Behavioural Brain Research 287 (2015) 73–81

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Increased palatable food intake and response to food cues in intrauterine growth-restricted rats are related to tyrosine hydroxylase content in the orbitofrontal cortex and nucleus accumbens Márcio Bonesso Alves a,∗ , Roberta Dalle Molle b , Mina Desai c,d , Michael G. Ross c,d , Patrícia Pelufo Silveira a,b a

Graduate Program in Neurosciences, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Graduate Program in Child and Adolescent Health, Hospital de Clínicas de Porto Alegre/Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil c Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Department of Obstetrics and Gynecology, Perinatal Research Laboratories, Torrance, CA, USA d Department of Obstetrics and Gynecology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA b

h i g h l i g h t s • • • •

Food restriction during gestation alters maternal behavior. Intrauterine growth restriction leads to altered feeding behavior. Intrauterine growth restriction causes alterations in the sensitivity to food cues. Intrauterine growth restriction causes sex-specific alterations in dopamine signaling.

a r t i c l e

i n f o

Article history: Received 19 November 2014 Received in revised form 6 March 2015 Accepted 10 March 2015 Available online 18 March 2015 Keywords: Fetal growth restriction Attention Feeding Dopamine Rats

a b s t r a c t Intrauterine growth restriction (IUGR) is associated with altered food preferences, which may contribute to increased risk of obesity. We evaluated the effects of IUGR on attention to a palatable food cue, as well as tyrosine hydroxylase (TH) content in the orbitofrontal cortex (OFC) and nucleus accumbens (NAcc) in response to sweet food intake. From day 10 of gestation and through lactation, Sprague–Dawley rats received either an ad libitum (Adlib) or a 50% food-restricted (FR) diet. At birth, pups were cross-fostered, generating four groups (gestation/lactation): Adlib/Adlib (control), FR/Adlib (intrauterine growth-restricted), Adlib/FR, and FR/FR. Adult attention to palatable food cues was measured using the Attentional Set-Shifting Task (ASST), which uses a sweet pellet as reward. TH content in the OFC and NAcc was measured at baseline and in response to palatable food intake. At 90 days of age, FR/Adlib males ate more sweet food than controls, without differences in females. However, when compared to Controls, FR/Adlib females needed fewer trials to reach criterion in the ASST (p = 0.04) and exhibited increased TH content in the OFC in response to sweet food (p = 0.03). In the NAcc, there was a differential response of TH content after sweet food intake in both FR/Adlib males and females (p < 0.05). Fetal programming of adult food preferences involves the central response to palatable food cues and intake, affecting dopamine release in select structures of the brain reward system. © 2015 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Departamento de Pediatria, FAMED, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, 2350, Largo Eduardo Zaccaro Faraco, 90035-903, Porto Alegre, RS, Brazil. Tel.: +55 51 3359 8000; fax: +55 51 3359 8001. E-mail address: [email protected] (M.B. Alves). http://dx.doi.org/10.1016/j.bbr.2015.03.019 0166-4328/© 2015 Elsevier B.V. All rights reserved.

