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Neuroscience 144 (2007) 344–355

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ESTRICTED FEEDING SCHEDULES PHASE SHIFT DAILY RHYTHMSF c-Fos AND PROTEIN Per1 IMMUNOREACTIVITY IN

ORTICOLIMBIC REGIONS IN RATS

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Departamento de Anatomía, Facultad de Medicina, Edificio “B” 4°iso, Universidad Nacional Autónoma de México, México DF 04510,exico

Neurobiologie des Rythmes, UMR7518 ULP/CNRS, IFR des Neuro-ciences, Strasbourg, France

Dirección General de Investigación, Universidad Veracruzana, Xalapa,exico

bstract—Entrainment by daily restricted feeding schedulesRFS) produces food anticipatory activity (FAA) which in-olves motivational processes which may be regulated byorticolimbic structures and the nucleus accumbens. Theresent study aimed first to determine whether corticolimbictructures participate in the expression of FAA, therefore-Fos immunoreactivity (Fos-IR) was employed as marker ofeuronal activity. The second goal was to characterize diur-al rhythms of the clock protein protein Per1 (PER1) in cor-icolimbic structures and to determine the influence of RFSn the diurnal temporal pattern.

Rats were maintained under RFS with food access for 2 haily, a control group was fed ad libitum. Food entrainmentroduced a pattern of increased Fos-IR during FAA and afterealtime in the two sub-regions of the nucleus accumbens

ACC), in the basolateral and central amygdala, in the beducleus of the stria terminalis (BNST), in the lateral septumLS), in the prefrontal cortex (PFC), and in the paraventricularhalamic nucleus (PVT). No increased Fos-IR was observed inhe hippocampus.

Under ad libitum conditions all structures studied showedaily oscillations of PER1, excluding both amygdalar nucleind the PFC. RFS shifted and set the daily peaks at zeitgeberime (ZT) 12 for both sub-regions in the accumbens, the hip-ocampus, lateral septum and PFC. RFS enhanced the ampli-ude at ZT12 of the BNST and shifted the peak of the PVT toT6. No changes were observed in the amygdalar nuclei.

Present data indicate that cellular activation of corticolim-ic structures is associated with behavioral events related toood anticipatory activity and that mealtime is a relevant

Correspondence to: C. Escobar, Departamento de Anatomía, Fac-ltad de Medicina, Edificio “B” 4° Piso, Universidad Nacional Au-ónoma de México, México DF 04510, México. Tel: �52-55-5623-422; fax: �52-55-5623-2425.-mail address: escocarolina@gmail.com (C. Escobar).bbreviations: ACC, nucleus accumbens; Acc-Core, nucleus accum-ens sub-region core; Acc-Shell, nucleus accumbens sub-region shell;L, ad libitum group; BLA, basolateral amygdala; BNST, bed nucleusf the stria terminalis; CeA, central amygdala; FAA, food anticipatoryctivity; Fos-IR, c-Fos immunoreactivity; Hip, hippocampus; LD, light/ark cycle; LS, lateral septum; PER1, protein Per1; PER1-IR, Per1

mmunohistochemistry; PFC, prefrontal cortex; PVT, paraventricularhalamic nucleus; RF, food-restricted group; RFS, restricted feeding

cchedules; SCN, suprachiasmatic nucleus; S.E., standard error of theean; ZT, zeitgeber time.

306-4522/07$30.00�0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2006.08.064

344

ignal that shifts daily oscillations of PER1 in corticolimbictructures. Data suggest a relevant role of corticolimbictructures as oscillators for FAA. © 2006 IBRO. Published bylsevier Ltd. All rights reserved.

ey words: food-entrainment, c-Fos, limbic system, circa-ian, motivation.

nimals exposed to daily restricted feeding schedulesRFS) exhibit food anticipatory activity (FAA) characterizedy behavioral arousal, augmented locomotion, explorationnd foraging starting 2–3 h prior to mealtime (Mistlberger,994; Stephan, 2001). Physiological, digestive and meta-olic diurnal rhythms also are entrained by RFS, thus theiraily peak, which is regularly associated with the light/darkycle (LD), is shifted toward mealtime (Davidson andtephan, 1999; Davidson et al., 2003; Krieger, 1979; Es-obar et al., 1998; Martínez-Merlos et al., 2004). It is wellccepted that both behavioral and metabolic anticipatoryrocesses depend on a time measuring system that canperate independent of the suprachiasmatic nucleusSCN), because SCN lesions do not abolish FAA or thenticipatory corticosterone peak (Krieger, 1979; Stephan,001). However, the functional and anatomical identity ofhe time keeping systems underlying FAA is not clear.

