to food between 12h00 (ZT6) and 14h00 (ZT8) (Fig. 1B), during which the rats received regular chow (SC groups) or chow and 10% (1.7 kJ/ml) sucrose (SS groups). On the fourth week, the groups of food-restricted rats were anesthetized and perfused during food-anticipation period before the access to food at ZT3 (9h00), ZT5 (11h00), and ZT6 (12h00). The SC-ZT7 and SS-ZT7 groups received their respective daily food at the usual time and ate during 1 h before being anesthetized and perfused. Four ad-libitum-fed groups were sacrificed simultaneously (at ZT3, ZT5, ZT6, and ZT7) with the respective groups of rats maintained on scheduled feeding. The time of sacrifice was fixed to the critical points detected in our previous study (Poulin and Timofeeva, 2008). This time course allowed tracking of early to maximum expressing of c-fos mRNA in the brain during food anticipation, as well as to compare the pattern of c-fos mRNA expression during food anticipation and feeding. Because the time points of sacrifice were fixed, on each consecutive day of the fourth week of the experiment (overall 5 days) one rat per group was sacrificed (from AL, SC, and SS groups simultaneously for each time point). Until the moment of sacrifice, all rats were maintained on their respective feeding regimens (AL, SC, or SS). One-way analysis of variance (ANOVA) confirmed that the day of sacrifice did not represent a significant factor for c-fos mRNA expression in all brain regions investigated in this study. This experiment included 12 groups (four time points for three feeding regimens) with five rats per group.

Food intake measurements Food intake was measured every day. Intake of chow and 10% sucrose solution was measured by subtracting the remaining amount of chow and sucrose solution from the fixed amount provided to the rats. Chow spillage was carefully calculated and accounted for in the measurements.

Locomotor activity and oxygen consumption An additional cohort of 16 rats was used to assess locomotor activity and oxygen consumption during food-anticipatory period. Rats were housed in individual calorimetric cages connected to a data acquisition system (AccuScan Instruments, Columbus, OH,

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Fig. 1. (A) Daily food intake of the rats fed ad libitum chow [AL (n⫽20); 24-h chow intake (Œ)] and the rats submitted to scheduled access to chow [SC (n⫽20); 2-h daily chow intake (Œ)] or scheduled access to chow and sucrose [SS (n⫽20); 2-h daily chow (⽧) and sucrose (⽧) intake]. Note that the SC rats increased their daily chow intake, while the SS rats escalated their sucrose intake. * Significantly different from the AL group on the same experimental day. †Significant difference between the amounts of consumed chow by the SC and SS groups during the same experimental day. (B) Schematic illustration of scheduled feeding (2 h per day, between ZT6 and ZT8) and time of sacrifice for the experimental groups 3 (at ZT3) and 1 (at ZT5) h before and at the expected meal time (at ZT6), and after 1 h of feeding (at ZT7). The control AL groups were sacrificed at the same time as the respective SC and SS groups.

USA) and maintained under 12:12-h dark–light cycle [light-on between 06h00 (ZT0) and 18h00 (ZT12)], with ambient temperature of 23⫾1 °C and free access to tap water. During the first week all rats were fed ad libitum with the standard laboratory rat chow. Thereafter, rats were randomly divided to groups that had scheduled 2-h daily (between ZT6 and ZT8) restricted access to chow (SC rats, n⫽8) or chow and 10% sucrose (SS rats, n⫽8) during 3 weeks. Total horizontal locomotor activity was monitored continuously with infrared detectors located on the walls of the cages and volume of oxygen consumption (VO2) was measured by an oxygen analyzer. Activity counts and VO2 were stored in 15-min intervals. Every day recordings were interrupted between ZT8 and ZT10 for recalibration of oxygen analyzer. The actograms for horizontal activity were created for each rat (as shown on the Fig. 2A). On the actogram the horizontal lines correspond to the day of the experiment, and the activity was plotted for each 15 min as a percentage of the absolute maximal activity recorded over entire experiment. The waveforms of the horizontal activity (Fig. 2B) and VO2 (Fig. 2C) average data for rats in the same feeding condition for selected blocks of days (AL: 1–7 day; SC and SS: 11–28 day of the experiment).

Brain preparation The brains were prepared as previously described (Timofeeva et al., 2005). Briefly, rats were rapidly anesthetized with a mixture of ketamine (60 mg/kg) plus xylazine (7.5 mg/kg), which produced deep anesthesia within 1–2 min. Without delay, rats were perfused intracardially with 200 ml of ice-cold isotonic saline followed by 500 ml of a paraformaldehyde (4%) solution. The brains were removed at the end of perfusion and kept in paraformaldehyde for an additional period of 7 days. They were then transferred to a solution containing paraformaldehyde (4%) and sucrose (10%) before being cut 12 h later, using a sliding microtome (Histoslide 2000, Reichert-Jung, Heidelberger, Germany). Brain sections

were taken from the olfactory bulb to the brainstem. Thirty-␮mthick sections were collected and stored at ⫺30 °C in a cold sterile cryoprotecting solution containing sodium phosphate buffer (50 mM, pH 7.2), ethylene glycol (30%), and glycerol (20%).

In situ hybridization histochemistry In situ hybridization histochemistry was used to localize c-fos mRNA on tissue sections taken from the entire brain. The protocol used was largely adapted from the technique described by Simmons et al. (Simmons et al., 1989). Briefly, the brain sections were mounted onto poly L-lysine coated slides and allowed to desiccate overnight under vacuum. They were then successively fixed for 20 min in paraformaldehyde (4%), digested for 25 min at 37 °C with proteinase K (10 ␮g/ml in 100 mM Tris–HCl containing 50 mM EDTA, pH 8.0), acetylated with acetic anhydride (0.25% in 0.1 M triethanolamine, pH 8.0), and dehydrated through graded concentrations (50, 70, 95, and 100%) of alcohol. After vacuum drying for at least 2 h, 90 ␮l of the hybridization mixture, which contains an antisense 35S-labeled cRNA probe (107 cpm/ml), was spotted on each slide. The slides were sealed under a coverslip and incubated overnight at 60 °C in a slide warmer. The next day, coverslips were removed and the slides were rinsed four times with 4⫻ standard saline citrate (SSC; 0.6 M NaCl, 60 mM trisodium citrate buffer, pH 7.0), digested for 30 min at 37 °C with RNase-A (20 ␮g/ml in 10 mM Tris–500 mM NaCl containing 1 mM EDTA), washed in descending concentrations of SSC (2⫻, 10 min; 1⫻, 5 min; 0.5⫻, 10 min; 0.1⫻, 30 min at 60 °C), and dehydrated through graded concentrations of alcohol. After a 2-h period of vacuum drying, the slides were exposed on an X Ray film (Eastman Kodak, Rochester, NY, USA) for 24 h. Once removed from the autoradiography cassettes, the slides were defatted in xylene and dipped in NTB2 nuclear emulsion. The slides were exposed for 7 days, before being developed in D19 developer for 3.5 min at 14 –15 °C and fixed in rapid fixer (Eastman Kodak, Rochester, NY,

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A. Mitra et al. / Neuroscience 192 (2011) 459 – 474 USA) for 5 min. Finally, tissues were rinsed in running distilled water for 1–2 h, counterstained with Thionin (0.25%), dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with DPX.

