Variations in the neonatal environment modulate adult behavioral and brain responses to palatable food withdrawal in adult female rats

Int. J. Devl Neuroscience 40 (2015) 70–75

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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu

Variations in the neonatal environment modulate adult behavioral and brain responses to palatable food withdrawal in adult female rats夽 Juliana Barcellos Colman a , Daniela Pereira Laureano b , Tatiane Madeira Reis a , Rachel Krolow c , Carla Dalmaz b,c , Carla da Silva Benetti a , Patrícia Pelufo Silveira a,b,∗ a

Programa de Pós-Graduac¸ão em Saúde da Crianc¸a e do Adolescente, Faculdade de Medicina, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b Programa de Pós-Graduac¸ão em Neurociências, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil c Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

a r t i c l e

i n f o

Article history: Received 13 August 2014 Received in revised form 11 November 2014 Accepted 11 November 2014 Available online 20 November 2014 Keywords: Neonatal handling Withdrawal Palatable food Chronic exposure

a b s t r a c t Background/objectives: Early handling alters adult behavioral responses to palatable food and to its withdrawal following a period of chronic exposure. However, the central mechanisms involved in this phenomenon are not known. Since neonatal handling has persistent effects on stress and anxiety responses, we hypothesized that its involvement in the aforementioned association may be associated with differential neuroadaptations in the amygdala during withdrawal periods. Methods: Litters were randomized into two groups: handled (H, removed from their dam for 10 min per day from the first to the tenth postnatal day and placed in an incubator at 32 ◦ C) and non-handled (NH). Experiment 1: on PNDs 80–100, females were assigned to receive palatable food + rat chow for 15 or 30 days, and these two groups were compared in terms of palatable food preference, body weight and abdominal fat deposition. In Experiment 2, H and NH rats were exposed to a chronic diet of palatable food + rat chow for 15 days, followed by (a) no withdrawal, (b) 24 h withdrawal from palatable food (receiving only rat chow) or (c) 7-day withdrawal from palatable food (receiving only rat chow). Body weight, 10-min rebound palatable food intake, abdominal fat deposition, serum corticosterone as well as TH and pCREB levels in the amygdala were then compared between groups. Results: Experiment 1—chronic exposure to palatable food induces comparable metabolic effects after 15 and 30 days. Experiment 2—neonatal handling is associated with a peculiar response to palatable food withdrawal following chronic exposure for 15 days. Rats exposed to early handling ingested less of this food after a 24 h withdrawal period, and displayed increased amygdala TH and pCREB levels. Conclusions: Variations in the neonatal environment affect both behavioral responses and amygdala neuroadaptation to acute withdrawal from a palatable diet. These findings contribute to the comprehension of the mechanisms that link early life events and altered feeding behavior and related morbidities such as obesity in adulthood. © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

1. Introduction

夽 Financial support: Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE/HCPA); Coordination for the Improvement of Higher Education Personnel (CAPES)–Brazil; PRONEX 2009, FAPERGS/CNPq 10/0018.3. ∗ Corresponding author at: Hospital de Clinicas de Porto, Departamento de Pediatria, Faculdade de Medicina, Ramiro Barcelos 2350, Largo Eduardo Zaccaro Faraco, 90035-003 Porto Alegre, RS, Brazil. Tel.: +55 51 3359 8000; fax: +55 51 3359 8001. E-mail addresses: [email protected], [email protected] (P.P. Silveira). http://dx.doi.org/10.1016/j.ijdevneu.2014.11.003 0736-5748/© 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

Eating is essential to survival, and the consumption of palatable foods is associated with additional advantages in the form of pleasurable sensations, which lead to the overindulgence on this type of food beyond homeostatic needs. The consumption of tasty foods can be triggered by exposure to specific cues (Grosshans et al., 2012; Wang et al., 2009) or stress (Adam and Epel, 2007; Epel et al., 2001). Both of these mechanisms are especially evident in obese individuals (Davids et al., 2010; Gibson, 2012). Currently, the increased