In recent years the use of clock gene reporters hashown that per1 and per2 mRNA and proteins exhibitircadian oscillations in peripheral tissues, coordinatedith the SCN. However, tissue explants monitored in vitroeripheral tissues can express daily oscillations indepen-ent of the SCN, in (Hara et al., 2001; Stokkan et al., 2001;amazaki et al., 2000; Yoo et al., 2004). In addition, underFS peripheral tissues uncouple from the SCN and exhibitscillatory patterns in phase with mealtime (Damiola et al.,000; Yamazaki et al., 2000; Kobayashi et al., 2004),

ndicating that peripheral oscillators are preferentially en-rained by feeding schedules. Likewise, some brain struc-ures exhibit daily clock gene oscillations, (Abe et al., 2002;mir et al., 2004; Masubuchi et al., 2000) and RFS entrainelectively the cerebral cortex and the oval nucleus of thetria terminalis (Lamont et al., 2005a; Wakamatsu et al.,001).

The signal to start the expression of FAA is not under-tood; it may be mediated by metabolic oscillations rele-ant for energy balance or by signals from the stomachue to prolonged fasting (Martínez-Merlos et al., 2004;atoh et al., 2006). This is supported by previous studies

hat describe entrainment of locomotor activity and liver

lock genes by daily delivery of adrenaline (Mendoza et al.,

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M. Ángeles-Castellanos et al. / Neuroscience 144 (2007) 344–355 345

003; Terazono et al., 2003) and induction of cellular os-illations by a serum shock or a single injection of dexa-ethasone (Balsalobre et al., 1998, 2000). The role ofetabolic signals for food entrainment is also supported by

he finding of c-Fos immunoreactivity (Fos-IR) entrainedscillatory patterns in hypothalamic nuclei involved in theomeostatic regulation of energy balance (Ángeles-Cas-ellanos et al., 2004) and brain stem structures that trans-it digestive and sensory signals to the brain (Ángeles-astellanos et al., 2005).

The signal to start FAA can also be elicited by anrousal state due to the daily and regular prolonged fasting

nterval imposed by RFSs. Studies describing cellular ac-ivation of structures involved in arousal during FAA sup-ort this possible relationship (Ángeles-Castellanos et al.,004; Inzunza et al., 2000; Valdés et al., 2005; Saper et al.,005). The signal to start FAA also may arise from moti-ational processes elicited by a reward or hedonic effectue to food arrival (Tuomisto et al., 1999; Kelley et al.,002; Kelley, 2004). It is now well studied that feedingehavior is strongly modulated by the incentive value ofood as well as by its hedonic impact or its aversive oreneficial outcomes (Kelley et al., 2005; Berthoud, 2004).he role of motivational and reward processes associatedith food entrainment was evidenced in rats fed ad libitumnd receiving daily a palatable meal, which also exhibitedAA (Mendoza et al., 2005c; Mistlberger and Rusak,987).

Motivation, hedonic and affective sensations are elic-ted by the corticolimbic system constituted by a complexetwork of interacting structures including the amygdala,

he bed nucleus of the stria terminalis (BNST), the hip-ocampus (Hip), the prefrontal cortex (PFC) and the sep-

um (Paxinos, 1995). Their interaction with neuroendo-rine, autonomic and sensory systems mediates behav-oral responses to ingestive behavior, reproduction andtress and thus is a key system for generating motivationor food search and expectation (Ahn and Phillips, 2002).

The nucleus accumbens (ACC), which is not classifieds part as the limbic system, but rather a constituent of theasal ganglia, is involved in the integration of hedonic andotivational responses, especially related to palatableeals, and with the elaboration of voluntary movements

Mogenson et al., 1980; Kelley et al., 2002). The ACC isubdivided in two sub-regions “core” and “shell.” The coreegion is associated with anticipatory responses, whichnclude the “wanting” component of motivation. In contrasthe shell region elicits reward responses consequent to atimulus and thus generates the “liking” effect. Recently weeported a food-entrained pattern of Fos-IR expression inoth sub-regions of the ACC, with peak values anticipatingealtime (Mendoza et al., 2005a). However, Mistlbergernd Mumby (1992) reported that lesions of the accumbensnd other structures of the limbic system do not abolishAA.

The paraventricular nucleus of the thalamus (PVT)lays a relevant role as interface for motivational andensory information from cortical, limbic and hypothalamic

tructures to the basal ganglia, especially to the ACC Á

Kelley et al., 2005; Su and Bentivoglio, 1990) The PVTlso establishes a relevant link between the limbic struc-ures and the SCN (Moga and Moore, 1997) and mayransmit non-photic stimuli to the SCN (Moga et al., 1995).

In order to better define the involvement of limbic struc-ures and the motivational systems during food entrain-ent, the present study was designed to identify those

orticolimbic structures that exhibit Fos-IR during FAA, forhich Fos-IR is a reporter of cellular activation as well asphase marker for circadian oscillators (Yan et al., 2005).herefore Fos-IR was quantified in the accumbens, amyg-ala, BNST, Hip, paraventricular thalamus, PFC and lat-ral septum (LS). In the same structures we aimed to

nvestigate the effect of food entrainment on the temporalattern of the protein Per1 (PER1) as a reporter of oscil-

atory mechanisms. This allowed us to determine whetherER1 expression in corticolimbic structures is entrained by

eeding schedules. Both parameters together will enabledentification of brain structures involved in hedonic and

otivational responses that may be related with food en-rained behavioral patterns.

