Progressive anticipation in behavior and brain activation of rats exposed to scheduled daily palatable food

Neuroscience 281 (2014) 44–53

PROGRESSIVE ANTICIPATION IN BEHAVIOR AND BRAIN ACTIVATION OF RATS EXPOSED TO SCHEDULED DAILY PALATABLE FOOD A. BLANCAS, S. D. GONZA´LEZ-GARCI´A, K. RODRI´GUEZ AND C. ESCOBAR *

INTRODUCTION Food-related signals are powerful time cues for physiological and behavioral systems. In rodents, scheduled daily access to food elicits anticipatory activity (FAA) to the daily meal. This FAA starts 2–3 h prior to food access and is characterized by behavioral arousal and activation, foraging, increased approach to a feeder and the search for food (Mistlberger, 1994). Associated with FAA plasma corticosterone and core temperature increase, indicating that feeding schedules also modify hormonal and metabolic functions (Krieger and Hauser, 1978; Honma et al., 1983). Likewise daily access to a palatable snack (5 g of chocolate) induces rodents to develop anticipatory activity and has shown to be a powerful stimulus to entrain behavior and neuronal activity in brain areas mediating motivation and reward (Mistlberger and Rusak, 1987; Mendoza et al., 2005; Hsu et al., 2010). Rats exposed to palatable food entrainment show a brief and precise anticipatory activation, and when the protocol is interrupted, rats continue anticipating precisely at the scheduled time for up to 5 days indicating the involvement of a clock mechanism (Angeles-Castellanos et al., 2008). Daily scheduled palatable food entrains c-Fos activation and PER1 and PER2 oscillations in corticolimbic areas, particularly in the nucleus accumbens (ACC), the prefrontal cortex (PFC), amygdala and arcuate nucleus (ARC), which are involved in producing motivational and reward responses (Angeles-Castellanos et al., 2008; Verwey et al., 2007). Such entrained temporal patterns of activation may underlie the time-keeping system that drives the search and craving for palatable food (Webb et al., 2009; Escobar et al., 2011). Visual inspection of actograms suggests that anticipation of a daily palatable snack (chocolate) requires several days before a clear anticipatory response is observed, which suggests that animals require several cycles to compute the time signal and possibly to induce daily oscillations in brain areas that will be involved in estimating time for anticipation. This evidence suggests that individuals monitor the time and phase when food is available in order to elaborate the anticipatory strategy (Mistlberger, 1994). The detection of brain areas involved in the progressive development of anticipation of a palatable snack may serve as a strategy to uncover structures that function as coordinators or pacemakers for palatable food anticipation. Based on this possibility, the present study aimed to explore the development of anticipatory activity and neuronal activation to palatable food because, our

Departamento de Anatomı´a, Facultad de Medicina, UNAM, Ciudad Universitaria 3000, Me´xico DF 04510, Mexico

Abstract—Scheduled and restricted access to a palatable snack, i.e. chocolate, elicits a brief and strong anticipatory activation and entrains brain areas related with reward and motivation. This behavioral and neuronal activation persists for more than 7 days when this protocol is interrupted, suggesting the participation of a time-keeping system. The process that initiates this anticipation may provide a further understanding of the time-keeping system underlying palatable food entrainment. The aim of this study was to analyze how this entraining protocol starts and to dissect neuronal structures that initiate a chocolate-entrained activation. We assessed the development of anticipation of 5 g of chocolate during the first 8 days of the entrainment protocol. General activity of control and chocolate-entrained rats was continuously monitored with movement sensors. Moreover, motivation to obtain the chocolate was assessed by measuring approaches and interaction responses toward a wire-mesh box containing chocolate. Neuronal activation was determined with c-Fos in reward-related brain areas. We report a progressive increase in the interaction with a box to obtain chocolate parallel to a progressive neuronal activation. A significant anticipatory activation was observed in the prefrontal cortex on day 3 of entrainment and in the nucleus accumbens on day 5, while the arcuate nucleus and pyriform cortex reached significant activation on day 8. The gradual response observed with this protocol indicates that anticipation of a rewarding food requires repetitive and predictable experiences in order to acquire a temporal estimation. We also confirm that anticipation of palatable food involves diverse brain regions. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: food entrainment, circadian rhythms, accumbens, prefrontal cortex, food anticipatory activity.

*Corresponding author. Address: Departamento de Anatomı´ a, Facultad de Medicina, UNAM, Edificio B 4° piso, Av Universidad 3000, Col. Copilco, Me´xico DF C.P. 04510, Mexico. Tel/fax: +52-5556232422. E-mail address: [email protected] (C. Escobar). Abbreviations: ACC, accumbens; ANOVA, analysis of variance; ARC, arcuate nucleus; BL, baseline; CH, chocolate-entrainment; FAA, food elicits anticipatory activity; PBS, phosphate-buffered saline; PBSGT, phosphate-buffered saline, 0.25% nutritive gelatin and 0.5% triton; PFC, prefrontal cortex; PiCX, pyriform cortex; SCN, suprachiasmatic nucleus. http://dx.doi.org/10.1016/j.neuroscience.2014.09.036 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 44