Antisense

35

S-labeled probe

The c-fos cRNA probe was generated from the EcoR I fragment of rat c-fos cDNA (Dr. I. Verma, The Salk Institute, La Jolla, CA, USA) sub-cloned into pBluescript SK-1 plasmid (Stratagene, La Jolla, CA, USA). Radioactive riboprobe were synthesized by incubation of 250 ng linearized plasmid in 10 mM NaCl, 10 mM dithiothreitol, 6 mM MgCl2, 40 mM Tris (pH 7.9), 0.2 mM ATP/ GTP/CTP, ␣-35S-UTP, 40 U RNasin (Promega, Madison, WI, USA), and 20 U of T7 or T3 RNA polymerase for, respectively, antisense and sense probes of c-fos RNA for 60 min at 37 °C. The DNA templates were treated with 100 ␮l of DNAse solution (1 ␮l DNAse, 5 ␮l of 5 mg/ml tRNA, 94 ␮l of 10 mM Tris/10 mM MgCl2). The purification of the riboprobes was accomplished by using a QIAGEN RNeasy Mini Kit (QIAGEN, Mississauga, ON, Canada). The specificity of the probe was confirmed by the absence of positive signal in sections hybridized with sense probe.

Quantitative analysis of hybridization signals

Fig. 2. (A) Actograms of the horizontal activity of two rats fed ad libitum chow for the first 7 d, and thereafter submitted for 3 wk to 2-h (between ZT6 and ZT8; shown by transparent rectangles) restricted scheduled access to chow (SC rat, left actogram) or chow and 10% sucrose (SS rat, right actogram). The night (ZT12–ZT24) period is shaded. (B) Average of the horizontal activity. (C) Average of the oxygen consumption (VO2). The waveforms (B, C) represent ad libitum chow feeding (Œ; corresponds to 1–7 days of the experiment), scheduled chow (Œ; corresponds to 11–28 days), or scheduled chow and sucrose (⽧; corresponds to 11–28 days). * AL significantly different from both SC and SS. † Significant difference between the SC and SS groups.

The hybridization signals revealed on NTB2-dipped nuclear emulsion slides were analyzed and quantified under a light microscope (Olympus BX 60, Markham, OV, Canada) equipped with video camera (RT Slider, model 2.3.0, Diagnostic Instruments Inc., Sterling Heights, MI, USA) using Image Pro-Plus software (version 6.0 for Windows). The intensity of the hybridization signal was measured under darkfield illumination at a magnification of 25⫻. Saturation of the hybridization signal was avoided by adjusting the exposure time for the image with the strongest hybridization signal sampled for each region in every series. The luminosity of system was set to the maximum and the saturation warning option was used to visualize saturated regions in the image preview. Thereafter, according to the pixel distribution histogram, the exposure time was adjusted to reduce to zero the number of saturated (pure white) pixels. The same luminosity and exposure time was conserved for the analysis of entire series. The analyses of expression of c-fos mRNA have been done for the brain regions demonstrating positive hybridization signal above background. The orbitofrontal cortex (OFC; 3.20 –2.70 mm rostral to bregma), infralimbic (IL) and piriform (Pir) cortex (3.70 mm rostral to bregma), the septohippocampal nucleus (SHi), cingulate prefrontal cortex (PFCg), medioventral part of the lateral septum (LSmv), nucleus accumbens shell (AcbSh), lateral and medial parts of the nucleus accumbens core (AcbCoL and AcbCoM, respectively) (1.40 –1.00 mm rostral to bregma), the anterior part of the paraventricular thalamic nucleus (PVTa) and SCN (0.92–1.30 mm caudal to bregma), the supraoptic nucleus (SON) (1.30 –1.80 mm caudal to bregma), the parvocellular and magnocellular paraventricular hypothalamic nucleus (PVHp and PVHm, respectively) and anterior part of the perifornical lateral hypothalamic area (LHapf) (1.60 –1.30 mm caudal to bregma), the compact and ventral parts of the dorsomedial hypothalamic nucleus (DMHc and DMHv, respectively) and caudal part of the perifornical lateral hypothalamic area (LHcpf) (3.14 –3.40 mm caudal to bregma), the rostral (11.96 –12.50 mm caudal to bregma), medial (13.20 –13.45 mm caudal to bregma), and caudal (13.70 –14.00 mm caudal to bregma) parts of the nucleus of the solitary tract (NTSr, NTSm, and NTSc, respectively), and the area postrema (AP) (13.70 –14.00 mm caudal to bregma) were outlined, and the measurements of the optical density (OD) of the hybridization signal were performed separately on each side of the brain on the 2– 4 sections for each animal assigned to each treatment. When no hybridization signal was visible under darkfield illumination, the brain structures of interest were outlined under brightfield illumi-

A. Mitra et al. / Neuroscience 192 (2011) 459 – 474 nation and then subjected to densitometric analysis under darkfield illumination. The OD for each specific region was corrected for the average background signal, which was determined by sampling unlabeled areas outside of the areas of interest.

Statistical analysis Results are presented as mean values⫾standard errors (SE) of the mean. Two-way (3⫻4) ANOVA was used to detect significant main and interaction effects of feeding conditions (AL, SC, and SS) and Zeitgeber time (ZT3, ZT5, ZT6, ZT7) on the measurements of the ODs of hybridization signals in the brain. A posteriori comparisons between groups were realized using the Fisher’s protected least significant difference (PLSD). Food intake, energy intake, body weight, horizontal activity, and VO2 were analyzed by one-way ANOVA followed by Fisher’s PLSD. Results were considered significant with P values ⬍0.05. Each experimental group for analyses of c-fos mRNA expression contained five animals. The experimental groups for analyses of horizontal activity and VO2 included eight animals. Statistical analysis was performed with StatView5 (SAS Institute, Natick, MA, USA) and MATLAB R2010b (The MathWorks, Natick, MA, USA).