EXPERIMENTAL PROCEDURES

ubjects and housing conditions

dult male Wistar rats weighing 250–300 g at the beginning of thetudy were maintained in a 12-h LD, lights on at 07:00 h, whichas defined as ZT0 (zeitgeber time 0). The temperature in thenimal room was maintained constant around 22°�1 °C andnimals had free access to tap water and regular laboratory ratiet (Rodent Laboratory Chow 5001; Purina, St. Louis, MO, USA),nless otherwise stated. Rats were acclimated to environmentalonditions for at least 1 week before starting the experimentalrocedures. Animal handling was conducted according to theational guide for care and use of animal experimentation inexico City (Decreto ley de protección a los animales del Distritoederal, Gaceta Oficial del Distrito Federal, 26/02/02, which com-lies with international guidelines on the ethical use of animals.he number of animals used was minimized, and measures wereonsidered to minimize animal suffering.)

roups and food entrainment

his study was performed in two stages. A first experiment ex-lored the Fos-IR in limbic structures during food entrainment.ats were randomly assigned to an ad libitum (AL) control or to a

ood-restricted group (RF) and were kept in groups of four to fiveats of the same condition for 3 weeks. The AL had free access toood throughout the experiment and provided information on thexpected Fos-IR under regular feeding conditions in a LD cycle.he RF group was maintained under RFS with food available daily

or 2 h, from 12:00 to 14:00 h (ZT5–ZT7). After 3 weeks in theeeding protocol rats were perfused and brains were obtained forhe time points 10:00,11:00,12:00, 14:00, 18:00 and 20:00 h,hich are further represented as ZT3, ZT4, ZT5, ZT7, ZT11 andT13, respectively (n�6 per group and phase). Sampling timeoints comprised the critical time window for cellular activationelated with anticipatory activity and with food ingestion. The dayf perfusion RF animals for the ZT5 time point, which corre-ponded to mealtime, remained unfed until anesthetized, the ratsor the following time points (ZT7, ZT11 and ZT13) received theiraily food at the usual time. The experimental groups and tissuessed for this experiment are the same as previously reported in

ngeles-Castellanos et al. (2004, 2005).

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M. Ángeles-Castellanos et al. / Neuroscience 144 (2007) 344–355346

A second experiment explored whether in the same corti-olimbic structures the diurnal cycle of PER1 immunohistochem-stry (PER-IR) was entrained by feeding schedules. A different setf rats was randomly assigned to the ad libitum feeding controlroup or to the RF group, as described above, food was sched-led at ZT6. Feeding protocols were followed for 3 weeks and the

ast day in such conditions rats were perfused at one of fouremporal points of the 24 h cycle to determine rhythmicity of PER1xpression (n�4 per phase) ZT0, ZT6, ZT12 and ZT18 (ZT0enotes time of lights on). Time points were chosen in order toample a complete 24 h cycle in regular intervals.

mmunohistochemistry

ats were anesthetized with an overdose of sodium pentobarbitalSedal-Vet 65 mg/ml), and were perfused transcardially with50 ml of 0.9% saline followed by 250 ml of fixative 4% parafor-aldehyde in phosphate buffer saline (PBS, 0.1 M, pH 7.2).rains were removed, post-fixed for 1 h and cryoprotected in 30%ucrose for 3–4 days. Brains were frozen and cut in sections of0 �m at �18 °C. Sections were serially collected in four sets, oneet was stained with Cresyl Violet acetate (Sigma Chemical Com-any, St. Louis, MO, USA) and a second set was processed foros-IR or for PER1 immunohistochemistry. Free floating sectionsere incubated in c-Fos antibody raised in rabbit (1:2500; Santaruz Biotechnology, Santa Cruz, CA, USA) or in PER1 raised inoat (1:1000; Santa Cruz Biotechnology) for 72 h. This was fol-

owed by incubation in secondary antibody processed accordingith the avidin–biotin method for immunohistochemistry as previ-usly described (Ángeles-Castellanos et al., 2004). Tissues wereeacted in diaminobenzidine with hydrogen peroxide (35 �l, 30%2O2), mounted, dehydrated and coverslipped with microscopystellan-new (Merck, Whitehouse Station, NJ, USA).