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1. Introduction The developmental origins of adult diseases theory proposes that an adverse fetal environment leads to adaptive physiological modifications to guarantee survival, but increases the risk for chronic diseases in the long term [1]. Indeed, there is growing epidemiological evidence demonstrating that individuals that do not grow at the expected rate during fetal life, whether due to maternal and/or fetal conditions or exposures, are at increased risk of developing type II diabetes [2,3], cardiovascular disease [4] and obesity [5,6]. As altered food intake and feeding behavior may also play a considerable role in the development of these diseases [66–69], it is interesting to suppose that intrauterine growth restriction (IUGR) may modify food preferences over the life course, thus leading to an adult obese phenotype and its metabolic consequences. Recent evidence in humans demonstrates that altered feeding behavior and preferences occur in IUGR individuals [7], persisting throughout different developmental phases [8–10] and tracking into altered adulthood food choices [11–15]. In addition, IUGR infants exhibit less mature neurodevelopmental and neurobehavioral profiles in childhood [16,17], poorer cognitive outcomes [16,18], and poorer executive functioning [19,20]. Furthermore, it has been shown that fetal growth interacts with the surrounding environment, especially maternal care, negatively affecting attentional skills in infants [21] and hippocampal volume in adulthood [22] in individuals born small for gestational age and raised by mothers with poor maternal care skills. Therefore, it seems that significant interactions exist between fetal and postnatal life concerning executive function in children. There are several domains of executive function, including inhibitory control, attention and mental flexibility, reward sensitivity, and working memory, mediated by the prefrontal cortex (PFC). The PFC and the amygdala are involved in the integration of valuation and comparison processes, such as the coding of rewards relative to other available rewards and negative valuations [23,24], that may affect food intake [25]. The neural response to food cues is a measure of the “incentive salience”, or the ability of the cue to trigger increased attention (salience) and wanting, a process that is mostly modulated by dopamine [26,27]. Interestingly, obese individuals are more prone to eating in response to external food cues [28–30]. Indeed, experimental studies show that individual variation in the attribution of incentive salience to reward-related cues seems to play an important role in the development of obesity [31]. Considering that IUGR is related to altered executive function and to a specific pattern of feeding behavior in humans, we hypothesized that sensitivity to food cues may be differentially modulated by dopamine in key structures of the mesocorticolimbic pathway in IUGR subjects, thus influencing food intake. Therefore, our objective was to ascertain whether a cognitive flexibility task using palatable food as a reward would be influenced by fetal growth restriction; if so, the very exposure to a challenge using palatable food would likely differentially affect measures of dopamine availability in the OFC and nucleus accumbens (NAcc) in IUGR rats.

2. Materials and methods

(45 cm × 30 cm × 19 cm) and kept in a controlled environment: temperature 22 ± 2 ◦ C, cage cleaning once a week, food and water provided ad libitum, and an altered dark/light cycle (lights on between 09:00 h and 19:00 h, due to maternal behavior observations in the dark phase of the cycle). At 10 days of gestation, dams were randomized by body weight and allocated into one of two dietary groups: Control (Adlib, n = 13), which received standard lab chow ad libitum (Nuvilab® : 2.94 kcal g−1 , 15% protein, 12% fat, 73% carbohydrate), or 50% food-restricted (FR, n = 12), based on the successful IUGR model described by Desai et al. [32], which received 50% of the ad libitum-fed dams’ intake (determined after quantification of the mean daily intake of the control group). These diets were provided from day 10 of pregnancy through day 21 of lactation. Within 24 h after birth, all pups were weighed and all litters were culled to eight (four males and four females) per litter and crossfostered to other dams, forming the following groups (considering the biological/adoptive mother, i.e., gestation/lactation maternal diet): Adlib/Adlib (n = 7), FR/Adlib (n = 7), FR/FR (n = 5), and Adlib/FR (n = 6). On postnatal day 21, pups were weaned, separated by sex, and allocated four to a cage. All animals were fed standard lab chow and water ad libitum and were kept in a controlled environment similar to that described above, except for the light cycle (lights on between 07:00 h and 19:00 h). From day 21 onwards, at the time of cage cleaning once a week, body weight was measured using a digital scale with 0.01 g precision (Marte® , Canoas, Brazil). 2.2. Maternal behavior observations The maternal behavior of each dam was observed for five 72min periods per day from PND2 to PND7, by observers who had received standardized training to carry out this task. Briefly, the observations occurred at regular times each day, with three observation periods during the light phase (10:00 h, 13:00 h, 17:00 h) and two periods during the dark phase (07:00 and 20:00 h) of the light/dark cycle. Within each observation period, the behavior of each dam was scored every 3 min. The following behaviors were scored: dam on or off the nest, dam licking and grooming (LG) any pup, dam nursing pups in either an arched-back posture or a passive posture in which the dam is lying either on her back or side while nursing the pups [33]. Behavioral categories were not mutually exclusive; for example, LG often occurred while the mother was nursing the pups. Nursing positions were classified according to the intensity of the arched back, which is related to milk let-down efficiency, from 1 (least efficient) to 4 (most efficient). The behavioral data for each female were analyzed as a percentage of the total number of observations for that female over the entire observation period. 2.3. Behavioral testing – general considerations Behavioral testing began with locomotor activity, at around 70 days of life. All behavioral tests were performed during the day, on two pups/sex per litter. The order of test performance was determined by the age of the animals (older animal first; however, groups were assigned in a balanced fashion during gestation).