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consumption of energy dense, high-fat, high-sugar ‘junk’/palatable foods is considered a major environmental contributor to weight gain and fat deposition. The phenomenon whereby animals and humans continue to consume high-fat, high-sugar foods even after satiety and despite their negative health consequences is similar to behaviors associated with drug dependence (Erlanson-Albertsson, 2005; Kelley and Berridge, 2002). In fact, studies have revealed substantial overlap between the brain circuitry underlying addictive behaviors and overeating; for instance, both substance-dependent and obese subjects exhibit decreased reward circuit activation in response to the drug of choice or palatable foods (Stice et al., 2008; Tomasi and Volkow, 2013; Volkow et al., 2013). Rats have also been found to self-administer sugar in ways which resemble substance abuse, involving loss of control, cross tolerance, failed attempts to quit, and the spontaneous production of withdrawal signs and symptoms following opiate antagonist administration (Avena et al., 2005; Gold and Avena, 2013; Hoebel et al., 2009). One factor which has been recently found to influence food preferences and eating behavior over the life-course is fetal/neonatal history (Portella et al., 2012; Silveira et al., 2006b, 2004, 2008). In addition to its known long-term effects on the programming of the hypothalamus–pituitary–adrenal (HPA) axis (Ader and Grota, 1969; Meaney et al., 1985a; Plotsky and Meaney, 1993), the early postnatal environment has been found to have several other effects on food intake. Neonatal handling is a form of early life stress induced in the pups by brief separations from their dam (Raineki et al., 2014) and has been found to increase the intake of palatable foods (Silveira et al., 2004), reduce plasma ghrelin levels (Silveira et al., 2006b) and dopamine metabolism in the nucleus accumbens in adult rats (Brake et al., 2004; Silveira et al., 2010). Although these animals show an increased intake of highly palatable foods, rich in sugar and fat (Benetti et al., 2007; Silveira et al., 2004), neonatally handled rats have also been shown to have better caloric efficiency, decreased levels of triglycerides and smaller abdominal fat depots when chronically exposed to this type of diet (Benetti et al., 2007). Additionally, after periods of chronic exposure followed by sudden withdrawal of the palatable diet, neonatally handled females display fewer behavioral signs of withdrawal (Benetti et al., 2010) but higher rebound intake in relation to controls (Benetti et al., 2013). Therefore, although such neonatal interventions are known to alter the adult behavioral response to palatable food withdrawal after periods of chronic exposure to this type of diet, the central mechanisms involved in this association are not known. Recent research has shown that heightened preference for highsucrose and high-fat foods and increased anxiety following the withdrawal of palatable high-fat diets was accompanied by a reduction in tyrosine hydroxylase (TH) and phospho-CREB (pCREB) expression in the amygdala (Sharma et al., 2013). Similar neuroadaptations have been observed following nicotine withdrawal (Pandey et al., 2001), suggesting that decreased CREB transcriptional activity in this region may be important in restoring palatable food intake after withdrawal. Given the persistent effects of neonatal handling on stress responses and anxiety (Meaney et al., 1993), we hypothesized that early-handled animals may display distinct neuroadaptation processes in the amygdala, which could be responsible for their altered behavioral responses to withdrawal after chronic exposure to palatable diets.

