Locomotor response to acute nicotine in adolescent mice is altered by maternal undernutrition during lactation

Int. J. Devl Neuroscience 47 (2015) 278–285

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Locomotor response to acute nicotine in adolescent mice is altered by maternal undernutrition during lactation Ana C. Dutra-Tavares a , Alex C. Manhães a , Juliana O. Silva a , André L. Nunes-Freitas a , Ellen P.S. Conceic¸ão a , Egberto G. Moura a , Patrícia C. Lisboa a , Cláudio C. Filgueiras a , Yael Abreu-Villac¸a a , Anderson Ribeiro-Carvalho a,b,∗ a Departamento de Ciências Fisiológicas, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Av. Prof. Manoel de Abreu 444, 5 andar – Vila Isabel, Rio de Janeiro, RJ 20550-170, Brazil b Departamento de Ciências, Faculdade de Formac¸ão de Professores da Universidade do Estado do Rio de Janeiro, Rua Dr. Francisco Portela 1470–Patronato, São Gonc¸alo, RJ 24435-005, Brazil

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Article history: Received 1 June 2015 Received in revised form 14 September 2015 Accepted 12 October 2015 Available online 19 October 2015 Keywords: Drugs of abuse Undernutrition Development Open field Corticosterone Catecholamine

a b s t r a c t Undernutrition during brain development causes long lasting alterations in different neurotransmitter systems that may alter responses to psychoactive drugs. Despite the recognized effects of early undernutrition on the cholinergic system, no evidence that demonstrates the influence of this insult on nicotine susceptibility has been reported. We investigated the effects of protein/calorie restriction during lactation on the susceptibility to nicotine in adolescent mice. Dams were randomly assigned to one of the following groups: Control (C, 20 litters)—free access to standard laboratory diet (23% protein); Protein Restricted (PR, 12 litters)—free access to a isoenergetic, 8% protein diet; Calorie Restricted (CR, 12 litters)—access to standard laboratory diet in restricted quantities (mean ingestion of PR: pair-fed group). Undernutrition extended from postnatal day 2 (PN2) to weaning (PN21). At PN30, animals either received an i.p. injection of nicotine (0.5 mg/Kg) or saline and were immediately placed in open field (OF). After the OF, adrenal glands and serum were collected for the analyses of stress-related endocrine parameters and leptin concentration. PR and CR offspring showed less body mass gain and visceral fat mass. PR offspring presented reduced serum leptin concentration. In the OF, nicotine increased locomotor activity of C and PR, but not of CR. CR and PR offspring showed decreased adrenal catecholamine content, which was not dependent on nicotine exposure. Our results indicate that early undernutrition interferes with nicotine-elicited locomotor effects in adolescent mice and suggest that endocrine parameters alterations in malnourished animals do not influence the behavioral response to nicotine. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Undernutrition remains the most prevalent form of nutritional disorder among children in developing countries (FAO et al., 2013), representing an important public health problem. Undernutrition during development has been associated with profound effects on health later in life. In fact, it was found to be associated with alterations such as high blood pressure (Roseboom et al., 1999), obesity (Ravelli et al., 1999), glucose intolerance (Ravelli et al., 1998), dyslipidemia (Roseboom et al., 2000a) and higher risk of coronary

∗ Corresponding author at: Departamento de Ciências, Faculdade de Formac¸ão de Professores, Universidade do Estado do Rio de Janeiro, Rua Dr. Francisco Portela, 1470, Patronato, São Gonc¸alo, RJ, 24435-005, Brazil. Fax: +55 21 2868 8029. E-mail address: ribeiro [email protected] (A. Ribeiro-Carvalho). http://dx.doi.org/10.1016/j.ijdevneu.2015.10.002 0736-5748/© 2015 Elsevier Ltd. All rights reserved.

disease (Roseboom et al., 2000b). Cognitive, behavioral and emotional impairments during adolescence and adulthood have also been reported (Galler et al., 2013). A critical period of brain development is lactation. In rodents, there is a surge in brain growth characterized by dendritic arborization, synaptogenesis and the migration of numerous neuronal populations during the first 2 weeks of postnatal life (Dobbing and Sands, 1979), rendering the brain particularly vulnerable to insults. In this sense, it is well established that nutritional insults during the lactation period promote several neurobiological disturbances, resulting in lifelong physiological consequences (Reyes-Castro et al., 2012). It has been described in animal models that protein restriction during lactation promotes alterations in anxiety levels and impairs learning behavior in the adult offspring (Almeida et al., 1993; Reyes-Castro et al., 2011). Several neurochemical systems could be involved in these behavioral