2.1. General Sprague–Dawley rats (CEMIB Laboratory Animal Reference Center, Campinas, SP, Brazil), approximately 70 days old, were time-mated at our animal facility after daily vaginal smearing visualization. Gestation was confirmed at day 1 by visualizing the presence of sperm on the vaginal smear. During pregnancy, animals were single-housed in Plexiglas home cages

2.3.1. Locomotor activity Locomotor activity was assessed in a rectangular arena (43 cm × 54 cm) divided into 12 squares by counting the number of times that the animal crossed the lines with all four paws within a 5-min period. This test was performed immediately before (on the same day) and in the same apparatus of the habituation phase of ASST.

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Table 1 Description of the attentional set-shifting task phases. Discriminations

Description

Simple discrimination (SD)

Two stimuli from the same dimension are presented (e.g.: odor), being one correct and the other one incorrect A second dimension (e.g.: digging media texture) is introduced, but this is irrelevant and the animal should keep discrimination between the two original stimuli The relevance of the stimuli is reversed and the correct stimuli is now incorrect, still within the same dimension New stimuli from both dimensions are introduced, but the correct dimension (e.g. odor) is still the same The relevant stimulus is again shifted within the same dimension New stimuli from both dimensions are introduced, but now the relevant dimension is shifted (e.g. from odor to texture) The relevance of the stimuli is again reversed

Compound discrimination (CD)

Compound discrimination reversal (REV1) Intradimensional shift (ID) Intradimensional shift reversal (REV2) Extradimensional shift (ED) Extradimensional shift reversal (REV3)

2.3.2. Attentional set shifting task (ASST) In this task, adapted from elsewhere [34], the animal’s attention is directed towards a certain stimulus (e.g., odor) while other irrelevant stimuli (e.g., texture) must be ignored. During the test, the relevant stimulus is changed, and the animal’s ability to both change its strategy and to keep it are evaluated. The task is based on the fact that rats rapidly learn how to dig in a bowl to search for a palatable food reward, as well as to use associated external cues to guide their decisions. In the previous week, rats were foodrestricted, receiving 80% of their habitual food consumption and a few pellets of Froot Loops® to gain familiarity with the palatable food. The animals were then exposed to habituation, training, and test sessions, with the entire test being performed for 2 or 3 days, depending on the animal’s performance during habituation. During habituation, animals were allowed to search for and eat the food reward inside the arena (the same as described in the locomotor activity test above). Thereafter, food rewards (1/4 of a Froot Loop) were placed within a ceramic bowl (diameter 7 cm, depth 4 cm) and progressively hidden under increasing quantities of wood chips. Habituation criteria were based on the speed and familiarity with which the animals dug and ate the food reward; if that did not happen in 3 days, the animal was excluded from the task. During the training phase, rats had access to two digging bowls, only one of which contained the food reward. The animal was required to complete two simple discriminations (SDs): odor and media. Here and in the test session, the first trials of each stage were discovery trials and were not recorded, regardless of whether the animal dug in the baited or in the unbaited bowl. From the second trial onward, an error was recorded if the rat dug first in the unbaited bowl, which was then quickly removed. If the rat dug in the baited bowl, it was allowed to eat the food pellet. This phase continued until the rat reached a criterion level of performance of six consecutive correct trials. In the test session, rats performed a series of discriminations that included: (a) SDs, in which the bowls differed on one of three dimensions (odor or texture or digging medium); (b) compound discrimination (CD), in which a second dimension was introduced, but the correct and incorrect exemplars remained constant; (c) compound discrimination reversal (Rev1); (d) intra-dimensional shift (ID), in which a new set of exemplars was introduced, but the relevant perceptual dimension remained the same (e.g., odor); (e) ID reversal (Rev2); (f) extra-dimensional shift (ED), in which a new pair of exemplars was again introduced, and the relevant perceptual dimension was then turned irrelevant; and (g) ED reversal (Rev3). The order of the discriminations was always the same, but the dimensions and pairs of exemplars were equally represented within groups and counterbalanced between groups as far as