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incubator was set up, cages were transported and dams were allowed to acclimate to the new room, pups were carefully removed from the nest, then handled for a period of time until they were returned to their dams. After a brief period, the cages were returned to the animal facility. Researchers also changed gloves before handling each litter to avoid the spread of any odors between nests. The day of birth was considered postnatal day (PND) 0, and after weaning occurred on PND 21, rats were placed in home cages similar to those described above, each of which housed two to three animals. A hundred and forty experimental female rats derived from 24 different litters, were used in the present experiments. Although no more than 2 pups from the same litter were used in the same experiment, all female pups were used either in Experiment 1 or in Experiment 2. The number of animals used was estimated from previous experiments (Benetti et al., 2013, 2010; Silveira et al., 2004). After weaning, rats had free access to food (standard lab rat chow or standard lab rat chow + palatable food, see below) and water. The rat chow used was Nuvilab® , having 2.95 kcal/g, 15% protein, 12% fat, 73% carbohydrate. All animal procedures followed national and international ethics guidelines (Brazilian Law 11.794/08, the Universal Declaration on Animal Welfare issued on January 27th, 1978, and the Council for International Organizations of Medical Sciences—CIOMS/WHO standards), and were approved by the Research Ethics Committee of the Hospital de Clínicas de Porto Alegre (GPPG/HCPA, project number 11-0025). The study was performed in climate-controlled rooms within our animal research facility (Unidade de Experimentac¸ão Animal/HCPA). 2.1. Neonatal stress model Non-handled group (NH): pups were left undisturbed with their dams until weaning. Dirty sawdust was carefully removed from one side of the cage, without disturbing the mother or the pups, and replaced by clean sawdust by the principal researcher. Neonatal handled group (H): pups were gently removed from their home cages and placed in a clean cage lined with clean paper towels, inside an incubator set to 32 ◦ C. After 10 min, pups were returned to their dams. This procedure was carried out in the first 10 days of life, after which pups were left undisturbed until PND 21. 2.2. Habituation to the palatable diet Starting at PND 60 days, rats were habituated to the palatable diet (4.82 kcal/g, 14% protein, 34% fat, 30.2% carbohydrate, 20% of which was derived from sucrose, manufactured by Prag Soluc¸ões® , Jaú, SP, Brazil). To decrease the neophobia to the new food, a previously weighted amount of this food was placed in a clean cage (similar to the animal’s own home cage), in which rats were individually placed for 3 min every day for 5 consecutive days (Silveira et al., 2004). For the rest of the day, rats were kept under very mild food restriction (receiving approximately 80% of the usual rat chow intake over 24 h). 2.3. Experiment 1—Comparison between 15 and 30 days of exposure to palatable food All animals were weighed between PNDs 80 and 100, before being randomly distributed between the following groups according to body weight: (a) 15 days of chronic exposure to palatable food: (a1) NH + rat chow + palatable food and (a2) H + rat chow + palatable food for 15 days in the home cage; (b) 30 days of chronic exposure to palatable food: (b1) NH + rat chow + palatable food and (b2) H + rat chow + palatable food for 30 days in the home cage. During this period, food intake was monitored by placing known quantities of the diets in each cage and measuring the amount remaining after each day. Since food intake was measured in each cage, data is represented as mean intake per rat, per cage (n = number of cages in each group), while preference for palatable food was measured by dividing the palatable food intake by total intake in kcal. Body weight was measured once a week using a scale with 0.01 g precision (Marte® , Canoas, Brazil). After decapitation, the two major portions of abdominal fat (gonadal and retroperitoneal adipose tissue depots) were dissected and weighed using a scale with 0.01 g resolution (Marte® , Canoas, Brazil). Results were expressed as percentage of body weight. 2.4. Experiment 2—Withdrawal from palatable food—Comparison between 0, 24 h and 7-day withdrawal periods

2. Methods Pregnant Wistar rats bred at our animal facility were randomly selected and housed individually in Plexiglas (65 × 25 × 15 cm) cages with sawdust-covered floors, which were kept in a controlled environment (lights on between 07:00 h and 19:00 h, temperature at 22 ± 2 ◦ C, cage cleaning twice a week, food and water provided) until parturition. Litters were kept intact save for handling procedures, which were carried out between 10:00 h and 11:00 h. During this period, the

Only the animals who underwent chronic exposure to the palatable food for 15 days were used in Experiment 2. After the 15-day exposure period, rats in the H and NH groups were assigned to one of the following: (a) no withdrawal (b) 24 h withdrawal from palatable food (receiving only rat chow) and (c) 7-day withdrawal from palatable food (receiving only rat chow). Each animal’s body weight as well as 10-min palatable food intake were measured in a manner identical to that described in Section 2.2. Rats then fasted for 4 h before being decapitated for tissue collection.