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Fig. 1. Effects of undernutrition during lactation on body mass and food intake for dams (A and C, respectively) and pups (B and D, respectively). E, visceral fat mass in the offspring at postnatal day (PN) 30. F, serum leptin levels in the offspring at PN30. Values are means ± S.E.M. Statistical differences between groups were assessed by FPLSD. C, Control group; PR, Protein restricted; CR, Calorie restricted; In A–C, * indicates significant differences between C and PR; @ indicates differences between C and CR; P values are indicated in the text of the results section. In E and F, * P < 0.05.

alterations (for review see (Almeida et al., 1996)). Previous studies also demonstrated that maternal protein/caloric restriction during lactation disturbs the pattern of food intake and the hypothalamic leptin signaling pathway later in the offspring’s life (Lisboa et al., 2012; Passos et al., 2004). These effects suggest that nutritional insults in this period promote changes in mechanisms of reward control. Of note, an association between early undernutrition and the response to psychoactive drugs has been reported in humans: a case-control study indicates a relationship between the prenatal famine during the Dutch hunger winter of 1944–45 and addiction later in life (Franzek et al., 2008). In parallel, animal models of perinatal undernutrition indicate an increased response of the mesocorticolimbic dopaminergic pathway to the rewarding effects of cocaine (Valdomero et al., 2006) and morphine (Velazquez et al., 2010). Among the drugs of abuse, tobacco consumption is one of the most important public health challenges. Nicotine, an important component of tobacco smoke, is known to be responsible for a wide range of nervous system effects resulting from tobacco use, including tobacco addiction (Benowitz, 1992). Smoking typically begins during adolescence (Centers for Disease Control Prevention, 2010) and, in animal models, it was demonstrated that nicotine exposure in adolescent mice elicits several distinct behavioral and biochemical effects (Ribeiro-Carvalho et al., 2009; RibeiroCarvalho et al., 2011; Abreu-Villaca et al., 2007; Abreu-Villaca et al., 2008; Ribeiro-Carvalho et al., 2008), characterizing adolescence as a period of vulnerability to nicotine effects. Interestingly, the cholinergic system, the primary target of nicotine, seems to be

particularly sensitive to early undernutrition, exhibiting longlasting alterations as well as altered responses to a variety of pharmacological treatments (Almeida et al., 1996). In spite of that, to our knowledge, there are no studies that investigate the influence of early undernutrition on nicotine susceptibility. Here, we investigated the effects of protein and/or caloric restriction during lactation on the acute effects of nicotine exposure on locomotor activity (in the open field test) of adolescent mice. An altered response to stress may influence the stimulatory response of nicotine in the mesolimbic dopaminergic system (Enrico et al., 2013). For this reason, we also evaluated ACTH and corticosterone serum levels, total catecholamine (adrenaline and noradrenaline) content as well as the expression of two catecholamine synthesising enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT), in the adrenal medulla. In addition, since it has been consistently demonstrated that undernutrition during development has both short and long-term effects on serum leptin concentration (Bonomo et al., 2007; Carvalho et al., 2014) and that leptin levels have been associated with locomotor activity (Fraga et al., 2011), we also assessed leptin serum concentration.

2. Materials and methods All experiments were carried out under institutional approval of the Animal Care and Use Committee of the Universidade do Estado do Rio de Janeiro (CEUA/016/2011), in accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National

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Fig. 2. Ambulation in the open field (OF): Effects of acute nicotine exposure during adolescence in mice malnourished during lactation. Values are means ± S.E.M. Statistical differences between groups were assessed by FPLSD. C-NIC, PR-NIC and CR-NIC indicated animals from Control (C), Protein restricted (PR) and Calorie restricted (CR) groups that received nicotine (0.5 mg/kg) immediately before being tested in the open field, respectively; C-SAL, PR-SAL and CR-SAL indicated animals from Control (C), Protein restricted (PR) and Calorie restricted (CR) groups that received saline, respectively. ** P < 0.01; *** P < 0.001.