Dimensions Relevant

Irrelevant

Odor

–

Odor

Texture

Odor

Texture

Odor

Texture

Odor

Texture

Texture

Odor

Texture

Odor

possible. The combination of exemplars into stimuli and the side of stimulus presentation were determined by an a priori pseudorandom list. To prevent the animals from smelling the reward, the odor of each digging medium was reinforced at the start of each testing day. The scents utilized were strawberry, cinnamon, vanilla, clove, paprika, caraway, thyme, and oregano. The digging media consisted of grained paper, satin strips, beads, buttons, paper clips, locking washers, and cardboard pieces (Table 1). 2.3.3. Acute exposure to sweet food for tissue collection Exposure to sweet food was initiated 1 week after all animals had completed the ASST. Animals were weighed and fasted for 4 h, with access to water ad libitum. At the end of this period, half of the animals were immediately decapitated for tissue collection, while the other half received approximately 30 g of Froot Loops for 1 h. At the end of this period, animals were immediately decapitated, also for tissue collection, and intake of Froot Loops was measured. 2.4. Tissue collection Brains were flash-frozen in isopentane under dry ice and stored at −80 ◦ C until analysis, at which time the brains were warmed to −20 ◦ C and the regions of interest (OFC and NAcc) macroscopically dissected through thick (0.25-cm) sections with the aid of an atlas [35]. At the time of acute exposure to sweet food and tissue collection, the animals were around 100 days old. 2.5. Western blotting Tissue samples were homogenized in a cytosolic extraction buffer with protease (Complete, Roche) and phosphatase inhibitors (Phostop, Roche). Total protein was quantified using a BCA kit with bovine serum albumin as standard (Thermo Scientific). Samples containing 20 ␮g of total protein were loaded on 4–15% polyacrylamide gradient gels (Invitrogen), subjected to electrophoresis, and transferred to a nitrocellulose membrane (GE Healthcare). A standard molecular weight marker (Magic Marker® , Invitrogen) was run simultaneously to compare the molecular weights of the visualized proteins. Blots were blocked in a Tris-Base solution containing 5% nonfat milk concentrate and 1% Tween-20. The membranes were incubated overnight at 4 ◦ C with appropriate primary antibodies against tyrosine hydroxylase (Sigma–Aldrich, T2928), 1:5000, followed by secondary anti-mouse antibody (1:2000) (Amersham ECL Anti-Mouse IgG, GE Healthcare, NA 931) at room temperature for 2 h, then film-developed (KODAK) using ECL (ECL Western blotting analysis system, GE Healthcare, RNP 2106). The intensity of Western blot bands was quantified by densitometric

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Table 2 Mean ± SEM of body weight gain during gestation and birth weight.

Body weight gain during gestation Birth weight *

Males Females

Adlib dams

FR dams

132.11 ± 5.06 6.80 ± 0.05 6.49 ± 0.06

63.55 ± 2.18 5.33 ± 0.06 5.18 ± 0.05

p < 0.001* p < 0.001 for group and for sex

Represents significant difference between groups.

Table 3 Maternal behavior variables in the different cross-fostering groups. Data is expressed as mean ± SEM. Values marked with different letters express statistically significant differences using Bonferroni Post hoc test. Groups

Adlib/Adlib

Adlib/FR

FR/FR

FR/adlib

Maternal behavior

n=7

n=6

n=6

n=6

Licking and grooming Out of the nest Nursing (total) Nursing positions 1 and 2 Nursing positions 3 and 4

50.14 ± 5.30a 38.7 ± 2.67a 45.18 ± 3.34a 36.07 ± 2.18a 9.10 ± 2.47a

57.5 ± 7.20a 41.1 ± 5.73a 42.4 ± 4.18a 33.3 ± 3.69a 9.1 ± 1.54a

33.6 ± 4.75b 46.1 ± 1.32a 38.5 ± 2.04a 29.6 ± 2.10a 8.91 ± 1.1a

25.0 ± 2.64b 45.6 ± 1.75a 39.7 ± 1.16a 33.1 ± 1.85a 6.7 ± 1.15a

Statistics

[F(3,24) = 7.81, p = 0.001] [F(3,21) = 1.00, p = 0.41] [F(3,21) = 1.02, p = 0.40] [F(3,21) = 1.11, p = 0.36] [F(3,21) = 3.67, p = 0.77]

analysis in the ImageJ software suite (National Institutes of Health, USA). Results were expressed as the ratio of intensity of the protein of interest to that of 1:2000 ␤-actin (muscle samples) (Sigma–Aldrich, A4700) from the same membrane.