A. Blancas et al. / Neuroscience 281 (2014) 44–53

automated recording system for general activity has indicated that anticipatory activity for chocolate is very brief (Angeles-Castellanos et al., 2008; Mendoza et al., 2005). We developed an additional strategy to evidence the motivation to obtain the daily snack. Based on a report by Valde´s et al. (2010) rats were challenged with a wiremesh box containing a piece of chocolate and the effort to obtain the chocolate was evaluated. Moreover core temperature was monitored with intraperitoneal sensors in order to further evidence a physiological response associated with FAA. Data here provided indicate that the development of anticipatory activity to chocolate is gradual, that behavioral markers of effort and general activity increased progressively associated with evolving neuronal activity in the PFC, ACC, ARC, and the pyriform cortex (PiCX). The prelimbic region of the PFC and the ACC, both involved in processing reward responses, exhibited early anticipatory activation, which indicates their relevant role for emergence of anticipation of a palatable snack.

EXPERIMENTAL PROCEDURES Subjects and general conditions Male Wistar rats weighting 250–300 g were obtained from the general bioterium in the Medical Faculty at the Universidad Nacional Auto´noma de Me´xico. Rats were housed in individual transparent acrylic cages with food (Rodent Laboratory Chow 5001) and water ad libitum throughout the experiments. Cages were placed in lockers (eight subjects/locker) with a controlled 12:12-h light/dark cycle (light onset at 0700), constant temperature (22 ± 1 °C) and continuous air flow. Rats were acclimated to environmental conditions for 1 week before starting the experimental procedures. Animal handling procedures were conducted according to the national guide for care and use of animal experimentation (Decreto ley de proteccio´n a los animales del Distrito Federal, Gaceta oficial del Distrito Federal 26/02/02), which is in agreement with international requirements for animal handling. Experimental design Rats were randomly assigned to one of two groups: (1) Control rats: remained in their cage with food and water ad libitum; (2) Chocolate-entrainment (CH) group: rats received daily in their home cage 5 g of chocolate at 13:00 h for 8 days (kinder maxiä; 5 g = 28.6 kcal; contains 10.3% proteins, 54.2% carbohydrates and 35.5% fat). A piece of chocolate was given to the rats and left in the cage until they finished, without time limitation. For assessment of neuronal activity, all rats were sacrificed at 14:00 h. Rats from the CH group were divided in two groups, one group was sacrificed 1 h after chocolate ingestion and the other group sacrificed anticipating chocolate. The time of sacrifice was projected based on the time of expression of c-Fos protein which shows a peak after 60 min and lasting around 90 min. Rats of the control and chocolateentrained groups shared lockers, thus at the moment of chocolate delivery, control rats were exposed to the

45

opening of the doors and the brief presence of the experimenter. Anticipatory activity to chocolate in general locomotion and core temperature One set of 12 rats was used to monitor general activity and eight of the same rats received an intraperitoneal temperature sensor. During the adaptation week, rats underwent surgery in order to place intra-abdominal temperature sensors (iButton Sensor-Temperature Logger; Maxim Integrated Products, Dallas, Semiconductor, USA). Rats were anaesthetized with an intramuscular administration of Xylazine (Procin 0.01 mL/100 g bodyweight) and Ketamine (Inoketam 0.02 mL/100 g body weight). Under deep anesthesia a small incision was cut in the abdominal cavity and a sterilized temperature sensor was introduced in the peritoneum. Anterior abdominal muscles and skin were sutured by layers with absorbable catgut 000 and rats were left to recover for 1 week before starting the baseline (BL). The iButtons were programed to collect temperature data every 30 min for the total handling interval and are shown as daily mean activity patterns for BL, days 1, 2, 3, 5 and 8 of chocolate entrainment. Also, the mean temperature exhibited 6, 4, and 2 h prior and 2 h after chocolate access was calculated. General activity was recorded with an automatic monitoring system using tilt switches placed under the individual cages as previously reported (Escobar et al., 2007). These sensors detected continuously the animal’s movements during the 24 h every day. Behavioral events were collected with a digitized system and were automatically stored every minute in a PC for further analysis The system for monitoring and collection of data was developed by the Mexican biomedical company Omnialva SA de CV. Analysis was performed with the program for PC SPAD9 (Sistema de Procesamiento y Adquisicio´n de Datos, version 1.1.1) designed for this system and based on Matlab (Omnialva SA de CV, Mexico City, Mexico). General activity counts were organized in 15-min bins, for which each time point represents the sum of the previous activity counts for the interval. Data are shown as mean activity temporal patterns for BL, days 1, 2, 3, 5 and 8 of chocolate entrainment. Evaluation of motivation for chocolate with the wire-mesh box A different set of control (C; n = 8) and chocolateentrained rats (CH; n = 8) were used to test the motivation for the palatable snack, C and CH rats were exposed during 5 min to a sealed wire-mesh box containing a piece of chocolate (based on the strategy reported by Valde´s et al. (2010) and the interaction with the box was monitored. First, during BL, all rats were exposed to an empty box. On days 1, 2, 3, 5 and 8 of chocolate entrainment C and CH rats were evaluated while exposed to a box containing 5 g of chocolate. An additional control group (n = 8) was tested with an empty box during the same days as a control for daily exposure to the box.