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2.5. Tissue collection, biochemical and neurochemical analysis Animals were sacrificed by fast decapitation at zeitgeber time 7, after which both brain tissue and blood were collected for analysis. For corticosterone measurements, blood was collected in tubes and centrifuged at +4 ◦ C at 4000 rpm for 10 min. The serum was then separated in aliquots and frozen at −20 ◦ C until analysis. Corticosterone levels were measured using a commercially available ELISA kit (ENZO, Life Science—Ann Arbor, Michigan, EUA). Brains were flash frozen in isopentane pre-cooled in liquid nitrogen and stored at −80 ◦ C until analysis. The brains were then warmed to −20 ◦ C and the amygdala was cut into thick sections of 0.1 cm with the aid of an Atlas (Paxinos and Watson, 2009) and macroscopically examined. The regions of interest were identified and carefully isolated, and tissue was collected from 2 mm diameter punches, which were processed for Western blot analysis as described below. Tissue samples were homogenized in cytosol extraction buffer supplemented with protease (Complete, Roche) and phosphatase inhibitors (Phostop, Roche). Total protein was quantified using a BCA kit with bovine serum albumin as a standard (Thermo Scientific). Samples containing 20 ␮g of total protein were subjected to electrophoresis using a 4 to 12% polyacrylamide gradient gel (Invitrogen), before being transferred to a nitrocellulose membrane (GE Helthcare). Another gradient containing standard molecular weight markers (Magic Marker , Invitrogen) was run in parallel for molecular weight estimation. Blots were blocked in Tris buffer saline containing 5% non-fat milk concentrate and 1% Tween-20. The membranes were incubated overnight at +4 ◦ C with anti-TH primary antibodies (1:5000) (anti-tyrosine hydroxylase, Sigma-Aldrich, cat: T2928), pCREB (1:500) (anti-phospho-CREB, INVITROGEN, cat: 368,600), followed by anti-mouse secondary antibodies (1:2000) (Anti-Mouse IgG, Cell Signalling, cat: 7076s) or anti-rabbit (1:2000) (Anti-Rabbit IgG, Cell Signalling, cat: 7074s) at room temperature for 1 h. The membrane was then exposed on a Kodak film using ECL (ECL western blotting analysis system, GE healthcare, RNP 2106). The intensity of Western blot bands was quantified by densitometry analysis using the ImageJ software (National Institute of Health, USA). Results were expressed as the ratio of intensity of the protein of interest to that of ␤-actin 1:1000 (Sigma-Aldrich, A4700) in the same membrane.

Fig. 1. Palatable food intake after different withdrawal lengths in H and NH animals. A two-way ANOVA showed an interaction between neonatal group × withdrawal length, P < 0.002, n = 7–19/group. * Handled rats without withdrawal ate more palatable food than non-handled rats (Tukey, P = 0.015). ** After a 24 h withdrawal period, handled animals displayed a greater decrease in their intake as compared to handled/non-withdrawal rats (P = 0.035). *** After 7 days of withdrawal, handled animals displayed an increase in food intake, and consumed a significantly higher amount of food than handled/24 h withdrawal animals (P = 0.028).

revealed no differences in body weight gain [two-way ANOVA, F(3, 70) = 0.507 P = 0.479 for group, F(3, 70) = 0.268 P = 0.607 for time of exposure, no interaction] or abdominal fat weight [two-way ANOVA, F(3, 70) = 0.298 P = 0.587 for group, F(3, 70) = 0.052 P = 0.291 for time of exposure, no interaction]. Rats exposed to the diet for 15 days ingested a greater amount of palatable food than those exposed to the diet for 30 days, regardless of neonatal group. This finding was expected, since preference naturally decreases over time when animals are given a choice between standard chow and palatable food (Benetti et al., 2007) [two-way ANOVA, F(3, 42) = 1.884 P = 0.178 for group, F(3, 42) = 17.367 P < 0.0001 for time of exposure, no interaction]. These results are displayed in Table 1.