for litter size showed that, at birth, FR pups weighed less than controls [F(1,336) = 292.56, p < 0.001], and females weighed less than males [F(1,336) = 16.03, p < 0.001], but no interaction was observed between group and sex [F(1,336) = 2.29, p = 0.13] (Table 2).

2.6. Ethics

3.2. Maternal care

All efforts were made to minimize pain or discomfort. All animal procedures followed national and international standards (Diretrizes e Normas Nacionais e Internacionais, Brazilian Law number 11.794 of October 8, 2008, and the Universal Declaration on Animal Welfare of January 27, 1978, as well as Council for International Organizations of Medical Sciences [CIOMS] standards), which are in agreement with the guidelines laid down by the U.S. National Institutes of Health, and were approved by the Research Ethics Committee of Hospital de Clínicas de Porto Alegre (GPPG/HCPA number 12-0353).

LG behavior was more frequent among Adlib dams [F(3,24) = 7.81, p = 0.001], independently of the adopted pup’s group. Post-hoc analysis showed that Adlib/Adlib dams performed LG behavior more often than FR/Adlib (p = 0.002) and FR/FR (p = 0.03) animals. The same was found in the Adlib/FR group (post-hoc analysis, p < 0.001 and p = 0.005 on comparison with the FR/Adlib and FR/FR groups respectively). On the other hand, time spent in nursing positions 3 and 4 did not differ between groups [F(3,24) = 3.67, p = 0.77], nor did time spent in nursing positions 1 and 2 [F(3,24) = 1.11, p = 0.36] or time the dam spent in body contact with the pups [F(3,24) = 1.00, p = 0.41] (Table 3).

2.7. Statistical analysis Weight during gestation was analyzed using repeated-measures ANOVA, adjusted for litter size. One-way ANOVA was used for maternal care variables. Except for sweet food intake in the home cage (which was analyzed, divided by sex, using one-way ANOVA followed by the least significant difference test), adult behavioral data focused on the two groups of interest (FR/Adlib and Adlib/Adlib) to investigate the effects of fetal programming. Twoway ANOVAs were used for birth weight (group and sex as factors, adjusted for litter size), ASST (performed for each phase of the task as reported elsewhere [36]), and Western blots (split by sex using group and metabolic status as factors). Data on NAcc TH content was not normally distributed (Kolmogorov–Smirnov test, p < 0.0001) and was thus log-transformed for analysis. All data were analyzed in SPSS 22.0 (SPSS Inc., IBM Company, Chicago, IL, USA). The significance level was set at p < 5% for all analyses. 3. Results 3.1. Body weight during gestation and birth weight Repeated-measures ANOVA showed that, from gestational days 1 to 21, FR dams gained less weight, as expected (time vs. group interaction [F(1,26) = 153.97, p < 0.001]). There was also an isolated effect of time [F(1,26) = 16.36, p < 0.001]. Two-way ANOVA adjusted

3.3. Sweet food intake in the home cage In females, there was no group effect [F(3,21) = 1.104, p = 0.373], whereas in males, a group effect was observed [F(3,18) = 3.536, p = 0.041]. Post-hoc least significant difference analysis showed that FR/Adlib males ate more sweet food than animals in all other groups, in a typical fetal programming effect (p = 0.031, p = 0.011, and p = 0.014 as compared with the Adlib/FR, FR/FR, and Adlib/Adlib groups, respectively) (Fig. 1). 3.4. Locomotor activity The mean number of crossings during 5 min in the Adlib/Adlib group was 66.6 ± 3.78 in males and 77.8 ± 4.29 in females. In the FR/Adlib group, the mean was 61.5 ± 3.36 crossings in males and 85.3 ± 4.69 in females. Females were more active than males, showing an increased number of crossings [two-way ANOVA, F(1,36) = 18.53, p < 0.001]. There was no effect of group allocation [F(1,36) = 0.087, p = 0.77] and no interaction [F(1,36) = 2.40, p = 0.13]. 3.5. Attentional set shifting task (ASST) Fig. 2 shows ASST data and analysis. An effect on extradimensional shift was observed, in which females needed