ell count

n order to quantify Fos-IR or PER1 expression in limbic nuclei,epresentative sections for each nucleus were selected in accor-ance with the stereotaxic atlas from Paxinos and Watson (1998).n anterior section (bregma 1.20) was selected to analyze theCC, which was then subdivided and evaluated separately in its

wo sub-regions core (Acc-Core) and shell (Acc-Shell). A secondnterior section (bregma 2.70) was used to analyze the PFC. TheS and the BNST were evaluated in bregma 0.20. The PVT wassed to analyze in bregma 1.30 and the amygdala was analyzed

n a caudal section (bregma 2.56). The amygdala was furtherubdivided in a basolateral (BLA) and a central region (CeA).inally the Hip was analyzed in a caudal section (bregma 4.16).he Hip was initially subdivided in three sub-regions, CA1, CA2nd CA3, however Fos-IR and PER1 data were identical for thehree regions and therefore will be reported as Hip. Images ofelected sections were obtained at a 10� magnification using aomputerized image analysis system (Meta Vue series 4.5, Uni-ersal Imaging Corporation, Downingtown, PA, USA) attached to

Nikon light microscope (Nikon Eclipse E600; Nikon, Tokyo,apan). To minimize the number of false positives, backgroundptic density was established in a nearby region lacking Fos-IR orER1, stained cells that reached or surpassed 3� the backgroundptic density were considered positive and were included,hereas cells under this staining threshold were discarded. Aingle examiner, who was blinded to treatment conditions, per-ormed all counts.

ata analysis

he numbers of cells for each sampled area are expressed asells/mm2. Data were classified for group and time and are rep-esented as mean�standard error of the mean (S.E.). Data were

ompared with a two-way ANOVA for independent measures with t

factor for group (two levels) and a factor for time (six levels foros-IR, four levels for PER1). In order to determine significant

ime effects for each curve of PER1 a one way ANOVA waserformed for individual groups. The one and two way ANOVAere followed by a Tukey post hoc test with significant values sett P�0.01. Statistical analysis was performed with the programtatistica version 4.5 (StatSoft, Inc., Chicago, IL, USA).

RESULTS

os-IR under food-entrainment

n the AL group time related Fos-IR fluctuations werebserved in the Acc-Core, Acc-Shell in the BLA and PFC.he other examined structures did not show significanthanges in time under ad libitum conditions. In the RFroup all limbic structures, excepting the Hip, showed sig-ificantly increased Fos-IR at in anticipation to mealtime,hich is the moment when rats exhibit FAA (Figs. 1 and 2).

In ad libitum conditions both Acc sub-regions (Fig. 1A,B) exhibited a significant peak previous to lights offP�0.001). In the RF group this temporal pattern washanged and rats exhibited significant high valuesP�0.001) at the three time points (ZT3, ZT4, ZT5) antic-pating mealtime. Significant high levels were also ob-erved at ZT7 (P�0.001), after the 2 h of food access. Thewo-way ANOVA confirmed significant difference betweeneeding conditions (Acc-Core: F(1,45)�35.09; P�0.0001;cc-Shell: F(1,44)�72.43) and the interaction of feedingondition and time (Acc-Core: F(5,45)�3.41; P�0.01; Acc-hell: F(5,44)�4.79; P�0.001).

In the amygdala Fos-IR was evaluated for two sub-egions, the BLA and CeA (Fig. 1D, 1E). In ad libitumonditions the BLA showed high values of Fos-IR in thearly time points (ZT3, ZT4) that were significantly differentP�0.01) from later time points (ZT5–ZT13). In AL rats theeA showed a peak at ZT11 which was significantly different

rom ZT5 and ZT13 (P�0.01). In RF rats both BLA and CeAhowed high values anticipating mealtime. In the CeA highalues were observed at ZT3, ZT4 and ZT5 and remained atT7 (P�0.001), while in the BLA significant high values werebserved at ZT5 and ZT7 (P�0.001). The evening timeoints, 6 and 8 h after mealtime showed similar values as theL controls. The two-way ANOVA confirmed significant dif-

erence between feeding conditions (BLA: F(1,60)�5.78;�0.01; CeA: F(1,60)�198.04; P�0.0001) and the interac-

ion of feeding condition and time (BLA: F(5,60)�17.64;�0.0001; CeA: F(5,60)�28.44; P�0.0001).

The BNST did not show significant changes in AL ratsor the sampled time points (Fig. 1C). Food entrainmentroduced an anticipating pattern with high values at ZT5 andT7 (P�0.01). The two-way ANOVA confirmed significantifference between feeding conditions (F(1,60)�18.62;�0.0001) and the interaction of feeding condition and

ime (F(5,60)�6.36; P�0.0001).In the Hip no significant time changes were observed in

he AL group and no significant differences were observedetween AL and RF in the time points anticipating orubsequent to food access (Fig. 1F). A significant increaseas observed at ZT13, posterior to lights off. In contradic-

ion the two-way ANOVA showed significant difference

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M. Ángeles-Castellanos et al. / Neuroscience 144 (2007) 344–355 347

etween groups (F(1,60)�4.18; P�0.04) and significantnteraction of feeding condition and time (F(5,60)�87.07;�0.02).