RESULTS Food intake, energy intake, and body weight Ad-libitum-fed rats showed a slow gradual increase of their daily chow intake over the 3 weeks of the experiment (for 1.34 times over 3 weeks from 23.52⫾0.86 g on the first day to 31.59⫾1.35 g on the last, 21st day of the experiment; P⫽0.001). The increase in the daily chow intake of rats maintained on scheduled access to chow was also sustained and gradual. In total, the SC rats increased their chow intake by 16 times over the 3 weeks (from 1.68⫾0.31 g on the first day to 25.19⫾1.36 g on the last day of the experiment; P⬍0.0001). Due to this considerable escalation of daily chow intake, at the end of experiment the amount of chow the SC rats ingested during their 2-h food access was non-significantly different from that of the AL rats (P⫽0.074 for the 20th day, and P⫽0.092 for the last, 21st day of the experiment), while at the beginning of the experiment the SC rats’ daily chow intake was significantly low compared to that of the AL rats (P⬍0.0001 for the first day of the experiment) (Fig. 1). In contrast to the slow or high but gradually sustained increase in daily chow intake of the AL and SC rats, respectively, the SS rats increased their 2-h chow intake during the first week of the experiment (from 0.88⫾0.17 g on the first day to 9.04⫾0.39 g on the seventh day of the experiment; P⬍0.0001). However, the SS rats did not increase their chow intake during the second and third weeks of the experiment. Indeed, the SS rats consumed 10.68⫾0.53 g of chow on the eighth day and 10.78⫾1.03 g of chow on the 21st day of the experiment, which was not significantly different (P⫽0.936). The chow intake of the SS rats was significantly low compared to that of the AL rats for all the days of the experiment (Fig. 1). Moreover, the chow intake of the SS rats was significantly lower compared to that of the SC rats from the eighth day (P⫽0.0009) to the last, 21st (P⬍0.0001) day of the experiment. In contrast to the low chow intake, the SS rats sustainably escalated their daily sucrose intake. Indeed, the SS rats 2-h sucrose intake increased by 4.4

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times over the 3 weeks (from 7.81⫾0.71 ml on the first day to 34.91⫾1.75 ml on the last, 21st day of the experiment; P⬍0.0001) (Fig. 1). Over 3 weeks of the experiment the daily energy intake of the AL group increased from 302⫾11.88 kJ (first day) to 427⫾9.75 kJ (21st day). The SC rats gradually increased energy intake from 21⫾3.75 kJ (first day) to 343.84⫾16.01 kJ (21st day). Increase in 10% sucrose (1 g of sucrose: 17 kJ; 1 ml of 10% sucrose: 1.7 kJ) but not chow (1 g of chow: 12.9 kJ) led to gradual increase in total energy intake of SS rats during 1–12 days (from 24.01⫾2.60 kJ to 211.62⫾10.71 kJ, respectively) but not during 13–21 days (from 211.13⫾12.84 kJ to 207⫾13.03 kJ, respectively). As a result, the energy intake of SS rats was not significantly different compared to that in SC rats for the 1–12 days, but was significantly lower for the 13–21 days of the experiment. At the beginning of the experiments all groups of rats had similar body weight (AL: 208.35⫾1.64 g; SC: 205.63⫾1.45 g; SS: 208.98⫾0.93 g). Over 3 weeks of the experiment the food-restricted rats (SC and SS rats) demonstrated lower increase in body weight compared to AL rats (body weight at the 21st day–AL: 370⫾6.42 g; SC: 321.05⫾8.84 g; SS: 235.32⫾12.14 g; P⫽0.0134 AL vs. SC; P⬍0.0001 AL vs. SS). Moreover, at the end of experiment the SS rats had significantly lower final body weight compared to SC animals (P⬍0.0001). Locomotor activity and oxygen consumption An additional cohort of rats (n⫽16) was used to detect how adding of sucrose to feeding schedules affects the motor activity and oxygen consumption. First, all rats were fed chow ad libitum for 7 days (Fig. 2A). Thereafter, rats were randomly divided to SC (n⫽8) and SS (n⫽8) groups with comparable body weight (SC: 302⫾4.44 g; SS: 299.77⫾4.32 g; P⫽0.6402). 2-h daily access to chow (SC rats) or chow and 10% sucrose (SS rats) was scheduled between ZT6 and ZT8 for 3 weeks (Fig. 2A). At the end of this experiment (at the 28th day) the SC rats had significantly higher body weight compared to SS animals (SC: 409.23⫾6.25; SS: 385.97⫾6.96; P⫽0.0475). In this experiment within-subjects (ad-libitum-fed versus restricted feeding) and between-subjects (SC versus SS feeding schedules) analyses have been used to characterize rats’ VO2 and horizontal activity. The waveforms for activity counts (Fig. 2B) and VO2 (Fig. 2C) represent the average for the 7 first days of ad libitum feeding (AL; n⫽16) and 18 days of scheduled feeding (11–28 days of the experiment; SC and SS groups; n⫽8). During the light period, the rats at ad-libitum-fed state showed significantly lower locomotor activity (between ZT3h15 and ZT11h45) and VO2 (between ZT3h45 and ZT11h45) compared to those at the restricted feeding states. Conversely, the rats at ad-libitum-fed state demonstrated higher locomotion (between ZT14h00 –ZT18h15 and ZT20h45–ZT24h00) and VO2 (between ZT14h15 and ZT24h00) during the night. Horizontal activity was significantly higher in SS rats compared to SC animals between ZT2h00 (P⬍0.0001) and ZT3h00 (P⬍ 0.0001); and between ZT4h00 (P⫽0.0231) and ZT6h15