46

A. Blancas et al. / Neuroscience 281 (2014) 44–53

The wire-mesh box (5 cm  5 cm square with 5 mm  5 mm holes) allowed the animals to see and smell the chocolate but hindered them to touch, scratch or bite it. The box was introduced at 12:50 in the home cage for 5 min. The main purpose of this device was to evaluate the interaction and effort the rats were willing to exert to obtain the snack. After 5 min the box was removed from the cage and at 13:00 CH rats received the daily chocolate portion. This procedure was performed daily for 8 days. Behavior during the 5-min test was recorded with a camera connected to a DVD recording device. The DVD recordings were later analyzed with an instantaneous sampling strategy (Martin and Bateson, 1993) to assess approaching and manipulating behaviors directed to the wire box. For this instantaneous sampling method videos were stopped every 5 s and the observed behavior was recorded, with this strategy 60 samples of behavior were obtained for the 5-min interval. The effort or motivation to obtain the chocolate was determined by counting the number of contacts and handling the box according to the following categories: Passive interaction with the box: smell or touch the cage; Active-effort interactions with the box: bite, pull/push, manipulate/turn the cage with forepaws; Total interaction: passive + effort interaction. Anticipatory activation in brain areas, immunohistochemistry for c-Fos A different set of rats control (C; n = 8) and chocolateentrained rats (CH; n = 8) were used to assess neuronal activation along the progression of chocolateentrainment. Brains were obtained from control and chocolateentrained rats. This last CH group was subdivided in two subgroups: Ingestion and Anticipation. The subgroup for ingestion was used to test the brain’s response after eating chocolate (n = 3–4/day), brains were obtained 1 h after chocolate ingestion (14:00 h) on BL, and days 1, 2, 3, 5 and 8. The subgroup for Anticipation was used to evaluate the brain’s response in anticipation of chocolate (n = 3–4/day) brains were obtained at 14:00 h, without chocolate delivery on BL, and days 2, 3 5 and 8. It is important to indicate that for this group brains collected on day 2 corresponded to rats that had ingested only once a chocolate piece and were sacrificed on day 2 before chocolate delivery, brains collected on day 3 corresponded to rats that had ingested a piece of chocolate for 2 days and were supposed to expect chocolate on day 3, and so on. Rats were anaesthetized with an overdose of pentobarbital and perfused intracardially with 250 mL saline (0.9%) followed by 250 mL cold 4% paraformaldehyde in 0.1 mM phosphate buffer (PB; pH 7.2). Brains were removed, postfixed for 24 h and cryoprotected in 30% sucrose solutions. Brain sections of 40 lm were cut with a cryostat. Free-floating sections were incubated for 72 h in rabbit polyclonal c-Fos protein primary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:2500 in phosphate-buffered saline (PBS), 0.25% nutritive gelatin and 0.5% triton (PBSGT). After rising with PBS, sections were

incubated with a biotinylated anti-rabbit immunoglobulin G (IgG) made in goat (Vector Laboratories, Burlingame, CA, USA) diluted 5:1000 in PBSGT for 90 min at room temperature. Sections were rinsed three times with PBS and incubated at room temperature in avidin–biotin– peroxidase complex (0.9% avidin and 0.9% biotin solutions; Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA). After 90 min, sections were rinsed three times with PBS and were reacted in a dilution of 3,3-diaminobenzidine (0.5 mg/mL) DAB; Sigma, St Luis, MO, USA); as the chromogen with 0.01% H2O2 and 10% nickel–cobalt. Sections were mounted on gelatin-coated slides, and dehydrated through a series of alcohols, cleared with xylene and coverslipped with Entellan (Merck, Darmstadt, Germany). We selected anterior, medial and posterior sections of each selected region for counting the number of c-Fospositive cells. Sections were identified with the atlas of Paxinos and Watson (1998). The prelimbic region of the PFC (3.5 mm, 3.2 and 2.5 mm anterior to bregma) ACC Core and Shell (1.9, 1.6 and 1.2 mm anterior to bregma); ARC (2.8. 3.14 and 3.3 mm posterior to bregma); and the suprachiasmatic nucleus (SCN; 0.98, 1.1 and 1.14 mm posterior to bregma). Sections were observed with an optical microscope (Olympus BX41), photographs were acquired in jpg format with a 20 magnification and with a digital camera (Evolution LC color) connected to a PC and using the program Image-Pro Plus 5.1. Immunopositive c-Fos neurons were counted with the Image J Launcher program setting an automatic threshold and contrast of pixels. Statistical analysis Temperature and general cage activity are presented as mean daily temporal patterns. Due to different sensitivity of the tilt switches placed under the individual cages 15-min activity bins were normalized to 100% of total daily counts. Also an analysis for anticipatory changes was performed by comparing between measurement days the 2-h interval preceding chocolate access. For BL and days 1, 2, 3, 5, and 8 of chocolate entrainment the 2-h intervals were compared with a one-way analysis of variance (ANOVA) for repeated measures followed by a Tukey post hoc test with a < 0.05. For the wire-mesh box, test data are presented as mean ± standard error of the mean and a two-way ANOVA with days as a variable of repeated measures was used to compare among groups the behavioral events. This was followed by, Duncan post hoc test with significant values set at P < 0.05. Cells positive to c-Fos in each region were classified by group and are presented as mean ± standard error of the mean. Data were compared with a one-way ANOVA followed by a Tukey post hoc multicomparisons test with a = 0.05. Statistical analysis was performed with the program Statistica for Windows version 4.5 (StatSoft, 1993).