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Fig. 1. Sweet food intake for 1 h in the homecage. Data are expressed as mean ± SEM (grams of sweet food ingested) in the different groups. In males, there was an effect of the group (*p = 0.041), in which FR/Adlib males eat more sweet food than all the other groups (p = 0.031, p = 0.011 and p = 0.014 in the comparison to Adlib/FR, FR/FR and Adlib/Adlib groups, respectively). In females, there was no effect of the group. n = 5/sex/group.

Fig. 2. Attentional set shifting task (ASST). Mean ± SEM of the number of trials to reach criterion in each phase in the different groups. There was effect of the sex in the extradimensional shift, in which females needed more trials to reach criterion than males (p = 0.042). In addition, FR/Adlib females needed less trials than Adlib/Adlib females to reach criterion in the Reversion 2 phase, without differences in males (interaction group vs. sex, p = 0.043). The insert table depicts the statistics for the different phases of the ASST test. n = 10/sex/group.

more trials to reach criterion than males [two-way ANOVA, F(1,36) = 4.480, p = 0.042]. In addition, there was an interaction between group and sex in the Reversion 2 phase, in which FR/Adlib females needed fewer trials than Adlib/Adlib females to reach criterion, without differences in males [two-way ANOVA, F(1,36) = 4.423, p = 0.043]. No other effects or interactions were observed. Adjusting the analysis for LG score and/or litter size did not alter results (data not shown).

3.6. Tyrosine hydroxylase (TH) content in the orbitofrontal cortex (OFC) and nucleus accumbens (NAcc) Fig. 3 illustrates TH content in the OFC of (A) males and (B) females. Two-way ANOVA using group and metabolic status (baseline or in response to sweet food) as factors showed that, in males, there was no group effect [F(1,21) = 0.122, p = 0.731] and no interaction [F(1,21) = 0.005, p = 0.943]. However, there was an isolated

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Fig. 3. Tyrosine hydroxylase (TH) content in the orbitofrontal cortex at baseline and in response to sweet food intake for 1 h in (A) males and (B) females. Data are expressed as % of controls of the relative OD TH/actin. In males, there was an isolated effect of the metabolic status (p = 0.004). In females, FR/Adlib group demonstrates a significant increase in TH in response to sweet food intake in comparison to all the other groups (interaction group vs. metabolic status, p = 0.033; *Post hoc analysis, p < 0.003). n = 5–6/sex/group.

effect of metabolic status [F(1,21) = 11.00, p = 0.004], whereby both groups showed a decrease in TH levels after Froot Loops intake (post-hoc analysis, p = 0.03). On the other hand, in females, we observed effects of group [F(1,21) = 4.946, p = 0.039], metabolic status [F(1,21) = 8.486, p = 0.009] and an interaction [F(1,21) = 5.337, p = 0.033], in which FR/Adlib animals exhibited a significant increase in TH in response to sweet food intake in comparison to all other groups (post-hoc analysis, p < 0.003). Fig. 4 illustrates TH content in the NAcc of (A) males and (B) females. In males, an interaction was observed between group and metabolic status [F(1,20) = 5.741, p = 0.028], in which FR/Adlib animals had increased TH in the NAcc at baseline when compared to animals in all other groups. These increased levels were not found after Froot Loops intake (post-hoc analysis, p < 0.05). There was also isolated effects of the group [F(1,20) = 7.990, p = 0.012], and metabolic status [F(1,20) = 4.609, p < 0.05]. In females, there was similarly an interaction between group and metabolic status [F(1,21) = 4.669, p = 0.04], in which FR/Adlib animals seemed to sustain an increase in TH in response to sweet food intake, with levels significantly higher than in the other groups (post-hoc analysis, p < 0.005). There was also isolated effects of group [F(1,21) = 6.527, p = 0.022] and metabolic status [F(1,21) = 6.297, p = 0.022].