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(P⫽0.0035) (Fig. 2B). VO2 was significantly higher in SS rats compared to SC animals between ZT1h15 (P⫽ 0.0337) and ZT3h30 (P⫽0.0472), as well as at ZT4h15 (P⫽0.0059), ZT4h30 (P⫽0.0381), ZT5h45 (P⫽0.0237), and ZT6h00 (P⫽0.0398) (Fig. 2C). Effects of scheduled feeding on c-fos mRNA expression in the hypothalamus The compact part of the DMH demonstrated early and sustained activation during the food anticipatory period in the SC rats. The expression of c-fos mRNA in the DMHc was significantly induced, compared to other groups, at 3 h (ZT3) and 1 h (ZT5) before the time of daily access to food and at the time when food was expected (ZT6) (Figs. 3 and 4). The c-fos mRNA expression remained at the plateau level after 1 h of feeding in the DMHc of the SC rats (P⫽0.6881 for ZT7 vs. ZT6) (Fig. 3). In contrast, the SS rats did not show any activation of c-fos mRNA expression in the DMHc during food anticipation or feeding (Fig. 4). In fact, at all estimated time points (ZT3, ZT5, ZT6, and ZT7), the levels of c-fos mRNA in the DMHc of the SS rats were considerably lower than those of the SC rats and were not significantly different compared to the c-fos mRNA levels in the control AL rats, in which the positive hybridization signal for c-fos mRNA was barely detectable (Figs. 3 and 4). The ventral DMH also showed significant increase in c-fos mRNA expression at the ZT3, ZT5, and ZT6 in the SC rats compared to those in the SS and AL animals (Fig. 3). One hour of feeding led to an additional increase in c-fos mRNA expression in the DMHv of the SC rats (P⫽0.008, ZT7 vs. ZT6). The levels of c-fos mRNA expression in the DMHv of the SS rats were not different from those in the ad libitum control rats at the ZT3, ZT5, and ZT6, but substantially increased during feeding (P⫽ 0.0002, SS vs. AL at ZT7). However, the induction of c-fos mRNA expression in the DMHv was significantly lower compared to that seen in the SC rats (P⬍0.0001, SC vs. SS at ZT7) (Figs. 3 and 4). ANOVA detected significant main and interactive effects of the feeding condition and Zeitgeber time for c-fos mRNA expression in the AL, SC, and SS rats, in the DMHc and the DMHv (Table 1). During food anticipation, an increase in c-fos mRNA expression was detected in the PVHp in the SC rats at ZT5 (P⫽0.001, SC vs. AL) and ZT6 (P⬍0.0001, SC vs. AL), but not in the PVHp of the SS animals (Figs. 3 and 5). Refeeding similarly stimulated c-fos mRNA expression in the PVHp in the SC and SS rats (P⫽0.083). In the magnocellular regions, the PVHm and SON, food anticipation did not induce c-fos expression in any experimental groups; however, refeeding significantly increased the levels of c-fos mRNA in the SC animals (P⬍0.0001, SC vs. AL; and P⬍0.0001, SC vs. SS, for the both PVHm and SON) but not in the SS (P⫽0.0555, SS vs. AL for the PVHm; P⫽0.943, SS vs. AL for the SON) animals (Figs. 3 and 5). ANOVA detected significant main and interactive effects of the feeding condition and Zeitgeber time for the c-fos mRNA expression in the AL, SC, and SS rats in the PVHp, PVHm, and SON (Table 1).

Fig. 3. The optical density (OD) of the c-fos mRNA hybridization signal in the hypothalamus of the AL (Œ), SC (Œ), and SS (⽧) rats at the ZT3, ZT5, ZT6, and ZT7. DMHc and DMHv, caudal and ventral parts, respectively, of the dorsomedial hypothalamic nucleus; PVHp and PVHm, parvocellular and magnocellular parts, respectively, of the paraventricular hypothalamic nucleus; LHcpf and LHapf, caudal and anterior parts, respectively, of the perifornical lateral hypothalamic area; SON, supraoptic nucleus. SCN, suprachiasmatic nucleus. * Significantly different from the AL group at the same time. †Significant difference between the SC and SS groups at the same time. Each group includes five rats.

The perifornical area of the lateral hypothalamus showed a differential response at the anterior and caudal levels. The LHcpf demonstrated a significant gradual increase in c-fos mRNA expression at the ZT5 (P⫽0.0067, SC vs. SS), ZT6 (P⬍0.0001, SC vs. SS), and ZT7 (P⬍0.0001, SC vs. SS) in the SC rats but not in other groups (Figs. 3 and 4). In the LHapf, the levels of c-fos mRNA expression in the SC rats were not significantly

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Fig. 4. Dark-field photomicrographs of coronal brain sections representing the c-fos mRNA hybridization signal in the compact and ventral parts of the dorsomedial hypothalamic nucleus (DMHc and DMHv, respectively) and in the caudal part of the perifornical lateral hypothalamic area (LHcpf). The brain sections (30-␮m thick) depict c-fos mRNA expression in the rats fed chow ad libitum (AL) at ZT3 and ZT6, and in the rats maintained on scheduled chow (SC) and scheduled chow and sucrose (SS) at the ZT3, ZT6 (3 and 0 h before expecting food), and ZT7 (after 1 h of refeeding). f, fornix. The scale bar corresponds to 300 ␮m.

In the SCN, the levels of c-fos mRNA expression were higher in the SS (P⫽0.0022) and SC (P⫽0.0155) rats compared to the AL animals at the ZT3, and in the SS (P⫽0.0100) rats compared to the AL animals at the ZT5. However, at the ZT6 and ZT7, the levels of c-fos mRNA in the AL groups increased (P⫽0.1250, AL vs. SC; and P⫽0.1186, AL vs. SS for the ZT6; P⫽0.1931, AL vs. SC; and P⫽0.1988, AL vs. SS for the ZT7) to levels comparable to those in the SC and SS rats and the difference between groups faded (Fig. 3). For the SCN, ANOVA revealed a

different from those of AL animals at all examined time points. Conversely, during food anticipation, c-fos mRNA expression in the LHapf in the SS rats was significantly higher compared to the AL and SC animals at the ZT5 (P⫽0.0006, SS vs. AL; P⫽0.0274, SS vs. SC) and ZT6 (P⬍0.0001, SS vs. AL; P⫽0.0047, SS vs. SC) (Figs. 3 and 5). ANOVA revealed significant main effects of the feeding conditions and Zeitgeber time for the LHcpf and the LHapf; however, the interactive effects of these factors was significant for the LHcpf but not for the LHapf (Table 1).

Table 1. F and P values of the two-way ANOVA for the main and interactive effects of feeding conditions (FC: AL, SC, SS) and Zeitgeber time (ZT: ZT3, ZT5, ZT6, ZT7) on the levels of c-fos mRNA expression in the brain nuclei of the rats fed ad libitum chow (AL), or submitted to schedules chow (SC) and scheduled chow and sucrose (SS) Nucleus

DMHc DMHv PVHp PVHm LHcpf LHapf SON SCN PVTa SHi PFCg IL OFC Pir LSmv AcbSh AcbCoL AcbCoM NTSr NTSm NTSc AP

Feeding conditions (FC)

Zeitgeber time (ZT)

F2,48

P

F3,48

P

F6,48

FC⫻ZT P

82.168 55.077 43.131 13.812 42.722 19.280 21.078 10.743 78.759 71.981 75.067 91.777 69.037 52.814 72.830 45.764 29.747 7.465 26.141 26.183 54.429 3.422

⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.0015 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.0408

3.043 9.577 15.522 7.844 3.174 4.328 17.785 1.045 2.035 2.076 3.000 4.920 10.330 5.698 4.536 6.075 4.209 9.198 84.508 67.666 182.817 10.325

0.0377 ⬍0.0001 ⬍0.0001 0.0002 0.0324 0.0089 ⬍0.0001 NS NS NS 0.0396 0.0046 ⬍0.0001 0.0020 0.0072 0.0014 0.0101 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001

2.355 2.826 6.739 6.706 9.035 1.753 17.947 0.475 0.438 0.875 1.709 4.582 2.688 0.800 1.241 2.932 1.701 7.023 20.420 18.228 51.404 2.253

0.0449 0.0195 ⬍0.0001 ⬍0.0001 0.0024 NS ⬍0.0001 NS NS NS NS 0.0009 0.0249 NS NS 0.0166 NS ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 NS