RESULTS On the first day of palatable food entrainment rats explored and smelled the piece of chocolate and took

A. Blancas et al. / Neuroscience 281 (2014) 44–53

up to 1 h to ingest the 5 g. As soon as day 2 all rats ingested the chocolate piece in less than 10 min and by day 5 they were ingesting 5 g in less than 5 min. This piece of chocolate contains 28.6 kcal which represents about 30% of the caloric intake of an adult rat (Johnson and Kenny, 2010). Anticipatory activity to chocolate in general locomotion and core temperature The continuous monitoring of general activity indicated during BL a clear daily rhythm, with low activity counts during the light phase (23% of total activity) and high values along the night (77% of total activity; Fig. 1 column A). Around chocolate delivery time, rats accumulated in a 2-h interval between 2.5% and 3.5% of their total daily activity counts (Fig. 1 column C). As

47

soon as day 3, during the 2-h interval preceding chocolate time, the activity counts increased significantly and were statistically different from the activation during BL (Fig. 1 column C, upper panel). This anticipatory activation was even more increased on days 5 and 8. The one-way ANOVA for repeated measures indicated significant effects between measurement days for the 2-h activation (F(5,55) = 5.45; p < 0.0003). For the registration of body temperature one of the thermistors produced uncalibrated data, thus results are presented for seven rats. During BL a consistent temperature rhythm synchronized to the light–dark cycle was observed in all rats (Fig. 1 column B top). The average temperature of rats during the light phase was 36.7 °C. Daily scheduled ingestion of 5 g of chocolate induced an immediate and brief increase in body temperature. Also, a progressive anticipatory decrease

Fig. 1. Mean activity temporal profiles for general activity (A; n = 10) and body temperature (B; n = 7) recorded for a baseline (BL) and for days 1, 2, 3, 5 and 8 of chocolate entrainment. The arrow and dotted lines indicate the moment of daily chocolate access. In (C) the daily increase of behavioral anticipation (top) and body temperature (bottom) are represented as change from the baseline mean (D). Asterisk represents statistical difference from baseline (p < 0.05). Plus indicates statistical difference from day 1 and 2 (p < 0.05).

48

A. Blancas et al. / Neuroscience 281 (2014) 44–53

of body temperature was observed starting about 6 h before chocolate delivery and an anticipatory rise in temperature developed between measurement days with peak values coinciding with the moment of chocolate delivery (Fig. 1, column C lower panel). This progressive increase reached statistical difference from BL on day 8. The ANOVA for repeated measures indicated a significant change along days (F(5,30) = 2.70; p < 0.03). Motivation for chocolate, the wire-mesh box test With the behavioral test using the wire-mesh box the twoway ANOVA for repeated measures indicated a

significant difference between groups for passive behaviors (F(2,18) = 12.28; p = 0.0004), for active interactions (F(2,18) = 87.52; p = 0.00000) and for total interaction with the box (F(2,18) = 59.17; p = 0.00008). Also it indicated a significant interaction of groups with days as a variable of repeated measurements for passive behaviors (F(10,90) = 1.95; p = 0.04); active interaction (F(10,90) = 2.45; p = 0.01) and total interaction (F(10,90) = 3.55; p = 0.0005). Between measurement days of assessment with the wire-mesh box control groups did not vary the number of interaction events with the box along the days, while CH rats receiving daily 5 g of chocolate progressively increased the events of interaction (Fig. 2). As early as

Fig. 2. The interaction with an empty (white bars) or chocolate containing (stripped bars) wire-mesh box placed for 5 min in the home cage of control- (white; n = 8/day) and chocolate-entrained rats (gray; n = 8/day). The number of passive, active/effort and total interaction events with the box is represented as mean ± standard error of the mean. One asterisk indicates significant difference from Ch group with the C with empty box; two asterisks indicate a statistical difference of the CH group with both C groups (empty and chocolate-filled box) (P < 0.05).