2.6. Statistical analysis Data were analyzed using a two-way ANOVA (palatable food intake, body weight, abdominal fat and corticosterone levels after withdrawal, TH and pCREB levels after withdrawal) with neonatal group and withdrawal time as independent variables. Palatable food intake was not normally distributed, and therefore, was log transformed for analysis. A repeated measures ANOVA was used in Experiment 1. ANOVAs were followed by Tukey post hoc tests when necessary. Data were analyzed using the Statistical Package for the Social Sciences (SPSS), version 20.0 (SPSS Inc., Chicago, IL, USA). Significance levels for all measures were set at P < 0.05.

3.3. Experiment 2—Withdrawal from chronic (15 day) exposure to palatable food—Comparison between 0, 24 h and 7-day withdrawal periods

3. Results 3.1. Initial body weight and habituation to palatable food

Experiment 1 showed that 15 and 30 days of chronic exposure to palatable food induced comparable body weight and adiposity. Therefore, Experiment 2 we used only the animals with 15-day periods of chronic exposure. Fig. 1 depicts the palatable food intake observed following withdrawal periods of different lengths. The results of a two-way ANOVA demonstrated an interaction between group and withdrawal length [F(5, 57) = 11.453 P = 0.002]. There was also a main effect of withdrawal length [F(5, 57) = 6.267 P = 0.026], but not of group [F(5, 57) = 0.605 P = 0.541], on palatable food intake. Post hoc analyses showed that handled rats ate more palatable food than non-handled animals after 0 days of withdrawal (Tukey, P = 0.015). After 24 h withdrawal, handled animals were found to consume less food than handled/non-withdrawn rats (P = 0.035). After 7 days of withdrawal, NH there was a trend toward higher food intake in

Student’s t tests revealed no differences in body weight between animals in the NH and H groups at 60 (NH = 186.13 ± 2.82 g, H = 191.88 ± 3.76 g, t(138) = 0.637 P = 0.525) and 90 days of life (NH = 224.04 ± 2.08 g, H = 225.99 ± 2.23 g, t(138) = 1.302 P = 0.195). During the 5-day period of habituation to the palatable diet, the two neonatal groups ingested comparable amounts of food (NH = 1.84 ± 0.25 g, H = 2.09 ± 0.20 g, t(137) = 0.785, P = 0.434). 3.2. Experiment 1—Comparison between 15 and 30 days of palatable food exposure Comparisons between rats in the NH and H groups who underwent chronic exposure to palatable food for 15 or 30 days

Table 1 Comparison between the effects of 15 and 30 days of palatable food exposure in H and NH animals. Two-way ANOVAs revealed no between-group differences regarding body weight, abdominal fat deposition or preference for palatable food over the course of the chronic exposure period, n = 17–26 rats/group for body weight and abdominal fat, n = 4–5 cages/group for palatable food preference. Variable

BW gain (final-initial) (g) Abdominal fat (% of total body weight) Preference for palatable food (kcal of palatable food/total kcal consumed) (%)

15 days of exposure to palatable diet

P

30 days of exposure to palatable diet

NH

H

NH

H

18.58 ± 4.44 3.27 ± 0.26 88.81 ± 2.31

22.92 ± 5.09 3.73 ± 0.29 91.12 ± 1.02

21.97 ± 4.93 3.62 ± 0.44 80.00 ± 2.83

24.62 ± 3.81 3.54 ± 0.29 83.22 ± 1.71

NS NS NS

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Table 2 Comparison between 0, 24 h and 7-day withdrawal from palatable food in H and NH animals. Two-Way ANOVAs revealed no between-group differences regarding body weight, abdominal fat deposition or serum corticosterone in response to withdrawal in the different groups, n = 7–19/group. Variables

Body weight (g) Abdominal fat (% of body weight) Corticosterone (pg/dl)