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Fig. 5. Dark-field photomicrographs of coronal brain sections depicting the c-fos mRNA hybridization signal in the parvocellular and magnocellular parts of the paraventricular hypothalamic nucleus (PVHp and PVHm, respectively), the anterior part of the perifornical lateral hypothalamic area (LHapf), and in the supraoptic nucleus (SON). The photomicrographs represent brain sections (30-␮m thick) of the ad-libitum-fed rats (AL) at the ZT3 and ZT6, and the rats maintained on scheduled chow (SC) or scheduled chow and sucrose (SS) at the ZT3, ZT6 (3 and 0 h before expecting food), and ZT7 (after 1 h of refeeding). f, fornix. The scale bar corresponds to 300 ␮m.

significant effect of the feeding conditions but not an effect of Zeitgeber time or the interactive effect of both factors (Table 1). c-fos mRNA expression in the extra-hypothalamic forebrain regions The PVTa demonstrated significant neuronal activation during food anticipation and following feeding in the SC and SS rats compared to the AL controls. However, the c-fos mRNA levels were not different between the SC and SS animals at all examined Zeitgeber time points (Fig. 6). The neuronal activation in the SHi was significantly higher in the SC and SS rats compared to the AL animals for all time points. In addition, the SC rats demonstrated significantly higher c-fos mRNA levels compared to the SS rats at the ZT3 (P⫽0.0050), ZT5 (P⫽0.0004), and ZT6 (P⫽0.0135) (Fig. 6). ANOVA revealed a significant effect of the feeding conditions but not an effect of Zeitgeber time or an interactive effect of both factors for c-fos mRNA in the PVTa and SHi (Table 1). In the PFCg, c-fos mRNA expression was significantly higher in the SS rats compared to the AL controls at all examined time points (P⬍0.0001 for the ZT3, ZT5, ZT6, and ZT7), while in the SC rats the c-fos transcript was significantly increased compared to the AL rats at the ZT5 (P⫽0.0262), ZT6 (P⫽0.0152), and ZT7 (P⫽0.0002). Importantly, the SS rats had significantly higher c-fos mRNA expression at all time points compared to the SC rats (Figs. 6 and 7). For the c-fos mRNA in the PFCg, ANOVA revealed significant effects of feeding conditions and Zeitgeber time; however, the interactive effects of both factors were not significant (Table 1). In the OFC, c-fos mRNA was increased at the ZT5 (P⫽0.0002), ZT6 (P⬍0.0001), and ZT7 (P⬍0.0001) in the SC rats and at all time points in the SS rats (P⫽0.0005 for the ZT3; and P⬍0.0001 for the ZT5, ZT6, and ZT7) compared to the AL control rats (Figs. 6 and 8). The SS rats had higher levels of c-fos transcript compared to the SC animals at the ZT3 (P⫽0.0273), ZT5 (P⫽0.0343), and ZT6

(P⫽0.0096) (Fig. 6). The main and interactive effects of feeding conditions and Zeitgeber time were significant for the c-fos mRNA in the OFC (Table 1). In the IL, c-fos mRNA expression was significantly higher in the SS rats compared to the AL controls at all examined time points (P⬍0.0001 for the ZT3, ZT5, ZT6, and ZT7), while in the SC rats the c-fos transcript was significantly increased compared to the AL rats at the ZT5 (P⫽0.0202), ZT6 (P⬍0.0001), and ZT7 (P⬍0.0001) (Fig. 8A, B). The main and interactive effects of feeding conditions and Zeitgeber time were significant for the c-fos mRNA in the IL (Table 1). In the Pir, the expression of c-fos mRNA was significantly higher in rats maintained on feeding schedules compared to ad-libitum-fed rats (Fig. 8A, C). However, the levels of c-fos transcripts in the Pir of SC and SS rats were not significantly different (P⫽0.2672 at ZT3; P⫽0.9191 at ZT5; P⫽0.8370 at ZT6; and P⫽0.5689 at ZT7). The expression of c-fos mRNA was significantly induced in the LSmv at the ZT3, ZT5, and ZT6 in the SS rats compared to the SC (P⫽0.0354, P⫽0.0002, and P⬍0.0001, respectively) and AL (P⫽0.0001, P⬍0.0001, and P⬍0.0001, respectively) animals. Feeding increased neuronal activation in the SS and SC rats, but the levels of c-fos mRNA expression were significantly higher in the SS rats compared to the SC animals (P⬍0.0001) (Figs. 6 and 9). The main, but not interactive, effects of feeding conditions and Zeitgeber time were significant for c-fos mRNA in the LSmv (Table 1). The levels of c-fos mRNA increased significantly at the ZT5, ZT6, and ZT7 in the AcbSh and AcbCoL in the SS rats but not in the other groups (Figs. 6 and 9). The main and interactive effects of feeding conditions and Zeitgeber time were significant for the AcbSh. The AcbCoL showed a significant effect of the main but not interactive effects of these factors (Table 1). A significant increase in the levels of c-fos transcript in the AcbCoM was seen only in the SC rats after feeding (P⬍0.0001, SC vs. both SS and AL

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the brainstem. One hour of feeding (ZT7) led to induction of c-fos mRNA expression in the SS and SC animals in the rostral, medial, and caudal parts of the NTS and in the AP (Figs. 10 and 11). In all parts of the NTS, the SS rats demonstrated significantly increased c-fos mRNA expression compared to the SC rats (P⬍0.0001 for the NTSc; P⫽0.0004 for the NTSm; and P⫽0.0336 for the NTSr). In the AP, the SC and SS refed rats showed a significant increase in c-fos mRNA levels compared to the AL rats (P⬍0.0001 for SC; P⫽0.0060 for SS), but no difference between the SC and SS refed animals was observed (P⫽0.1384). All parts of the NTS showed significant main and interactive effects of the feeding conditions and Zeitgeber time on c-fos mRNA expression. The main but not interactive effects of these factors were significant for the AP (Table 1).