Table 1 Comparisons between control and protein restricted diets. Controla Ingredients (g/kg) Soybean + wheat Cornstarch Soybean oil Vitamin mixc Mineral mixc Macronutrient composition (%) Protein Carbohydrate Fat Total energy (kJ/kg)

Protein-restrictedb

230.0 676.0 50.0 4.0 40.0

80.0 826.0 50.0 4.0 40.0

23.0 66.0 11.0

8.0 81.0 11.0

17138.7

17138.7

to a severe protein restriction, also have a reduced caloric intake. In the present study, this reduction was of about 30% throughout the restriction period when compared to the C group (total food intake during the lactation period is shown in the supplementary material). Milk composition in this model reflects the protein/caloric undernutrition of dams: Passos et al. (2000) demonstrated that undernutrition during lactation impairs milk production in both PR and CR dams, but only PR milk presents reduction of protein concentration(Passos et al., 2000). The PR diet was prepared in our laboratory by replacing part of the protein in the standard diet with cornstarch. The amount of starch was calculated to make up for the decrease in energy content resulting from the protein reduction. Vitamin and mineral mixtures were formulated to meet the American Institute of Nutrition AIN93G recommendation for rodent diets. Macro and micronutrients from each batch were properly analyzed to ensure that the required quantities of all components were present. The chow used throughout the experiment was stored in freezers until use. Comparisons between Control and Protein Restricted diets are shown in Table 1. To minimize the influence of the litter size, we only used litters with 8–12 pups. Litters with 11 or 12 pups were culled to 10 pups. Undernutrition extended from PN2 to weaning (PN21). After weaning, mice were separated by sex and housed in groups of two or three animals per cage. During the lactation period, the body mass of dams and pups were monitored every third day, and the food intake of dams was monitored every day. After weaning, pups had free access to standard laboratory diet and their body mass and food intake were assessed every third day. At PN30, mice from each litter were tested in the open field (OF). 2.2. Open field (OF)

a

Standard laboratory diet for rodents (Nuvilab-NUVITAL Nutrientes LTDA, Paraná, Brazil). b The protein-restricted diet was prepared in our laboratory using the control diet and replacing part of its protein with cornstarch. c Vitamin and mineral mixtures were formulated according the AIN-93G recommendation for rodent diets.

Institutes of Health. All Swiss mice were bred and maintained in our animal facility with controlled temperature (21 ± 1 ◦ C) on a 12:12 h light/dark cycle (lights on at 1:00 a.m.). All behavioral tests were carried out in a sound-attenuated room next to the animal facility. 2.1. Model of maternal undernutrition during lactation Adult nulliparous female mice were housed with male mice and, after mating, each female was placed in an individual cage with free access to water and food until parturition. On the 2nd postnatal day (PN2), mouse dams were randomly assigned to one of the following groups: (1) Control (C, 20 litters)—which had free access to a standard laboratory diet containing 23% protein (Nuvilab–NUVITAL Nutrientes LTDA, Paraná, Brazil, Table 1); (2) Protein restricted (PR, 12 litters)—which had free access to an isoenergetic protein restricted diet, containing only 8% protein; (3) Calorie restricted (CR, 12 litters)—which received standard laboratory diet in restricted quantities that corresponded to the mean ingestion of the PR dams in the previous day. Thus, the amount of food consumed by CR and PR groups was about the same throughout the period of undernutrition. The inclusion of this third group (pair-feeding) in the experimental design was based on previous findings that demonstrate that rodent dams submitted to protein restriction present a reduction in food intake when compared to control dams (Correa et al., 2011). As a result, PR dams, in addition

The arena (Insight, SP, Brazil) consists of a transparent acrylic box (46 cm length × 46 cm width × 43 cm height) that is equipped with 2 perpendicular arrays of 16 paired infrared emitters and detectors positioned at 1.5 cm above the floor. Each emitter is aligned with a single detector placed directly across the arena. Interruptions of the infrared beams during animal locomotion are detected by a computer system and used to locate the animal within the field and track its movements. At PN30, each animal received an i.p. injection of nicotine (0.5 mg/Kg) or saline, a procedure that resulted in six experimental groups: animals from the C, PR and CR groups that received nicotine (C-NIC, n = 11 females and 12 males; PR-NIC, n = 10 females and 9 males; and CR-NIC, 10 females and n = 10 males) and animals from the C, PR and CR groups that received saline (C-SAL, n = 10 females and 12 males; PR-SAL, n = 10 females and 9 males; and CR-SAL, n = 11 females and 10 males). This nicotine administration protocol was chosen because it was previously shown to produce increased locomotor activity in rodents (Celik et al., 2006). Immediately after the injection, mice were individually placed in the corner of the open field arena and were allowed to explore for 5 min. Spontaneous locomotor activity (Total Ambulation) was determined on the basis of the traversed distance. All sessions were performed between 2:00–6:00 p.m. and the arena was cleaned with paper towels soaked in 70% ethanol and dried before each test. 2.3. Endocrine measurements To minimize the effect of circadian cycles on the endocrine variables, mice were decapitated between 2:00–6:00 p.m., immediately after the behavioral tests (at PN30). Visceral fat was excised and its mass was assessed. Trunk blood was collected for the evaluation of total corticosterone (C, n = 12 females and 12 males; PR, n = 12 females and 11 males; and CR, n = 11 females and 11 males), leptin (C, n = 6 females and 6 males; PR, n = 6 females and 6 males;