No withdrawal

24 h withdrawal

7 d withdrawal

NH

H

NH

H

NH

H

251.84 ± 8.50 3.25 ± 0.28 197.66 ± 76.32

256.78 ± 3.38 3.73 ± 0.29 262.23 ± 34.99

257.40 ± 9.82 2.83 ± 0.22 243.84 ± 79.29

256.88 ± 6.72 3.57 ± 0.30 300.55 ± 89.77

261.10 ± 8.26 2.91 ± 0.41 186.68 ± 57.30

257.62 ± 11.81 2.43 ± 0.28 185.91 ± 45.63

handled animals than in NH/non-withdrawn rats (P = 0.055). Additionally, after 7 days of withdrawal, handled animals increased their intake again, and consumed significantly more food than H/24 h animals (P = 0.028), but similar levels of food to H/non-withdrawal rats (P = 0.990). Table 2 shows the results of body weight, abdominal fat and serum corticosterone measurements in the different withdrawal periods. There were no differences in abdominal fat deposition [two-way ANOVA using neonatal group and withdrawal time as variables, F(5, 62) = 0.761 P = 0.387 for group, F(5, 62) = 3.052 P = 0.055 for withdrawal time, no interaction], body weight [F(5, 62) = 0.021 P = 0.884 for group, F(5, 62) = 0.190 P = 0.827 for withdrawal time, no interaction], or serum corticosterone concentrations [F(5, 62) = 0.543 P = 0.464 for group, F(5, 62) = 0.726 P = 0.488 for withdrawal time, no interaction] between neonatal groups or withdrawal lengths. A Two-Way ANOVA comparing TH expression in the amygdala of rats in each experimental group revealed no main effect of group [F(5, 32) = 1.679 P = 0.206], but a significant effect of withdrawal length [F(5, 32) = 3.682 P = 0.464], as well as an interaction between group and withdrawal length [F(5, 32) = 4.705 P = 0.018]. Post hoc analyses showed that, after a 24 h-withdrawal period, handled animals expressed higher levels of TH than all other groups, except for NH rats exposed to a 7-day withdrawal period. Amygdala pCREB levels were found to be influenced by the interaction between group and withdrawal length [F(5, 32) = 7.676 P = 0.002] and by withdrawal alone [F(5, 32) = 3.567 P = 0.042], although no main effects of group were identified[F(5, 32) = 3.398 P = 0.076]. Post hoc analyses showed that, after a 24 h withdrawal period, handled rats expressed higher pCREB levels than NH/24 h animals (Tukey, P = 0.018), rats in the H/no withdrawal group (P = 0.006) and subjects in the NH group exposed to a 7-day withdrawal period (P = 0.001). No other between-group differences were observed. These results are displayed in Fig. 2.

stress (Marcolin Mde et al., 2012). These findings were confirmed in our study, which found that handled rats ate more palatable food than their non-handled counterparts in the absence of withdrawal. It is interesting to note that, according to Sharma and colleagues (Sharma et al., 2013), exclusive and chronic exposure to the palatable diet is associated with progressively increasing caloric intake. This differs from what we observed in our study, in which the palatable diet was offered as an alternative to another type of food. This suggests that, regardless of neonatal exposure to the palatable diet, having the option to ingest rat chow as an alternative to palatable food leads to a decrease in the intake of palatable foods over time. Some studies have suggested that chronic and exclusive exposure to a high fat diet induces a state of chronic stress (Shin

4. Discussion Our main objective was to verify whether variations in the neonatal environment are associated with different behavioral responses to the withdrawal of a palatable food diet offered chronically in adult life, as well as to investigate the mechanisms that may be responsible for this association. We found that neonatal handling was associated with specific behavioral and neurochemical responses to withdrawal, corroborating our previous studies (Benetti et al., 2013, 2010). Although we did not find statistical differences in palatable food intake between the groups exposed to different lengths of chronic exposure to a palatable diet, as described in our previous experiments (Benetti et al., 2007, 2010, 2013), mean food intake was higher in the H group as compared to the NH group. The current findings may be explained by the different type of diet offered, since our previous studies used other types of palatable foods such as chocolate or Froot Loops (Benetti et al., 2007; Silveira et al., 2004). In general, it seems that the handling procedure had permanent effects on palatable food intake (Benetti et al., 2007, 2010, 2013; Silveira et al., 2004, 2006b, 2008, 2010) or on dietary responses to