DISCUSSION

Fig. 6. The optical density (OD) of c-fos mRNA hybridization signal in the extra-hypothalamic forebrain regions in the rats that were fed chow ad libitum (AL: Œ), or were maintained on scheduled chow (SC: Œ) or scheduled chow and sucrose (SS: ⽧) at the ZT3, ZT5, ZT6, and ZT7. PVTa, anterior part of the paraventricular thalamic nucleus; SHi, septohippocampal nucleus; PFCg, cingulate prefrontal cortex; OFC, orbitofrontal cortex; LSmv, medioventral part of the lateral septum; AcbSh, nucleus accumbens shell; AcbCoL and AcbCoM, lateral and medial parts, respectively, of the nucleus accumbens core. * Significantly different from the AL group at the same time. † Significant difference between the SC and SS groups at the same time. Each group includes five rats.

groups) (Figs. 6 and 9). The main and interactive effects of feeding conditions and Zeitgeber time were significant for c-fos mRNA expression in the AcbCoM (Table 1). Effects of scheduled feeding on c-fos mRNA expression in the brainstem During food anticipation (ZT3, ZT5, and ZT6), no significant induction of c-fos mRNA expression was detected in

The present data provide evidence that adding sucrose to feeding schedules alters the pattern of neuronal activation during food anticipation and following feeding. We recently described the pattern of induction of c-fos mRNA expression in the brains of rats maintained on scheduled daily access to chow (Poulin and Timofeeva, 2008). This treatment led to the activation of the DMH, PVTa, and SHi as early as 3 h before the expected meal, the time when the animals start to express FAA. As the time of access to chow approached, the neuronal activation propagated in the hypothalamus to the PVH and LH (Poulin and Timofeeva, 2008). This increase in induction of anticipatory c-fos mRNA expression in the hypothalamus of rats maintained on scheduled chow was in agreement with the reported induction of Fos protein expression during food anticipation in the DMH and LH and by following refeeding in the PVH in rats maintained on scheduled access to chow (Angeles-Castellanos et al., 2004). The apparent discrepancy for the timing of c-fos activation in the PVH (during the last 2 h of food anticipation for c-fos mRNA and during the first 2 h following feeding for the Fos protein) may be attributed to the different time course for the expression of c-fos mRNA (in minutes) and the Fos protein (in hours) after activation. The activation of the hypothalamic structures during food anticipation in rats maintained on restricted feeding schedules was not reported in the studies, implicating the limited daily access to palatable food in free-feeding rats (Angeles-Castellanos et al., 2008; Mendoza et al., 2005b). In fact, rats anticipating daily access to chocolate, demonstrated induced Fos protein expression in the limbic regions, such as AcbCo, AcbSh, and PFC, but not in the DMH, LH, and PVH (Mendoza et al., 2005b). These results have suggested the existence of at least two neuronal circuitries entrained by the alleviation of the metabolic negative state in restricted feeding schedules and by the incentive and nutritive value of palatable food in freefeeding rats. The nutritive value of palatable food is an important factor for entrainment of FAA. Several studies have demonstrated that restricted daily access to palatable, nutrient-rich (such as sugar, chocolate, glucose), but

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Fig. 7. Dark-field photomicrographs of coronal brain sections demonstrating the c-fos mRNA hybridization signal in the cingulate prefrontal cortex (PFCg) in the brain sections (30-␮m thick) of the ad-libitum-fed rats (AL) at the ZT3 and ZT6, and the rats maintained on scheduled chow (SC) or scheduled chow and sucrose (SS) at the ZT3, ZT6 (3 and 0 h before expecting food), and ZT7 (after 1 h of refeeding). cc, corpus callosum. The scale bar corresponds to 300 ␮m.

not to palatable non-nutritive (such as saccharin) food in free-feeding rats can engender FAA (Mistlberger and Rusak, 1987; Stephan and Davidson, 1998). It seems that in free-feeding rats the amount of palatable nutritive food has to be high enough to create the nutrient “pulses” at the metabolic level. The minimum threshold to shift circadian activity was achieved with 4 g of palatable mash (Mistlberger and Rusak, 1987), 6 g of glucose (Stephan and Davidson, 1998), 6 g of standard chow (Stephan, 1997), or 30% of the daily intake (Mistlberger, 1994). Another study demonstrated that brief (⬃5 min) daily access to sucrose increased anticipatory wheel running activity, body temperature, and plasma corticosterone in food-restricted (to 85% of the body weight of the control rats) but not in ad-libitumfed rats (Pecoraro et al., 2002). It has been suggested that the combination of a deprivation regimen and sucrose

access may give rise to a more global anticipatory mechanism because the temporal cycles of energy balance may be strongly modulated by the incentive properties of sucrose (Pecoraro et al., 2002). The present experimental design combines the negative metabolic state with scheduled access to palatable sucrose. Because we used 10% sucrose containing only 1.7 kJ/ml, increase of sucrose intake but not more calorie-dense chow (12.9 kJ/g) by SS rats led to lowering of total energy intake and body weight of these rats compared to other groups. The SS rats demonstrated higher food-anticipatory locomotor activity and activity-dependent increase in oxygen consumption compared to SC rats, which could be due to elevated negative metabolic state in SS rats, as total calorie consumption in this group was significantly lower compared to SC rats during the second half of the experiment.

Fig. 8. (A) X-ray film autoradiograms of the coronal brain sections depicting positive hybridization signal of c-fos mRNA in the infralimbic prefrontal cortex (IL), orbitofrontal cortex (OFC), and piriform cortex (Pir). The graphs represent the optical density (OD) of c-fos mRNA hybridization signal in the IL (B) and Pir (C). The rats were fed chow ad libitum (AL: Œ), or were maintained on scheduled chow (SC: Œ) or scheduled chow and sucrose (SS: ⽧) and sacrificed at the ZT3, ZT5, ZT6, and ZT7. * Significantly different from the AL group at the same time. † Significant difference between the SC and SS groups at the same time. Each group includes five rats.

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Fig. 9. Dark-field photomicrographs of coronal brain sections depicting the c-fos mRNA hybridization signal in the medioventral sub-division of the lateral septum (LSmv), nucleus accumbens shell (AcbSh), and the medial and lateral parts of the nucleus accumbens core (AcbCoM and AcbCoL, respectively). The photomicrographs represent brain sections (30-␮m thick) of the ad-libitum-fed rats (AL) at the ZT3 and ZT6, and the rats maintained on scheduled chow (SC) or scheduled chow and sucrose (SS) at the ZT3, ZT6 (3 and 0 h before expecting food), and ZT7 (after 1 h of refeeding). ac, anterior commissure. The scale bar corresponds to 300 ␮m.

The pattern of neuronal activation in rats maintained on restricted feeding schedules in which the negative metabolic state is temporally alleviated by scheduled access to palatable food may include the hypothalamic and limbic structures. To test this hypothesis, we compared the pattern of c-fos mRNA expression in rats maintained on restricted feeding schedules with regular chow or restricted feeding schedules with chow and 10% sucrose. The daily chow intake was gradually increased in the SC rats over all

Fig. 10. The optical density (OD) of c-fos mRNA hybridization signal in the brainstem of the rats that were fed chow ad libitum (AL: Œ), or were maintained on scheduled chow (SC: Œ) or scheduled chow and sucrose (SS: ⽧) at the ZT3, ZT5, ZT6, and ZT7. NTSr, NTSm, and NTSc—rostral, medial, and caudal parts, respectively, of the nucleus of solitary tract; AP, area postrema. * Significantly different from the AL group at the same time. †Significant difference between the SC and SS groups at the same time. Each group includes five rats.