Fig. 4. Tyrosine hydroxylase (TH) content in the nucleus accumbens at baseline and in response to sweet food intake for 1 h in (A) males and (B) females. Data are expressed as % of controls of the relative OD TH/actin. Both in males (p = 0.028) and females (p = 0.044), there is an interaction between group and metabolic status. *Post-hoc analysis demonstrates statistically significant differences in comparison to the other groups, P < 0.05. n = 5–6/sex/group.

4. Discussion In this study, 50% food restriction during gestation induced intrauterine growth restriction, as previously reported [32,37,38], and altered maternal care. In addition, this intervention led to sex-specific alterations in food preferences and behavioral/neurochemical responses to palatable food cues in the offspring. Thus, the fetal programming of adult food preferences involves a central response to palatable food cues and intake, affecting parameters of dopaminergic signaling in select structures of the brain reward system. In addition, FR dams exhibited decreased levels of licking and grooming towards their pups, independently of the nutritional status of the adopted pup after the cross-fostering procedure. Chronic stress during gestation has been shown to diminish the quality of maternal care in primates and rodents [39,40]. Regarding dietary manipulations, malnutrition during gestation is linked to altered maternal care towards the offspring [41–44]. On the other hand, a high-fat diet during gestation also seems to affect this behavior [45]. Although it is known that variations in maternal care are associated with several behavioral, neuroendocrine, and neurochemical effects in adulthood [46–48], the licking and grooming score did not influence ASST performance in this study. It is important to highlight, however, that the groups of interest (Adlib/Adlib and

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FR/Adlib) were both nurtured by Adlib mothers (hence, with similar LG scores). FR/Adlib males ingested more Froot Loops when acutely exposed to this food, in a typical fetal programming effect. As stated in the Section 1, several studies demonstrate that altered feeding behavior and preferences seem to be the result of fetal programming in human IUGR individuals [7], persisting throughout different ages [8–10] onto adulthood [11–15]. In rodents, food restriction during gestation is associated with hyperphagia later in life [32]. In addition, protein restriction during pregnancy in rats promotes an increased preference for foods rich in fat in adult female offspring [49,50]. The lack of behavioral differences in the FR/FR group regarding palatable food intake suggests that match/mismatch mechanisms could be involved [1]. The mismatch concept suggests that the degree of disparity between the environment experienced during development and that experienced later in life affects the risk of disease. During prenatal and early postnatal life, predicted adaptive responses produce phenotypic attributes best suited for the environment in which the organism will probably live later in life [1]. Individuals would be more likely to suffer from disease if a mismatch occurs between the early programming environment and the later adult environment [70]. Hence, while in the FR/Adlib group there was a large mismatch between the pre- and postnatal environments, in the FR/FR group predictive adaptive responses were appropriate to the neonatal environment; therefore, behavioral findings were not specific in this group as they were in the FR/Adlib group. In addition, the timing of malnutrition during gestation and postnatal catch-up growth induce different phenotypes in the offspring [32,51,52], consistent with our results. The apparent discrepancy between studies regarding sex specificity may well be due to differences in the protocol of food preference used. Hence, it is possible that our 1-h food intake test was not sufficiently sensitive to detect behavioral differences between FR/Adlib and Adlib/Adlib females, though we found evidence of specific neurochemical functioning at baseline and in response to intake of this type of food in FR/Adlib females through measurement of TH content in the OFC and NAcc respectively. FR/Adlib females demonstrated a considerable increase in OFC TH content after exposure to sweet food. This marked (approximately fivefold) increase could indicate that the sweet food cue used in the ASST task is perceived differently by FR/Adlib females, with concomitant increased dopaminergic signaling in the OFC, and possibly favoring their performance in this reversal learning phase of the task. On the other hand, quantification of OFC TH content in males showed that both Adlib/Adlib and FR/Adlib animals have a decrease in TH levels in response to sweet food. Recent studies highlight the role of OFC dopamine in attention and impulsivity-related behaviors. For instance, injection of dopamine antagonists into the mPFC or OFC increases impulsivity in rats [53], and OFC dopamine blockade reverts the beneficial effects of methylphenidate on reversal learning in an animal model of attention deficit [36]. Therefore, it is possible that increased TH content in response to the food cue in females had a favorable effect on the performance of FR/Adlib rats through modulation of impulsive behavior. Other studies indicate that impaired performance on a reversal stage could arise due to a reduced ability to focus on relevant information or due to greater distraction by irrelevant cues [71–73]. Apparently, the ability of the cue to trigger increased attention (salience) and wanting was greater in IUGR females, causing them to become more focused on the task and, consequently, to perform better. More than a crucial region for behavioral alteration/adaptation when faced with unexpected outcomes, the OFC is a highly integrative area, particularly important for the processing of information related to reward-associated value [54,55] and learning [56]. OFC