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and CR, n = 6 females and 6 males) and ACTH (C, n = 8 females and 8 males; PR, n = 8 females and 8 males; and CR, n = 8 females and 8 males) concentrations in serum. The adrenal medullas were dissected for total catecholamine (adrenaline and noradrenaline) content evaluations (C, n = 12 females and 12 males; PR, n = 12 females and 11 males; and CR, n = 11 females and 11 males). In addition, the adrenal medullas were used for the quantification of tyrosine hydroxylase (TH) (C, n = 7 females and 9 males; PR, n = 8 females and 9 males; and CR, n = 9 females and 6 males), phenylethanolamine N-methyltransferase (PNMT) (C, n = 6 females and 9 males; PR, n = 8 females and 8 males; and CR, n = 8 females and 6 males) and melanocortin 2 receptor (MC2R) (only in males; C, n = 9; PR, n = 9; and CR, n = 8) expression. For each variable, the sample was equally distributed into SAL and NIC sub-groups. 2.3.1. Serum hormone measurements Trunk blood was obtained, centrifuged (1000 × g, 4 ◦ C for 20 min) and the serum was stored at −20 ◦ C. Total corticosterone was measured using a radioimmunoassay kit (ICN Biomedicals Inc., Cleveland, OH, USA) with a detection range from 25 to 1000 ng/mL and an intra-assay variation coefficient of 7%. ACTH concentration was determined using a specific enzyme immunoassay kit (Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) with a detection range from 0.04 to 25 ng/mL and an intra-assay variation of 2%. Leptin was measured with a specific radioimmunoassay kit (Linco Research Inc., St Charles, MO, USA) with a detection range from 0.5 to 50 ng/mL; the intra-assay variation was 2.9%. All measurements were determined in only one assay. 2.3.2. Adrenal medulla catecholamine evaluation The total adrenaline and noradrenaline content was measured by the trihydroxyindole fluorescence method. Right adrenal glands were homogenized in 200 ␮l 10% acetic acid and centrifuged (1120 × g, 5 min). Briefly, 50 ␮l of the supernatant fraction was mixed with 250 ␮l 0.5 M buffer phosphate (pH 7.0) and 25 ␮l 0.5% potassium ferricyanide, followed by incubation (20 min in ice bath). Next, 500 ␮l ascorbic acid 5 M NaOH (1:19) was used to stop the reaction. The fluorometer (Plate Chameleon V, Hidex, Turku, Finland) parameters were: 420 nm to excitation and 510 nm to emission. Results were obtained by plotting the values into a linear regression of the standard adrenaline curve. Data were expressed as ␮M of total catecholamines. 2.3.3. Tyrosine hydroxylase (TH) and phenylethanolamine N methyltransferase (PNMT) and melanocortin 2 receptor (MC2R) Western blot analysis The left adrenal glands were homogenized in 250 ␮l phosphate buffer (pH 7.4), containing a protease inhibitor cocktail (Complete Protease Inhibitor Cocktail Tablet, EDTA-free, Roche Applied Science, Mannheim, DE). After centrifugation (7500 × g for 5 min), homogenates were stored at −20 ◦ C until the SDS-PAGE assay. The total protein content of homogenate was determined by the Bicinchoninic Acid (BCA) protein assay kit (Thermo Scientific, Inc., Barrington, IL, USA). The adrenal protein content of TH, PNMT, MC2R and ␤-tubulin were evaluated using adequate primary antibodies incubation (overnight; 1:500 antibody from Sigma–Aldrich Co., MO, USA; Abcam plc, Cambridge, UK; and Santa Cruz Inc., CA, USA, respectively) followed by proper secondary antibodies incubation (1 h; 1:10,000; antibody from Sigma–Aldrich Co., MO, USA) and streptavidin (1 h; 1:10,000; Zymed, CA, USA) as previously described (Conceicao et al., 2013). The protein bands were visualized by chemiluminescence by Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Bio-Sciences Uppsala, SE) followed by exposure to autoradiographic film (Hyperfilm ECL, GE Healthcare Bio-Sciences Uppsala, SE). Area and density of the bands were quantified by Image J software (Wayne Rasband