3 weeks of the experiment. Conversely, the SS rats showed an initial increase in chow intake during the first week, but during the second and third weeks, the SS rats did not increase their chow intake. Instead, the SS rats demonstrated a gradual significant escalation of their daily sucrose intake over all 3 weeks of the experiment. This differential sucrose and chow intake may be explained by the fact that intermittent access to palatable food promotes the increased intake of palatable ingredients of food (Hagan and Moss, 1997; Martin and Timofeeva, 2010), while reducing the reinforcing efficacy of regular chow (Cottone et al., 2008). The SC and SS rats demonstrated fairly similar activation of the anterior PVT during food anticipation and following feeding. The PVT has been recognized as an integrative relay structure that may convey information related to the metabolic state from the hypothalamus and brainstem to the limbic structures (Bhatnagar et al., 2000; Kelley et al., 2005). Although the PVT seems not to be an exclusive FEO, because lesion studies demonstrated controversial results on the role of the PVT in the generation of FAA (Landry et al., 2007a; Nakahara et al., 2004), the activation of the PVT can be important for coordinating behavioral and metabolic activity during FAA in the SC and SS rats. The induction of c-fos mRNA expression during food anticipation and refeeding was seen in the SHi, in the SC and SS rats, although the SC group demonstrated significantly higher levels of c-fos transcript expression in this structure during the anticipatory period compared to the SS animals. The activation of the SHi may be important for entrainment and memorization of behavioral patterns. The SHi contains cholinergic neurons that increase their activity during training and memorization (Park et al., 1992; Yamamuro et al., 1995). Release of acetylcholine that stimulates arousal and behavioral activity is increased in

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Fig. 11. Dark-field photomicrographs of coronal brain sections demonstrating the c-fos mRNA hybridization signal in the rostral, medial, and caudal parts of the nucleus of the solitary tract (NTSr, NTSm, and NTSc, respectively) and in the area postrema (AP) in the brain sections (30-␮m thick) of the rats maintained on scheduled chow (SC) or scheduled chow and sucrose (SS) at the ZT7 (after 1 h of refeeding). 4v, fourth ventricle. The scale bar corresponds to 300 ␮m.

the prefrontal cortex and hippocampus during FAA (Ghiani et al., 1998; Inglis et al., 1994). Similar to the PVT, the SHi is not an exclusive FEO, as FAA persists after ablation of the SHi (Mistlberger and Mumby, 1992). In contrast to comparable activation of the PVT and SHi, the SC and SS rats showed a striking difference in the levels of c-fos mRNA expression in the hypothalamus. The induction of c-fos mRNA expression in the compact part of the DMH during food anticipation and following feeding was significantly increased in the SC rats, but not in the SS animals. The SS rats also did not demonstrate increased c-fos mRNA expression during food anticipation in the ventral part of the DMH. Refeeding stimulated c-fos mRNA expression in the DMHv of the SS rats, but again, this increase in the levels of c-fos mRNA was significantly lower in the SS rats compared to their SC counterparts. This absence of c-fos mRNA expression during food anticipation in the DMH in the rats maintained on scheduled sucrose, is in agreement with the reported absence of Fos protein expression in the DMH in free-feeding rats maintained on scheduled access to chocolate (Mendoza et al., 2005b). The apparent discrepancy in the presence and absence of c-fos mRNA and the Fos protein, respectively, in the DMHv in sucrose- and chocolate-refed rats may be attributed to different temporal dynamics for mRNA and protein expression, or to the different feeding conditions used in these studies. The DMH is important for regulating feeding (Bellinger and Bernardis, 2002; Zigman and Elmquist, 2003) and circadian activity (Bellinger et al., 1976). The differential activation of the compact and ventral DMH during food anticipation and feeding suggests particular role of these sub-regions in feeding behavior. The compact, but not ventral, part of the DMH synchronize the expression of Period gene Per2 with the food-anticipatory activity (Mieda et al., 2006). Conversely, the direct ascending projections of the viscerosensory regions of the parabrachial nucleus and the nucleus of the solitary tract enters the ventral DMH (Ter Horst et al., 1989; Thompson and Swanson, 1998). However, although the DMH is in a good position to integrate food-related signals and syn-

chronize its activity with food anticipation, the DMH seems not to be an exclusive FEO (Gooley et al., 2006; Landry et al., 2006, 2007a,b). Similar to the DMHc, the caudal part of the perifornical LH was activated during food anticipation and refeeding in the SC rats but not in the SS animals. Conversely, the anterior part of the perifornical LH was significantly activated 1 h before feeding and at the time when food was expected by the SS but not by the SC rats. The perifornical LH contains neuropeptides hypocretin 1 and 2 which are important for arousal and feeding (Saper, 2006). Although saporin-induced depletion of LH hypocretin-2 neurons in rats did not affect FAA (Mistlberger et al., 2003), genetic ablation of all hypocretin neurons in mice attenuated FAA expression (Akiyama et al., 2004) suggesting that hypocretins may be important regulators of FAA. Entrainment on chow or chow and sucrose differentially activated posterior and anterior parts of the perifornical LH. This difference may depend on the particular differential projections of the ventral striatum to the LH. The GABAergic medial spiny neurons of the nucleus accumbens specifically project to the anterior but not posterior LH (Sano and Yokoi, 2007). Interestingly, these inhibitory accumbens neurons do not directly connect hypocretin LH neurons (Sano and Yokoi, 2007), that may implicate the local LH interneurons and the mechanisms of disinhibition of particular population of the anterior perifornical LH neurons. The expression of c-fos mRNA in the PVN was analyzed separately in the parvocellular and magnocellular parts because of specificity of these sub-nuclei in regard to their particular neurochemistry, neuroanatomy, and physiological implication (Richard and Timofeeva, 2009; Swanson and Kuypers, 1980). The key neuropeptide expressing by the parvocellular PVH is corticotropin releasing factor, which, by reaching the anterior pituitary, regulates the activity of the hypothalamic-pituitary adrenal (HPA) axis (Rivier et al., 1983; Timofeeva, 2010). Conversely, the magnocellular PVH, by producing and secreting vasopressin to the posterior pituitary, regulates water–salt balance (Swanson and Sawchenko, 1983). In the parvocellular