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neurons respond to the presentation of rewarding foods or simply to stimuli that signal future reward, and the strategic position of this structure in the most ventral portion of the prefrontal cortex facilitates its role in behavioral motivation [57,58]. Neuroimaging studies in humans suggest that the OFC and NAcc demonstrate high connectivity during the processing of reward information [59]. States such as obesity and drug dependency are characterized by alterations in the functional connectivity between the OFC and regions related to inhibitory control, such as the medial prefrontal gyrus [60], as well as with the NAcc [61,62]. It has been proposed that alterations in this neurocircuitry can make individuals more susceptible to environmental food cues (for instance, the presence of Froot Loops during the task in this study). Therefore, improper feedback between the OFC and NAcc due to decreased functional integration could lead to an aberrant evaluation of reward, resulting in abnormally intense initial learning (as seen in the FR/Adlib females) and resistance to behavior change in the long-term traits, characteristically seen in drug dependence. Lesion studies in rats and mice have shown that intradimensional and extra-dimensional shifts are closely related with the anterior cingulate cortex (ACC) and the medial prefrontal cortex (mPFC) respectively [34,74]. The lack of differences in these stages suggests that IUGR animals do not show deficits in rule-based learning, working memory, forming associations between stimuli, or encoding correct and incorrect responses [34,75–77]. This speaks in favor of our hypothesis that IUGR animals performed better simply because they were more focused on the task due to the increased value assigned to the sweet reward. Moreover, it could be suggested that the ACC and mPFC are not as sensitive in IUGR individuals as the OFC is. Quantitation of TH content in the NAcc revealed more complex alterations, although consistent with behavioral findings. As compared to all other groups, FR/Adlib males had increased NAcc TH levels. Interestingly, after exposure to sweet food, this increase disappeared. Elevated DA levels in the NAcc have been shown to promote increased intake of palatable foods, at the same time that such intake itself leads to DA release in these areas, in a feedforward process [63–65]. Thus, the higher level of TH observed at baseline in IUGR males could explain, at least in part, the higher consumption of sweet food observed in these animals. Microdialysis studies have shown that feeding-evoked increases in DA release in the NAcc last only a few minutes, with levels returning to baseline within approximately 60 min [63,78]. Therefore, it is possible that, between the initial exposure to sweet food and the time of tissue collection (a 60-min gap), dopamine levels will have peaked and returned to baseline, precluding capture of this increase in the groups, except in FR/Adlib females, in which TH levels remained high even after this period. As DA in the NAcc is involved in the rewarding properties of food [79–81], this again suggests that the reward used in the ASST was most highly valued by these females. Moreover, the sustained increase in NAcc TH levels observed could be involved in long-term behavioral changes we believe to exist in FR/Adlib females. Although we did not evaluate connectivity between the OFC and the NAcc in this study, the differences in TH found in FR/Adlib animals in these two brain structures at baseline and in response to sweet food intake, both in males and females, suggest that sensitivity and/or food reward value attribution are different in these animals. We propose that this characteristic influences the performance of FR/Adlib rats in cognitive flexibility tasks, as seen in this study. In summary, IUGR causes sex-specific alterations in feeding behavior and in sensitivity to food cues in adulthood, and altered dopaminergic signaling in key areas involved in these behaviors seems to be implicated in these findings. Knowledge about the mechanisms involved in the increased vulnerability to overweight

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