Fig. 2. (A) TH levels in the amygdala of H and NH animals after no withdrawal, 24 h withdrawal or 7-day withdrawal. A two-way ANOVA showed an interaction between group vs. length of withdrawal period (P = 0.018, n = 4–6/group), in which handled animals after 24 h withdrawal had higher amygdala TH levels than those in all other groups, save for NH 7-day withdrawal rats. (B) pCREB concentration in the amygdala of H and NH animals after no withdrawal, 24 h withdrawal or 7-day withdrawal. An interaction was observed between group and duration of withdrawal period (P = 0.002, n = 4–7/group), in which handled rats exposed to 24 h withdrawal had higher pCREB levels than those exposed to NH/24 h withdrawal, H/no withdrawal and NH/7-day withdrawal. Different letters represent statistically significant differences using Tukey Post hoc test.

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et al., 2010; Tannenbaum et al., 1997), and this was not found in our protocol. Other studies involving chronic and exclusive exposure to highly palatable foods have also reported increases in peripheral or central corticosterone levels in response to withdrawal (Rabbani et al., 2009; Sharma et al., 2013). However, when similar analyses were performed in our sample, only a non-significant increase in corticosterone levels in the 24 h withdrawal group was found. Pankevich et al. (2010) showed that withdrawal from palatable food might lead to compulsive eating when the diet is offered again. However, in the present study, handled animals had a lower palatable food intake following a 24 h withdrawal period than nonhandled animals or subjects exposed to withdrawal periods of different lengths. This is an interesting finding, since our research group has consistently shown that handled animals have significantly greater palatable food intake than control animals (Benetti et al., 2007, 2010, 2013; Silveira et al., 2004, 2006b, 2010). The present results regarding responses to acute withdrawal are the first to demonstrate decreased palatable food intake in handled H animals in adulthood, although we have already described this finding in periadolescent rats (Silveira et al., 2006a). Two putative explanations to this finding were proposed: (1) acute withdrawal from the palatable food was much more stressful to neonatally handled animals, leading to food intake reduction or (2) neonatally handled animals respond differently to palatable food withdrawal, demonstrating decreased binge eating for this type of food when re-exposed, and this could possibly be tracked onto specific neuroadaptations to food withdrawal. These two possibilities are explored below. The fact that this decrease in food intake is not accompanied by an increase in corticosterone levels suggests that it is not likely to be caused by an acute stress response or anhedonia. This finding is in agreement with the vast literature showing that handled animals have a more discrete neuroendocrine response to acute stress (Ader and Grota, 1969; Levine, 1957; Meaney et al., 1985b). In fact, neonatal handling is a form of early life stressor induced by short periods of separation from the dam, as reviewed in (Raineki et al., 2014), that leads to long term adaptations in central regulation of the stress responses. For instance, these animals have reduced CRH release from the median eminence following stress (Francis et al., 1999), as well as increased glucocorticoid receptor density binding in the hippocampus (Meaney and Aitken, 1985; Meaney et al., 1985a), which potentiate the efficacy of the HPA axis’ negative feedback, reducing the ACTH and corticosterone response to stress (Meaney et al., 1993). We have previously shown that the increased intake of sweet foods observed in handled animals is not reversed by the use of anxiolytic drugs (Silveira et al., 2005), unlike other forms of stress-induced palatable food intake (Ely et al., 1997). Interestingly, our research had previously shown that acute withdrawal from chronic chocolate exposure induced a more pronounced body weight decline in handled animals than in their non-handled counterparts (Benetti et al., 2010), possibly due to the fact the former exhibit a more significant decrease in food intake in response to withdrawal, as observed in the present study, although the current experiments did not reveal differences in body weight between the two groups. It is important to highlight that increased levels of TH and pCREB in the amygdala accompanied the decrease in palatable food intake following withdrawal in handled animals. This finding was also in agreement with the literature. According to Sharma et al. (2013) and Jang et al. (2011), both the withdrawal and the subsequent craving for high-fat foods is associated with diminished TH concentration in the amygdala, suggesting that a decrease in dopamine biosynthesis may play a role in the behavioral and neuroendocrine responses to withdrawal. While signs of resilience to acute stress in handled animals have been widely reported (Ader and Grota, 1969;