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PVH, sucrose blocked the activation of c-fos mRNA expression during the anticipatory period. The SC rats demonstrated significantly increased, compared to other groups, c-fos mRNA expression in the PVHp 1 h before feeding and at the food-expecting time. Refeeding increased neuronal activation in both the SC and SS groups. Activation of the PVHp in the SC rats occurred during food anticipation, when behavioral activation coincides with an increase in plasma corticosterone (Belda et al., 2005; Diaz-Munoz et al., 2000; Krieger, 1974; Moberg et al., 1975; Morimoto et al., 1977; Poulin and Timofeeva, 2008). The absence of neuronal activation in the PVHp during food anticipation in the SS rats may be attributed to the damping effects of sucrose intake on the activity of the PVHp neurons (Dallman et al., 2003; Foster et al., 2009; Laugero et al., 2001; Martin and Timofeeva, 2010). The magnocellular regions, the PVHm and SON, were not activated in any experimental group during food anticipation. Following refeeding, induction of c-fos mRNA expression was seen in the magnocellular regions in the SC rats but not in the other rats. The different responses of the magnocellular regions of the SS and SC rats to feeding may depend on the different alteration in plasma osmolarity and sodium concentration after chow and sucrose intake (Gutman and Krausz, 1969; Haddy, 1987). The levels of c-fos mRNA expression in the SCN were higher in the SC rats at 3 h and in SS rats at 3 and 1 h before feeding, compared to the ad libitum controls. However, at the expected feeding time and after 1 h of refeeding, the levels of c-fos mRNA expression were not different in the SCN across the experimental groups. This result is in agreement with previous observations that c-fos mRNA and Fos immunoreactivity in the SCN may be altered by the circadian rhythm and scheduled food entrainment (Angeles-Castellanos et al., 2004; Mendoza et al., 2005b; Poulin and Timofeeva, 2008). In the present study, we did not divide the SCN to sub-nuclei. Further investigation is required to specify the activation of the ventral versus dorsal SCN in the rats anticipating palatable food. Early activation, at 3 h before feeding, was seen in the orbitofrontal, cingulate, and infralimbic prefrontal cortex in the SS rats but not in the SC animals. As the feeding time approached (1 h before feeding and at the moment of feeding), neuronal activation in these cortical regions also increased in the SC rats, but this increase was significantly lower compared to the levels of c-fos mRNA expression seen in the SS animals. The higher activation of the OFC in the SS rats was expected because the OFC is the secondary taste cortex that processes signals related to the reward value of taste (O’Doherty et al., 2000; Rolls, 2000). The prefrontal cortex is involved in processing foodand reward-related information (D’Angio and Scatton, 1989; Kolb et al., 1978; Schalomon et al., 1994). In addition, the prefrontal cortex is implicated in the consolidation of interference learning associated with the relative reward value of different concentrations of sucrose solutions (Pecoraro et al., 2008). In fact, anticipation and consumption of sucrose solutions activate the neurons of the OFC and the

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medial prefrontal cortex (Gutierrez et al., 2006; Petyko et al., 2009; van Wingerden et al., 2010). The OFC innervates the accumbens (Schilman et al., 2008), while the medial prefrontal cortex sends the efferents to the nucleus accumbens and lateral septum (Sesack et al., 1989). The LS, AcbSh, and AcbCoL demonstrated a significant increase in c-fos mRNA expression during food anticipation in the SS rats but not in the SC rats. Conversely, the AcbCoM showed neuronal activation in response to feeding in the SC rats but not the SS animals. The activation of the septum and accumbens regions in the SS rats may be related to the ability of these areas to reinforce positive reward stimuli. The interconnection of the LS with the mesolimbic dopamine system makes the stimulation of the LS extremely rewarding (Olds and Milner, 1954; Sheehan et al., 2004; Swanson, 1999). The activation of the AcbCo during food anticipation (in the SS rats) and feeding (in the SC rats) may be relevant to the motor aspect of anticipatory and consummatory behavior (Mendoza et al., 2005a). The present results do not show activation of the AcbCo in the chow-anticipating rats. These data differ from those demonstrating increased Fos immunoreactivity in the AcbCo during anticipation of regular food (Mendoza et al., 2005a). The controversy on the expression of c-fos mRNA and Fos protein may arise due to different dynamics of expression and stability of these molecules. The activation of the AcbSh in the SS rats may be mainly related to the motivational and reward aspects of anticipation and feeding (Mendoza et al., 2005a,b). Indeed, the dopaminergic input to the AcbSh from the ventral tegmental area (Carelli, 2002; Roitman et al., 2004) and the AcbSh output to the lateral hypothalamus (Stratford and Kelley, 1999) are involved in regulating palatable food intake. The LS and the AcbSh contain GABAergic projecting neurons, and both structures directly innervate the hypothalamus (Risold and Swanson, 1997; Sano and Yokoi, 2007; Varoqueaux and Poulain, 1994). However, whether these regions are implicated in the reorganization of neuronal activity in the SS rats requires further clarification. In the brainstem, neuronal activation was seen in the NTS and AP in the refed SC and SS rats. Analysis of c-fos mRNA expression in the NTS has been done separately in the rostral, medial, and caudal sub-divisions because the taste, glossopharyngeal, and gastro-intestinal sensory afferents demonstrate rostro-caudal viscerotopic mapping of the NTS (Altschuler et al., 1989; Hamilton and Norgren, 1984; Travers and Norgren, 1995). The levels of c-fos mRNA expression were significantly higher in the rostral, medial, and caudal NTS in the SS rats compared to their SC counterparts. Conversely, the neuronal activation seen in the AP was comparable between the SS and SC groups. These results are in agreement with a reported increase in postprandial Fos immunoreactivity in the NTS but not in the AP of rats subjected to repeated schedules of caloric restriction and access to palatable food compared to rats maintained on continuous palatable feeding or chow-restricted rats (Bello et al., 2009). The NTS may integrate the metabolic signals and information related to food taste,

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while the AP mainly mediates the metabolic postprandial signals.

CONCLUSION The present results have demonstrated that adding sucrose to daily scheduled feeding displaced neuronal anticipatory activity from the medial and lateral tuberal hypothalamus to the lateral anterior hypothalamus as well as to the prefrontal cortex and the ventral striatum. The dorsomedial paraventricular thalamus, which interconnects the hypothalamic and limbic regions, was similarly activated by both treatments. The present data provide evidence that the dorsomedial thalamo-hypothalamic loop is entrained by the chow feeding schedules, and the activation from this oscillatory loop is expanded to other forebrain structures as the time for expected food approaches. Adding sucrose to feeding schedules entrains the dorsomedial thalamusprefrontal cortex loop, which reorganizes brain activity by activating the limbic-related regions but not the dorsomedial and paraventricular hypothalamic nuclei. The present data are in agreement with the evidence that the brain may contain different oscillatory systems (Abe et al., 2002; Angeles-Castellanos et al., 2008; Mendoza et al., 2005b) that are specifically entrained according to food palatability and metabolic state. Acknowledgments—This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and Canadian Institutes of Health Research. E.T. is a scholar of Fonds de la Recherche en Santé du Québec (FRSQ) and A.M. is a scholar of the Medicine Faculty of Laval University. We thank Julie Plamondon and Pierre Samson for technical assistance.

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(Accepted 10 June 2011) (Available online 17 June 2011)