A. Blancas et al. / Neuroscience 281 (2014) 44–53

day 1 a significant increase was observed in the active and total interaction with the box, which reached the highest expression on day 8 of chocolate access. General exploration in the cage during the test ranged between 40 and 28 daily events for both C groups (empty box and box with chocolate; data not shown) and decreased along the days for the C empty box and for the groups exposed to the chocolate box (F(5,42) = 3.35; P < 0.01; F(5,39) = 5.63; P < 0.0005, respectively), while no changes were observed between measurement days for the C exposed to the chocolate box (F(5,36) = 1.30; P = 0.28). Anticipatory activation in brain areas Representative microphotographs of c-Fos immunoreactivity in brain areas explored in this study are shown in Fig. 3 (left columns). For the anticipatory response to chocolate in c-Fos expression the one-way ANOVA indicated a significant effect between measurement days for the PFC (F(4,15) = 8.73; P = 0.007); for the ACCShell (F(4,12) = 9.91; P = 0.0008); for the ACC-Core (F(4,16) = 10.51; P = 0.0002); for the PiCX (F(4,13) = 4.16; P = 0.021); and for the ARC (F(4,15) = 8.55; P = 0.0008), while no significant effects were observed in the SCN (F(4,12) = 1.63; P = 0.22). In brains obtained from rats expecting chocolate (Fig. 3 1st column) a gradual increase of c-Fos was observed with the progression of the chocolate-entrainment protocol. As early as day 3 of the entrainment protocol a significant anticipatory increase of c-Fos-positive cells was observed in the PCF, while significant increase was observed for both ACC subregions on day 5 and for the PiCX and ARC on day 8. For the effects of chocolate ingestion the one-way ANOVA indicated a significant effect on c-Fos expression in the PFC (F(5,13) = 4.53; P = 0.013); in the ACC-Core (F(5,15) = 5.94; P = 0.0031); the ACC-Shell (F(5,16) = 3.54; P = 0.023); the PiCX (F(5,16) = 3.57; P = 0.023) and in the ARC (F(5,17) = 9.13; P = 0.0002) and no significant changes in the SCN (F(5,18) = 0.39; P = 0.84). As early as day one, chocolate ingestion produced c-Fos expression in the PFC, in both subregions of the nucleus ACC, Core and Shell, in the PiCX and in the ARC but not in the SCN (Fig. 3 2nd column) and this activation was similar along the 8 days of chocolate ingestion and statistically different from the control conditions (Fig. 3). For the PiCX this activation decreased with the progression of the protocol.

DISCUSSION Present data describe in rats progressive changes in behavioral, neuronal and physiological variables under a chocolate-entrainment protocol. This study provides evidence that as soon as day 3 of daily scheduled chocolate access, rats develop a significant anticipatory activation which is associated with increased active interaction with a wire-mesh box containing chocolate and increased anticipatory c-Fos expression in the PFC. Moreover, a trend to show increased values was also

49

observed to increase in body temperature neuronal activation in the ACC. Previously we had described that restricted access to palatable food is a powerful zeitgeber for locomotor activity, c-Fos and Per1 expression in reward-related areas (Angeles-Castellanos et al., 2008). In the present study, chocolate, as a palatable stimulus, was a strong drive to modify behavior, brain activation and core temperature. It is important to note that rats were in ad libitum-satiated conditions and the behavioral activation for chocolate was during the light phase, when rats are usually sleeping. The progressive behavioral activation has been previously described for anticipation of a scheduled regular chow food (Pitts et al., 2003). In this study we provide evidence of the gradual anticipation regarding motivational behaviors directed to a palatable snack. This anticipation evidenced in general activity, and in effort-like behaviors directed to the wire-mesh box, suggests a learning-related process. The mechanisms underlying anticipatory activity have been amply discussed and different models underlying the 24-h time-estimating process have been suggested (Mistlberger, 1994). The timing process may depend on oscillating structures that function as a food-entrained pacemaker, a continuously consulted clock or both (Stephan et al., 1979; Boulos and Terman, 1980; Aschoff, 1986; Mistlberger, 1994, 2009; Escobar et al., 2011). For this later system the gradual response here described may reflect the necessary cycles for entrainment to the periodic event, this relationship between learning and circadian entrainment has been previously discussed (i.e. Mistlberger, 1994; Amir and Stewart, 1996; Daan, 2000). Learning and memory have been proposed to be necessary in order to learn the time for the occurrence of a significant event (Antle and Silver, 2009) such as the access to a palatable reward as in this study. Present data confirm that anticipation of a rewarding food requires repetitive and predictable experiences in order to acquire a temporal estimation. In a previous study we described that when access to food is restricted to unpredictable schedules, rodents are unable to elaborate anticipatory activation (Escobar et al., 2007), moreover, unpredictable access to a palatable food also did not synchronize temporal patterns of PER2 expression in corticolimbic regions involved in reward and motivation to food (Verwey and Amir, 2012). The gradual anticipation of chocolate was observed in the activation of brain structures related with the reward system and this gradual change was not observed in rats after chocolate ingestion, which indicates that neuronal activation in anticipation or after chocolate ingestion was indeed different despite the fact that all rats were sacrificed at 14:00 h. A previous study using functional brain mapping also determined a gradual anticipatory activation in reward-related areas due to intraperitoneal ethanol infusion and reported a significant anticipation after 5–6 days of infusion in the anterior cingulated cortex, ventral pallidum and the medial preoptic area; (Tsurugizawa et al., 2012). Different from our study, preceding the ethanol infusion a conditioned stimulus (CS) was presented to elicit the anticipation while

50

A. Blancas et al. / Neuroscience 281 (2014) 44–53

Fig. 3. A gradual increase of c-Fos expression was observed in anticipation of the daily chocolate access (column BEFORE/ANTICIPATION). A significant increase was first observed in the prefrontal cortex and the two subregions in the nucleus accumbens. A later increase was observed in the pyriform cortex and the arcuate nucleus, while no changes were observed in the suprachiasmatic nucleus. Chocolate ingestion induced enhanced c-Fos-positive cells in limbic and hypothalamic structures as early as day 1 (column AFTER). Black bars represent the control group (n = 4/day) and gray bars represent the chocolate-entrained group (CH; n = 3–4/day). The X axis indicates the number of days that rats were exposed to chocolate. Asterisks indicate significant difference with the control (P < 0.05). Representative microphotographs correspond to the control condition (left) and to day 8 during anticipation (middle) or after chocolate ingestion (right). The scale bar = 200 lm.