The LS showed no significant variations among timeoints in ad libitum conditions (Fig. 1G), but showed aood-entrained pattern in RF rats, with significantly highalues at ZT4, ZT5 and ZT7 (P�0.001), anticipating meal-ime and subsequent to feeding. The two-way ANOVAonfirmed significant difference between feeding condi-ions (F(1,60)�160.71; P�0.0001) and the interaction ofeeding condition and time (F(5,60)�35.34; P�0.0001).

In ad libitum conditions the PFC showed high values ofos-IR in the early time points (ZT3, ZT4) which wereignificantly different (P�0.01) from later time points (ZT5–T13; Fig. 1H). Food entrainment modified the diurnalattern and induced high levels anticipating mealtime atT4 and ZT5 (P�0.001), and high levels subsequent to

eeding at ZT7 (P�0.001). Later time points (ZT11 and

ig. 1. Temporal patterns for Fos-IR cells in corticolimbic structures inctivation can be observed in all structures during the time when rats ahat does not show cellular activation associated with FAA and feedean�S.E. * Significant difference between AL and RF group obtaine

ow time points, and � for RF.

T13) returned to similar values as controls. The two-way a

NOVA confirmed significant difference between feedingonditions (F(1,60)�57.90; P�0.0001) and the interactionf feeding condition and time (F(5,60)�24.30; P�0.0001).

The PVT did not show time changes in ad libitumonditions (Fig. 1I) but showed a significant food entrainedattern with high levels at ZT4 (P�0.001) and ZT7P�0.001). The two-way ANOVA confirmed significant dif-erence between feeding conditions (F(1,60)�35.87;�0.0001) and the interaction of feeding condition and

ime (F(5,60)�12.74; P�0.0001).

ircadian expression of PER1

n ad libitum conditions daily PER1-IR oscillations werebserved in both sub-regions of the Acc, in the Hip, LS,FC and PVT. Food entrainment modified daily PER1-IRscillations in all structures, either by producing a phase shiftr by enhancing the amplitude of the curve (Figs. 3, 4

rained rats (Œ) and their ad libitum controls AL (�). Significant cellularating food access and after mealtime. The Hip (F) is the only structurehorizontal bar on the abscissa represents food access. Values areTukey post hoc test (P�0.01), � for the AL group between peak and

food-entre anticiping. The

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M. Ángeles-Castellanos et al. / Neuroscience 144 (2007) 344–355348

ig. 2. Representative photomicrographs of Fos-IR in Acc-Core and Acc-Shell, Hip, PFC and PVT, during FAA (ZT5). On the left imagesorresponding to AL group and on right images of the RF group. The scale bar�100 �m; aca, anterior commissure anterior part; cc, corpus callosum;

V, third ventricle.

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M. Ángeles-Castellanos et al. / Neuroscience 144 (2007) 344–355 349

Both sub-regions of the Acc, exhibited robust dailyscillations of PER1-IR in ad libitum conditions (Fig. 3And B), with highest values around ZT18 (Acc-Core:(3,11)�342.47; P�0.0001; Acc-Shell: F(3,11)�244.58;�0.0001). Food-entrainment shifted the daily rhythmicity ofoth regions, resulting robust and significant oscillations withpeak at ZT12 (Acc-Core: F(3,11)�76.17; P�0.0001; Acc-hell: F(3,11)�10.37; P�0.001). The two way ANOVA con-rmed a significant difference between both feeding con-itions (Acc-Core: F(1,22)�22.26; P�0.0001; Acc-Shell:(1,22)�38.35; P�0.01) and a significant interaction (Acc-ore: F(3,22)�227.51; P�0.0001; Acc-Shell: F(3,22)�8.35; P�0.0001).

In the amygdala of AL rats no significant daily PER1-IRscillations were observed (BLA: F(3,11)�0.13; P�NS;eA: F(3,11)�2.27; P�NS; Fig. 3D and E). Restricted

eeding enhanced daily oscillations, especially in the BLA,owever, they reached no statistical significance (BLA:(3,11)�2.75; P�NS; CeA: F(3,11)�1.02; P�NS). The

ig. 3. Daily rhythm of PER1 expression in corticolimbic structures of fre observed in AL and RF rats produced a main shift of PER1 towardbscissa represents food access. * Significant difference between AL afor RF group for difference between the peak and low time points. O

wo-way ANOVA confirmed no significant difference be- t

ween AL and RF groups (BLA: F(1,22)�0.12; P�NS;eA: F(1,22)�0.84; P�NS) it also indicated no effect due

o the interaction of the factor feeding condition and time.In the BNST temporal changes were observed in AL

ats, with high values at ZT12 and ZT18 (F(3,12)�6.62;�0.01; Fig. 3C). Food entrainment induced an increase

n the amplitude with a significant peak value at ZT12F(3,12)�25.33; P�0.001), however trough values were atT6 in both feeding conditions. The two-way ANOVA indi-ated no significant difference between groups (F(1,23)�.82; P�NS), however it indicated a significant interactionf the factors feeding condition and time (F(3,23)�5.41;�0.005).