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National Institute of Health, MA, USA). The results were normalized by ␤-tubulin content and were expressed as relative (%) to the control group. 2.4. Statistical analysis Repeated measures analyses of variance (rANOVA) were performed for body mass and food intake. Treatment (C, PR and CR) was used as between-subjects factor. In order to minimize the influence of litter effects, we considered the average of values from mice of the same litter instead of using individual values. Day was considered the within-subjects factor. Separate univariate analyses of variance (uANOVA) were performed for the OF, visceral fat mass and endocrine measures. Treatment, Exposure (nicotine or saline) and Sex were used as between-subjects factors. For both uANOVAs and rANOVAs, significant Treatment interactions were followed by lower order ANOVAs and by Fisher’s Protected Least Significant Difference (FPLSD) tests. Effects were considered significant when P < 0.05 (two-tailed). No more than one male and one female from each litter were assigned to each behavioral test condition. For the sake of simplicity, we will report results based only on the averaged univariate F tests. The univariate approach is considered more powerful than the multivariate criteria. However, each univariate test requires that the variances of all transformed variables for an effect to be equal and their covariances to be zero. Therefore, the extent to which the covariance matrices deviate from sphericity was estimated by Mauchly’s test. Whenever the sphericity assumption was violated, we used the Greenhouse–Geisser correction, which adjusts the degrees of freedom, in order to avoid Type I errors. Figures were only segmented by sex or exposure when significant Treatment × Sex or Treatment × Exposure interactions were respectively observed. 3. Results 3.1. Body mass, visceral fat mass, leptin levels and food intake Diet affected body mass during the lactation period (Treatment × Day: F8.0,140.9 = 13.2, P < 0.001). As depicted in Fig. 1A, from the eighth lactation day onwards, both PR and CR dams had lower body mass (P < 0.001 and P < 0.01, respectively) when compared to controls. Diet also affected body mass gain in the offspring (Treatment × Day: F4.4,147.7 = 20.5, P < 0.001). In spite of the fact that body mass increased in all groups (Day: F2.2,147.7 = 464.2, P < 0.001), PR and CR offspring showed less mass gain when compared with the controls (Fig. 1B). Malnutrition affected visceral fat mass on PN30 (Treatment: F2,103 = 3.2, P = 0.04): Both PR and CR mice presented less visceral fat mass than controls (PR and CR < C: P < 0.05) (Fig. 1E). For serum leptin concentration (Fig. 1F), the effect of undernutrition (Treatment: F2,32 = 3.3, P = 0.05) was significant in the PR group, which presented reduced levels when compared to control mice (P = 0.02). The reduction in leptin levels in the CR group did not reach statistical significance. The food intake was corrected for body mass per day (total food intake (g)/body mass (g)). As depicted in Fig. 1C, food intake increased throughout the lactation period (Day: F2.8,99.6 = 13.1, P < 0.001). However, the average intake was significantly different among groups (Treatment: F2,36 = 4.8, P < 0.014). In fact, PR dams had reduced food intake when compared with controls on the 2nd (P < 0.05) and 5th (P < 0.05) days and the CR group had reduced food intake on the 2nd (P < 0.05), 5th (P < 0.01), 11th (P < 0.01) and 14th (P = 0.001) days. No differences in food intake were observed between CR and PR groups, indicating a successful pair-feeding. After weaning (PN24 to PN30), no significant differences in food intake were identified in the offspring (Fig. 1D).