our study did not provide evident external cues to inform rats of the upcoming chocolate reward. Interestingly, here we show that the first neuronal structures active during anticipation were the PFC and the ACC. The PFC is involved in higher cognitive functions such as learning

and memory and the regions forming the PFC, namely anterior cingulate area, prelimbic and infralimbic cortex (Kesner and Churchwell, 2011) participate as part of the reward system (Chandler et al., 2013) and modulate DA release (Patton et al., 2013). Daily activity rhythms as

51

A. Blancas et al. / Neuroscience 281 (2014) 44–53

measured with c-Fos have been described for the PFC and ACC. Lesions of the PFC including the prelimbic and infralimbic regions resulted in loss of daily oscillations in the ACC, suggesting that PFC mediates neural activation in the ACC (Baltazar et al., 2013). Therefore, we hypothesize that the interaction between the PFC and ACC may promote cyclic activation in the reward system for the expression of anticipatory activity. Moreover we described in rats that the PFC and ACC continue exhibiting chocolateentrained oscillations of the clock gene Per1 after interruption of chocolate access suggesting their participation in a time-keeping system (Angeles-Castellanos et al., 2008). We and others have reported that hypothalamic nuclei involved in food ingestion and metabolic regulation exhibit c-Fos anticipatory activation in food-entrained rats (Angeles-Castellanos et al., 2004; Gooley et al., 2006; Jime´nez et al., 2013; Mitra et al., 2011), however Per1 a gene reflecting the circadian clock machinery, is not entrained by a palatable snack (Mendoza et al., 2005; Angeles-Castellanos et al., 2008). Here we report significant anticipatory c-Fos expression in the ARC on day 8, corresponding to 7 days of scheduled ingestion of chocolate, suggesting that the ARC may be affected by metabolic or behavioral changes necessary for anticipation. This process requires further studies since Bake et al. (2013) did not find changes in the RNA production of peptides involved in energy balance in rodents anticipating palatable food. In contrast, during this short 8-day protocol the SCN did not exhibit a change in the c-Fos expression confirming that it does not mediate anticipatory activation to chocolate. Temporal patterns for core temperature also reflected a gradual change associated with chocolate entrainment, we observed mainly a steep increase in response to chocolate ingestion when 2-h intervals were compared. This progressive increase mirrors the anticipatory response observed in behavioral activation. A similar temporal pattern was reported by Merkestein et al. (2012) after 5 weeks of chocolate entrainment. A previous study reported in rats exposed to daily access to sucrose an anticipatory decrease in body temperature followed by a significant increase following sucrose access (Pecoraro et al., 2002). This temperature decrease is suggested to reflect an addiction-like process and has been observed in rodents exposed regularly to opioids (Spangler et al., 2004). Moreover, Gallardo et al. (2012) only reported a moderate increase in body temperature in mice in response to the daily access to a scheduled palatable high-fat meal, however, the preceding handling to record the behavior of the animals complicated the interpretation of results. In contrast, with palatable entrainment, during a food restriction protocol body temperature increases anticipating meal-time and remains high after food ingestion (Recabarre´n et al., 2005; Angeles-Castellanos et al., 2010). In this condition a strong anticipatory activation is expressed and 100% of the daily caloric ingestion occurs in a 2–3-h interval. We conclude that associated with the progressive behavioral activation elicited by the daily scheduled access to chocolate an anticipatory increase in core temperature is elicited possibly due to locomotor activation.

Motivation to obtain a rewarding stimulus is commonly assessed with classical operant chambers in which motivation is defined by determining the break point, which is the maximal number of lever pressings a rat is willing to perform in order to obtain the reward (Zhang et al., 2003; D’Souza and Markou, 2010). This classical approach requires a previous conditioning procedure and the exposition to the rewarding stimulus during the training protocol. Other tests proposed for measuring motivation and appetitive behavior for a rewarding stimulus require rodents to jump over obstacles or barriers, or to cross electrified bars in order to exhibit the motivation to obtain a rewarding stimulus (Borgland et al., 2009; Oswald et al., 2011; Bai et al., 2014). Conditioned place preference has also been used as an indicator of the rewarding effects of a determined stimulus such as drugs of abuse or natural rewards (Abarca et al., 2002; Di Sebastiano et al., 2011; Me´ndez-Dı´ az et al., 2012). For this study we developed a behavioral test to evidence motivation to obtain the piece of chocolate. This simple and inexpensive procedure does not require training, and allows assessment of behavior in naı¨ ve rats. This method allowed rats to see and smell the chocolate and evidenced the effort-related behavior to obtain the rewarding stimulus. The strategy proved to be sensitive enough to evidence effort behaviors toward the wiremesh box and significantly differentiated the response to an empty box from a box containing chocolate. Moreover this response underwent the same progressive pattern as that observed in the neuronal activation of reward-related areas.