In the Hip a robust daily rhythm was observed in ALats, with peak values at ZT18 (F(3,11)�14.25; P�0.001;ig. 3F). Food entrainment produced an increase in themplitude of the daily oscillation and shifted the highesteak to ZT12 (F(3,11)�119.34; P�0.0001). The two wayNOVA indicated no difference between feeding condi-

ined rats (Œ) and their ad libitum controls AL (�). Diurnal fluctuationse PVT is the exception with a peak at ZT6. The horizontal bar on theoup obtained with the Tukey post hoc test (P�0.01); � for the AL andications as in Fig. 1.

ood-entraZT12. Th

ions (F(1,22)�3.78; P�NS), however indicated a signifi-

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M. Ángeles-Castellanos et al. / Neuroscience 144 (2007) 344–355350

ant interaction of feeding condition and time (F(1,22)�1.12; P�0.0001).

The LS showed a robust daily oscillation of PER1-IRith highest values at ZT18 (F(3,11)�13.89; P�0.001;ig. 3G). This daily oscillation was shifted to ZT12 in RF

ats, the amplitude was lower but the time effect remainedignificant (F(3,12)�6.35; P�0.01). The two-way ANOVAndicated no significant difference between feeding condi-ions (F(1,23)�0.49; P�NS), but the interaction of feedingonditions and time indicated a significant effect (F(3,23)�.61; P�0.01).

In the PFC PER1-IR exhibited a daily oscillation with aow amplitude however statistically significant (F(3,11)�.51; P�0.01; Fig. 3H). Food restriction induced a highermplitude and a significant peak at ZT12 (F(3,11)�16.04;�0.001). The two way ANOVA indicated no significant

ig. 4. Representative photomicrographs of PER1 immunoreactivity inar�100 �m; aca, anterior commissure anterior part; cc, corpus callo

ifference between AL and RF groups (F(1,22)�0.84; w

�NS), but a significant effect due to the interaction feed-ng condition and time (F(3,22)�7.41; P�0.001).

The PVT exhibited a low amplitude daily oscillation ofER1-IR, which was statistically significant (F(3,11)�9.93;�0.001; Fig. 3I). Food restriction imposed a phase shiftnd increased amplitude, peak value was observed at ZT6F(3,12)�51.75; P�0.0001). The two-way ANOVA con-rmed a significant difference between both groupsF(1,23)�33.94; P�0.0001) and a significant effect to thenteraction of the feeding condition and time (F(3,23)�1.02; P�0.0001).

DISCUSSION

he present study reports that during FAA and during theollowing two hours after food access, all limbic structures,

e and Acc-Shell, Hip, PFC and PVT in AL group for a 24 h cycle. Scalethird ventricle.

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M. Ángeles-Castellanos et al. / Neuroscience 144 (2007) 344–355 351

During FAA the animals exhibit arousal, increased for-ging, exploration, sniffing locomotion and increased ap-roaches to a food cup (Stephan, 2001). Due to this be-avioral complex it can be assumed that during FAA ratsndergo a high food-motivated state. Studies using a par-digm of daily palatable food delivery have confirmed thatotivation plays a relevant role for food-entrainment.ood-deprived hamsters anticipate a palatable meal (Abend Rusak, 1992), as well as free-feeding rats offered amall piece of chocolate (Mendoza et al., 2005c,d) andree-feeding rats offered a daily meal containing a concen-rated amount of nutrients (Mistlberger and Rusak,1987).ood-deprived rats exposed to hypocaloric diets or to a lowrotein diet, both of which induce a negative energy bal-nce and therefore a high food-motivated state, show in-reased FAA (Salazar-Juarez et al., 2003; Challet et al.,003). In addition, under constant lighting conditions daily

ig. 5. Representative photomicrographs of PER1 immunoreactivity inar�100 �m; aca, anterior commissure anterior part; cc, corpus callo

ccess to deficient diets or to a palatable food attains the 2

apacity to entrain the SCN (Mendoza et al., 2005d;alazar-Juarez et al., 2003). Altogether, feeding conditions

hat impose a high food-motivated state are potent entrain-ng signals. Present data add up evidence indicating thatorticolimbic structures and the ACC, which are involved inenerating a motivational and an appetitive state are sig-ificantly active when rats are exhibiting FAA.

The circuitry mediating motivation with behavioral ex-ression has been extensively discussed and differentodels highlight diverse components of the limbic systems key structures for the generation of food motivation. Inrecent review, Kelley et al. (2005) have suggested a

ircuit constituted by the hypothalamus–thalamus–striatalxis, as the interface between energy homeostasis, hedo-ism and action. They have indicated a main role of theVT and ACC to promote and mediate hunger and energyalance with motivation and behavioral expression (Kelley

and Acc-Shell, Hip, PFC and PVT, in RF group for a 24 h cycle. Scalethird ventricle.