Levine, 1957; Meaney et al., 1985b), the present results regarding decreased palatable food intake and increased TH levels in handled animals after a 24 h withdrawal period could be seen as a specific adaptation to palatable food withdrawal. A similar conclusion could be drawn from the increased amygdala pCREB levels seen in handled animals after 24 h of withdrawal. The concentration of pCREB in the amygdala has been found to decrease in response to nicotine (Pandey et al., 2001), ethanol (Pandey et al., 2003) and palatable food withdrawal (Sharma et al., 2013). Sharma et al. (2013) have suggested that the diminished expression of pCREB in the amygdala may contribute to the increase in palatable food intake observed following withdrawal by increasing its reward value. Interestingly, after 7 days, neurochemical differences were no longer identified between groups in the study. The present findings suggested that neonatal handling affects the behavioral and neurochemical response to palatable food withdrawal in adult life. The most important interpretation of this study is that an apparently innocuous and brief neonatal intervention is able to persistently modulate the behavioral and neurochemical response to palatable food withdrawal. In other words, the individual’s fetal and neonatal history seems important when planning dietary interventions, corroborating the findings of several studies in humans (Ayres et al., 2012; Barbieri et al., 2009; Portella and Silveira, 2014; Silveira et al., 2012). This conclusion has significant implications for the comprehension of the mechanisms involved in altered feeding behavior and related morbidities such as obesity, as well as the development prevention strategies for humans. Acknowledgements This research received financial support from: PRONEX 2009, FAPERGS/CNPq 10/0018.3, Projeto IVAPSA—Impacto das Variac¸ões do Ambiente Perinatal sobre a Saúde do Adulto; Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE/HCPA). Colman JB received an MSc grant from the Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.ijdevneu.2014.11.003. References Adam, T.C., Epel, E.S., 2007. Stress, eating and the reward system. Physiol. Behav. 91, 449–458. Ader, R., Grota, L.J., 1969. Effects of early experience on adrenocortical reactivity. Physiol. Behav. 4, 3. Avena, N.M., Long, K.A., Hoebel, B.G., 2005. Sugar-dependent rats show enhanced responding for sugar after abstinence: evidence of a sugar deprivation effect. Physiol. Behav. 84, 359–362. Ayres, C., Agranonik, M., Portella, A.K., Filion, F., Johnston, C.C., Silveira, P.P., 2012. Intrauterine growth restriction and the fetal programming of the hedonic response to sweet taste in newborn infants. Int. J. Pediat. 2012, 657379. Barbieri, M.A., Portella, A.K., Silveira, P.P., Bettiol, H., Agranonik, M., Silva, A.A., Goldani, M.Z., 2009. Severe intrauterine growth restriction is associated with higher spontaneous carbohydrate intake in young women. Pediatr. Res. 65, 215–220. Benetti, C.d.S., Silveira, P.P., Wyse, A.T.S., Scherer, E.B.S., Ferreira, A.G.K., Dalmaz, C., Zubaran Goldani, M., 2013. Neonatal environmental intervention alters the vulnerability to the metabolic effects of chronic palatable diet exposure in adulthood. Nutr. Neurosci. 17 (3), 127–137 (140123093232009). Benetti, C.S., Silveira, P.P., Matte, C., Stefanello, F.M., Leite, M.C., Goncalves, C.A., Wyse, A.T., Dalmaz, C., Goldani, M.Z., 2010. Effects of a chronic exposure to a highly palatable diet and its withdrawal, in adulthood, on cerebral Na+,K+ATPase and plasma S100B in neonatally handled rats. Int. J. Dev. Neurosci. 28, 153–159. Benetti, C.S., Silveira, P.P., Portella, A.K., Diehl, L.A., Nunes, E., de Oliveira, V.S., Dalmaz, C., Goldani, M.Z., 2007. Could preference for palatable foods in neonatally handled rats alter metabolic patterns in adult life? Pediatr. Res. 62, 405–411.

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