CONCLUSION This study has provided evidence that entrainment to a palatable food is a gradual process which can be observed at the behavioral and neuronal levels simultaneously. We report a simple and inexpensive method to assess motivation and for the first time in this entrainment protocol, we show that rats are willing to exhibit effortful behavior in order to get chocolate out of a sealed wire-mesh box, evidencing that this entrainment protocol induces a highly motivational state to food. The anticipatory activation of the PFC and ACC as early as day 3 and 5 of the entrainment protocol coincided with the expression of anticipatory activity to palatable food. This neuronal activation may underlie increased arousal, increased locomotion, foraging or other behavioral states related with food anticipation. Further studies will need to explore whether this process of palatable food entrainment in a long-term can lead to an addiction-like process. Acknowledgments—This study was supported by grants PAPIIT IN-224911; IG-200314 and CONACYT 82462.

REFERENCES Abarca C, Albrecht U, Spanagel R (2002) Cocaine sensitization and reward are under the influence of circadian genes and rhythm. PNAS 99(13):9026–9030.

52

A. Blancas et al. / Neuroscience 281 (2014) 44–53

Amir S, Stewart J (1996) Resetting of the circadian clock by a conditioned stimulus. Nature 8(379):542–545. Angeles-Castellanos M, Aguilar-Roblero R, Escobar C (2004) C-Fos expression in hypothalamic nuclei of food-entrained rats. Am J Physiol Regul Integr Comp Physiol 286(1):R158–R165. Angeles-Castellanos M, Salgado-Delgado R, Rodriguez K, Buijs RM, Escobar C (2008) Expectancy for food or expectancy for chocolate reveals timing systems for metabolism and reward. Neuroscience 155:297–307. Angeles-Castellanos M, Salgado-Delgado R, Rodriguez K, Buijs RM, Escobar C (2010) The suprachiasmatic nucleus participates in food entrainment: a lesion study. Neuroscience 165(4):1115–1126. Antle MC, Sliver R (2009) Neural basis of timing and anticipatory behaviors. Eur J Neurosci 30(9):1643–1649. Aschoff J (1986) Anticipation of a daily meal: a process of ‘‘learning’’ due to entrainment. Ital J Zool 20(2):195–219. Bai Y, Li Y, Lv Y, Liu Z, Zheng X (2014) Complex motivated behaviors for natural rewards following a binge-like regimen of morphine administration: mixed phenotypes of anhedonia and craving after short-term withdrawal. Front Behav Neurosci 3(8):23. Bake T, Duncan JS, Morgan DG, Mercer JG (2013) Arcuate nucleus homeostatic systems are not altered immediately prior to the scheduled consumption of large, binge-type meals of palatable solid or liquid diet in rats and Mice. J Neuroendocrinol 25(4):357–371. Baltazar RM, Coolen LM, Webb IC (2013) Diurnal rhythms in neural activation in the mesolimbic reward system: critical role of the medial prefrontal cortex. Eur J Neurosci 38:2319–2327. Borgland SL, Chang SJ, Bowers MS, Thompson JL, Vittoz N, Floresco SB, Chou J, Chen BT, Bonci A (2009) Orexin A/hypocretin-1 selectively promotes motivation for positive reinforcers. J Neurosci 29(36):11215–11225. Boulos Z, Terman M (1980) Food availability and daily biological rhythms. Neurosci Biobehav Rev 4(2):119–131. Chandler DJ, Lampersky CS, Waterhouse BD (2013) Identification and distribution of projections from monoaminergic and cholinergic nuclei to functionally differentiated subregions of prefrontal cortex. Brain Research 1522:38–58. D’Souza MS, Markou A (2010) Neural substrates of psychostimulant withdrawal-induced anhedonia. In: Self DW, Staley JK, editors. Behav Neurosci Drug Addict:120–157. Daan S (2000) Learning and circadian behavior. J Biol Rhythms 15(4):296–299. Di Sebastiano AR, Wilson Pe´rez HE, Lehman MN, Coolen LM (2011) Lesions of orexin neurons block conditioned place preference for sexual behavior in male rats. Horm Behav 59(1):1–8. Escobar C, Martı´ nez-Merlos MT, Angeles-Castellanos M, del Carmen Min˜ana M, Buijs RM (2007) Unpredictable feeding schedules unmask a system for daily resetting of behavioural and metabolic food entrainment. Eur J Neurosci 26(10):2804–2814. Escobar C, Salgado R, Rodriguez K, Blancas Va´zquez AS, AngelesCastellanos M, Buijs RM (2011) Scheduled meals and scheduled palatable snacks synchronize circadian rhythms: consequences for ingestive behavior. Physiol Behav 104(4):555–561. Gallardo CM, Gunapala KM, King OD, Steele AD (2012) Daily scheduled high fat meals moderately entrain behavioral anticipatory activity, body temperature, and hypothalamic c-Fos activation. PLoS ONE 7(7):e41161. Gooley JJ, Schomer A, Saper CB (2006) The dorsomedial hypothalamic nucleus is critical for the expression of foodentrainable circadian rhythms. Nat Neurosci 9(3):398–407. Honma KI, Honma S, Hiroshige T (1983) Critical role of food amount for prefeeding corticosterone peak in rats. Am J Physiol 245(3):R339–R344. Hsu CT, Patton DF, Mistlberger RE, Steele AD (2010) Palatable meal anticipation in mice. PLoS one 5(9):1–13.