Acc-Core

004, Kelley et al., 2005; Mogenson et al., 1980). This is

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M. Ángeles-Castellanos et al. / Neuroscience 144 (2007) 344–355352

urther supported by data reporting an increased Fos-IR inoth ACC regions in rats expecting a daily meal as well asaily scheduled chocolate (Mendoza et al., 2005c). Phillipst al. (2003) indicate that the amygdala plays a relevantole with the PFC in order to select and co-ordinate specificequences of behavior toward incentive stimuli. The pre-rontal and infralimbic cortex exhibit increased Fos-IRhen rats are expecting food and have shown to be a

elevant element to start arousal during an appetitive be-avior (Valdés et al., 2006). In addition, increased Fos-IRas been reported in the BNST, PFC and amygdala asso-iated with high motivational states during food or rewardxpectation (Moncho-Bogani et al., 2005; Valdés et al.,006). Data here reported are in agreement with thoserevious studies.

Because FAA comprises diverse behavioral patterns, itannot be discarded that Fos-IR observed during FAA maye due to arousal or stress. It is possible that during FAAnimals undergo a stressful motivational state especiallyue to the long fasting intervals between meals. This isossible especially because FAA coincides with high lev-ls of corticosterone anticipating food access (Krieger,979; Díaz-Muñoz et al., 2000). In animals exposed totress Figueiredo et al. (2003) have described Fos-IR inhe ventral septum, PFC, BNST and PVT. In addition, thenatomical circuit formed by the amygdala the basal fore-rain and hypothalamus has shown to influence the hypo-halamic–pituitary–adrenal system and thus may promotentrainment of the corticosterone peak (Swanson andetrovich, 1998; Herman et al., 2005) observed duringAA. The PFC also shows Fos-IR when rats undergo atate of arousal (Valdés et al., 2006). However, the partic-

pation of corticolimbic structures during FAA to a palatableeal without food restriction suggests that rats may un-ergo a high motivated state related to a hedonic stimulusMendoza et al., 2005c). Thus, it is likely that corticolimbicctivation observed in present data may be related mainlyo a motivational state.

Diurnal rhythms of the clock protein PER1 were ob-erved in corticolimbic structures, but not in the amygdalaBLA and CeA). In AL rats daily peak values for the rhyth-ic structures were observed at ZT12 or at ZT18,werebserved at thus associated with the onset of nocturnalctivity. The Acc-Core, Acc-Shell, BNST, Hip, LS, PFC andVT showed phase adjustments due to the RFS, suggest-

ng entrainment of the clock gene machinery. In addition inome structures food entrainment increased the amplituden the daily rhythm. Interestingly, in all structures peakalues were shifted toward ZT12, with the exception of theVT for which the peak was shifted to ZT6.

The entraining effect of feeding schedules on PER1 inhe Acc-Core and Acc-Shell associated with the intenseos-IR observed previous to mealtime suggests a relevantole of this structure for generation of FAA. This is furtherupported by evidence indicating that Acc-Core is a con-tituent of a circuit mediating anticipatory actions for foodriented behaviors, better known as “wanting” responseBerridge and Robinson, 2003; Cardinal and Everitt, 2004)

nd that Acc-Shell contributes to the hedonic response r

nd motivation for sweet and palatable food (Peciña anderridge, 2005). Because we now describe that daily feed-

ng can impose a phase on the oscillations of PER1 in thisircuitry, we suggest that cellular oscillations in this neuralircuit may keep track of mealtime in order to predict thexpected stimulus and to generate the corresponding mo-ivational and/or arousal state for anticipation.

No significant oscillations of PER1 were observed inhe BLA or in the CeA in ad libitum or food-entrainedonditions. These data are not consistent with a study thateported diurnal oscillations of PER2 in the BLA and CeALamont et al., 2005b). The difference of this previoustudy and present data may rely on the clock gene proteinsed to monitor rhythmicity. In recent years evidence hasmerged indicating that certain entraining stimuli modifyhe phase of clock genes in a different manner. Such is thease of the study by Mendoza et al. (2005b) in which aypocaloric diet changed the phase of Per1 and Cry2 inhe SCN, but not of Per2 and Bmal1. Likewise Yan andkamura (2002) indicated that Per1 and Per2 are ex-ressed in a topographic manner in the SCN and theirxpression is not uniform in time nor among the sub-egions after a light pulse. A similar phenomenon has beenbserved in peripheral tissue explants in which the expres-ion of Per1 m RNA dampens after a few cycles, whileer2 mRNA persists oscillating for more than 20 cycles

Stokkan et al., 2001; Yamazaki et al., 2000; Yoo et al.,004). Therefore, the effects of RFS on PER2 in the amyg-ala, as well as in other brain structures remain to bexplored.

The Hip did not exhibit a FOS-IR response at theealtime points, indicating no activation associated with

eeding or FAA, but the changes in PER1 indicate thatlock genes in this structure are responsive to entrainingignals elicited by RFS. A previous study using in situybridization described diurnal rhythms of mPer1 mRNA inhe Hip, pyriform cortex, striatum and parietal cortex witheak values around ZT12 (Wakamatsu et al., 2001). Dailyhythms were entrained by RFSs shifting peak values toT6. Present data are in agreement with that study indi-ating a shift of PER1 protein in the Hip, and PFC in RFats toward ZT12, 6 h after the peak expression of theRNA (ZT6).

A previous study using transfected luciferase as aeporter of the clock gene per1 indicated that 14 of 27nalyzed brain areas exhibit circadian rhythms of per1RNA in culture (Abe et al., 2002). Authors observedeak rhythmicity in the PVT and no rhythmicity in telence-halic structures including the ACC, the BNST, the Hip andhe medial septum. Because the study was performed withultured brain explants it is difficult to conclude if the same

s expected for an intact animal. In contrast, Amir et al.2004) using the clock protein PER2 as reporter of rhyth-icity, described a diurnal rhythm in the BNST with a peakt ZT12, which faded after a bilateral lesion of the SCN

ndicating that rhythms in the BNST are dependent ofignals emerging from the master circadian clock. How-ver, dampened rhythmicity of PER2 in the BNST was

ecovered with RFSs (Lamont et al., 2005a). Present data

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M. Ángeles-Castellanos et al. / Neuroscience 144 (2007) 344–355 353

onfirm that in the rat the BNST exhibits diurnal oscillationsf PER1 and that RFS can modify its daily rhythms.

To the present it is not clear how the clock genescillations are transduced into cellular function (Okamura,004; Yamamoto et al., 2005). It is also not clear what cane the role of PER1 for the physiology for peripheral os-illators (Cailotto et al., 2005); and PER1 has mainly beensed as a reporter of cellular or tissue rhythmicity. Theature of the main entraining signals to peripheral clocks islso uncertain. In the SCN light triggers Per1 mRNA ex-ression, that is translated 6 h later to a peak of PER1rotein expression (Lowrey and Takahashi, 2000). How-ver, peripheral oscillators are not entrained by light whenhey are uncoupled from the SCN (Yamazaki et al., 2000).ata obtained from peripheral cells and tissues in culturend in vivo indicate that humoral signals, like hormones oretabolites, that modify the energetic state of the cell, can

eset clock gene oscillations (Balsalobre et al., 1998, 2000;ou et al., 2005). Clock genes in peripheral tissues arelso entrained by restricted feeding (Damiola et al., 2000;avidson et al., 2003; Hara et al., 2001). However, forscillators in the brain the nature of entraining signals hasot been explored. Evidence indicates that chronic treat-ent with methamphetamine entrains activity rhythms and

hifts Per1, Per2 and Per3 rhythms selectively in the cau-ate-putamen and parietal cortex (Masubuchi et al., 2000).resent data suggest that feeding stimuli or related events

nvolved in food entrainment are potent synchronizers forER1 in corticolimbic structures and the ACC and shift thehase of PER1 to ZT12, 6 h after feeding.

In recent years a series of data has pointed out thateripheral oscillators are mainly entrained by feedingchedules, including peripheral tissues like liver, pancreas,igestive organs, and central integrators in the brain stemnd hypothalamus involved in energy balance and main-

enance of body weight. Present data indicate that limbictructures involved in motivational and appetitive pro-esses also are modified by food entrainment. Thus, wean conclude that RFSs entrain behavior and physiologicalariables by entraining simultaneously diverse regulatoryystems. The markers of cellular activation used for thistudy point out two different pathways through which RFSodify the cells, Fos-IR indicates significant activation dur-

ng FAA and PER1-IR indicates an influence of restrictedeeding on the clock machinery of corticolimbic structures.t remains to be established whether energetic fluctuationsre the signal to modify those cellular markers, or whether

hey are affected by behavioral or motivational factorsssociated with food entrainment.

Present data add up another piece of information abouthe substrates involved in food-entrainment. Due to theelevant and powerful entraining force of feeding sched-les, future studies should explore their use to reentrain

ndividuals in conditions of internal desynchronization oross of rhythmicity.

cknowledgments—This study was supported by grants DGAPAN-203803 and CONACyT 43950-M. Authors wish to thank Dr.

uud Buijs and the three reviewers for valuable comments on this

anuscript, María Isabel Pérez Montfort for revision of the Englishrammar and style and Dr. Miguel Pérez de la Mora and his groupor providing a cryostat to obtain the second set of sections forER1 staining.

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(Accepted 29 August 2006)(Available online 11 October 2006)