Jime´nez A, Caba M, Escobar C (2013) Food-entrained patterns in orexin cells reveal subregion differential activation. Brain Res 1513:41–50. Johnson PM, Kenny PJ (2010) Dopamine D2 receptors in addictionlike reward dysfunction and compulsive eating in obese rats. Nat Neurosci 13:635–641. Kesner RP, Churchwell JC (2011) An analysis of rat prefrontal cortex in mediating executive function. Neurobiol Learn Mem 96(3):317–431. Krieger DT, Hauser H (1978) Comparison of synchronization of circadian corticosteroid rhythms by photoperiod and food. Neurobiology 75(3):1577–1581. Martin P, Bateson P (1993) Measuring behavior. An introductory guide. UK: Cambridge University Press. p. 84–100. Me´ndez-Dı´ az M, Rueda-Orozco PE, Ruiz-Contreras AE, Prospe´roGarcı´ a O (2012) The endocannabinoid system modulates the valence of the emotion associated to food ingestion. Addict Biol 17(04):725–735. Mendoza J, Angeles-Castellanos M, Escobar C (2005) Entrainment by a palatable meal induces food-anticipatory activity and c-Fos expression in reward related areas of the brain. Neuroscience 133:293–303. Merkestein M, Brans MA, Luijwndijk MC, de Jong JW, Egecioglu E, Dickson SL, Adan RA (2012) Ghrelin mediates anticipation to a palatable meal in rats. Obesity 20(5):963–971. Mistlberger R (1994) Circadian food-anticipatory activity: formal models and physiological mechanisms. Neurosci Biobehav Rev 18(2):171–195. Mistlberger RE (2009) Food anticipatory circadian rhythms: concepts and methods. Eur J Neurosci 30(9):1718–1729. Mistlberger R, Rusak B (1987) Palatable daily meals entrain anticipatory activity rhythms in free-feeding rats: dependence on meal size and nutrient content. Physiol Behav 41(3):219–226. Mitra A, Lenglos C, Martin J, Mbende A, Gagne´ A, Timofeeva E (2011) Sucrose modifies c-fos mRNA expression in the brain of rats maintained in feeding schedules. Neuroscience 19: 459–474. Oswald KD, Murdaugh DL, King VL, Boggiano MM (2011) Motivation for palatable food despite consequences in an animal model of binge eating. Int J Eat Disord 44(3):203–211. Patton MH, Bizup BT, Grace AA (2013) The infralimbic cortex bidirectionally modulates mesolimbic dopamine neuron activity via distinct neuronal pathways. J Neurosci 33(43):16865–16873. Paxinos G, Watson C (1998) The rat brain stereotaxic coordinates. 4th ed. San Diego: Academic Press. Pecoraro N, Gomez F, Laugero K, Dallman MF (2002) Brief access to sucrose engages food-entrainable rhythms in food-deprived rats. Behav Neurosci 116(5):757–776. Pitts S, Perone E, Silver R (2003) Food-entrained circadian rhythms are sustained in arrhythmic Clk/Clk mutant mice. Am J Physiol Regul Integr Comp Physiol 285(1):R57–R67. Recabarre´n MP, Valde´s JL, Farı´ as P, Sero´n-Ferre´ M, Torrealba F (2005) Differential effects of infralimbic cortical lesions on temperature and locomotor activity responses to feeding in rats. Neuroscience 134(4):1413–1422. Spangler R, Wittkowsky KM, Goddard NL, Avena N, Hoebel BG (2004) Opiate-like effects of sugar on gene expression in reward areas of the rat brain. Brain Res Mol Brain Res 124(2):134–142. Stephan FK, Swann JM, Sisk CL (1979) Entrainment of circadian rhythms by feeding schedules in rats with suprachiasmatic lesions. Behav Neural Biol 25(4):545–554. Tsurugizawa T, Uematsu A, Hisayuky U, Torii K (2012) Functional brain mapping of conscious rats during reward anticipation. J Neurosci Methods 206(2):132–137. Valde´s JL, Sa´nchez C, Riveros ME, Blandina P, Contreras M, Farı´ as P, Torrealba F (2010) The histaminergic tuberomammillary nucleus is critical for motivated arousal. Eur J Neurosci 31(11):2073–2085.

A. Blancas et al. / Neuroscience 281 (2014) 44–53 Verwey M, Amir S (2012) Variable restricted feeding disrupts the daily oscillations of period2 expression in the limbic forebrain and dorsal striatum in rats. J Mol Neuroscience 46(2):258–264. Verwey M, Khoja Z, Stewart J, Amir S (2007) Differential regulation of the expression of period2 protein in the limbic forebrain and dorsomedial hypothalamus by daily limited access to highly palatable food in food-deprived and free-fed rats. Neuroscience 147(2):277–285.

53

Webb IC, Baltazar RM, Lehman MN, Coolen LM (2009) Bidirectional interactions between the circadian and reward systems: is restricted food access a unique zeitgeber? Eur J Neurosci 30(9):1739–1748. Zhang M, Balmadrid C, Kelley AE (2003) Nucleus accumbens opioid, GABAergic, and dopaminergic modulation of palatable food motivation: contrasting effects revealed by a progressive ratio study in the rat. Behav Neurosci 117(2):202–211.

(Accepted 12 September 2014) (Available online 27 September 2014)