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Fig. 3. Endocrine parameters after the open field test: effects of undernutrition during lactation on adolescent mice. A, serum ACTH levels in male and female offspring respectively; B, serum corticosterone levels; C, melanocortin 2 receptor (MC2R) western blot analysis of the adrenal gland; D, catecholamine total content in the adrenal gland; E and F, tyrosine hydroxylase (TH) and phenylethanolamine N methyltransferase (PNMT) western blot analysis of the adrenal gland respectively; G, representative blots of TH, PNMT and MC2R; Values are means ± S.E.M. Statistical differences between groups were assessed by FPLSD. C-NIC, PR-NIC and CR-NIC indicated, respectively, animals from Control (C), Protein restricted (PR) and Calorie restricted (CR) groups that received nicotine (0.5 mg/kg) immediately before being tested in the open field; C-SAL, PR-SAL and CR-SAL indicated, respectively, animals from Control (C), Protein restricted (PR) and Calorie restricted (CR) groups that received saline. @@@ P < 0.001: male C-SAL vs. male C-NIC in A. * P < 0.05; ** P < 0.01.

3.2. Open field The uANOVA indicated that locomotor activity was affected by nicotine administration (Exposure: F1,99 = 9.3; P = 0.003). In addition, a Treatment × Exposure interaction was observed (F2,99 = 4.1; P = 0.02). A nicotine-elicited increase in locomotor activity was evident in C (C-NIC > C-SAL: P < 0.001) and PR offspring (PRNIC > PR-SAL: P < 0.05), but not in CR mice (Fig. 2). Caloric restriction per se was associated with a trend toward increased activity (CR-SAL > C-SAL: P = 0.069). 3.3. Stress-related endocrine evaluations Regarding the stress-related endocrine measures, only ACTH levels were affected by nicotine administration

(Treatment × Exposure × Sex: F2,47 = 3.7, P = 0.03). The effects were restricted to control males (Fig. 3A), in which nicotine elicited increased ACTH levels (C-NIC > C-SAL: P < 0.001). The lack of nicotine-elicited effects in PR and CR male mice could be due to the ACTH hypersecretion identified in both saline-exposed malnourished groups (PR-SAL > C-SAL: P = 0.004 and CR-SAL > C-SAL: P = 0.013). Despite the ACTH findings, corticosterone levels were not altered (Fig. 3B). In order to investigate why the hypersecretion of ACTH in malnourished male mice was not accompanied by increases in circulating corticosterone levels, we evaluated the expression of melanocortin 2 receptor (MC2R) in male mice by western blot analysis. The MC2R is the ACTH receptor in adrenal glands. No differences in MC2R were observed among the groups (Fig. 3C). Regarding the adrenal catecholaminergic system, both PR and CR groups showed a decrease in adrenal catecholamine

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content when compared to controls (P < 0.01 in both pairwise comparisons) (Fig. 3D). In order to explain the reduction in adrenal catecholamine content, we evaluated the expression of enzymes involved in the catecholamine biosynthetic pathway: TH and PNMT. TH levels were similar among groups (Fig. 3E). The PNMT analysis indicated an effect of the dams’ diet (Treatment: F2,40 = 3.9, P = 0.03): The PR group presented higher levels than the CR one (P = 0.008). However, malnourished groups differed from the control group (Fig. 3F). Representative blots of TH, PNMT and MC2R are shown in Fig. 3G. 4. Discussion Previous studies suggest an association between a deficient nutritional status during early life and an increased responsiveness to psychoactive substances later in life (Franzek et al., 2008; Valdomero et al., 2006; Velazquez et al., 2010). Here, undernutrition during lactation did not elicit an increase in nicotine susceptibility in adolescent mice. In addition, alterations in endocrine parameters associated with stress response in undernourished animals did not seem to play a role in the behavioral response to nicotine. 4.1. Effects of undernutrition during lactation on body mass, visceral fat mass, serum leptin concentration and food intake Lactation is a critical period during the development of mammals and it is important in the establishment of body composition and metabolic programming (Correa et al., 2011; Moura et al., 2008). Accordingly, our data indicate that both PR and CR offspring presented a reduction in body mass gain throughout the lactation period that, by the beginning of adolescence, resulted in lighter animals when compared to controls. These results and the reduced visceral fat mass in PR and CR mice demonstrate that our models were efficient in eliciting undernutrition, and corroborate previous findings that show reduced body mass and body fat mass in rat offspring whose dams were submitted to protein restriction during lactation (Correa et al., 2011; Moura et al., 2008). The fact that both PR and CR offspring were lighter than controls even after the lactation period seems to be related to the fact that animals in these two groups did not present an increase in food intake after weaning. Since caloric restriction during the lactation period result in obese adult offspring while lighter-than-normal adult offspring is the usual outcome of protein restriction during the same period (Correa et al., 2011), our results suggest that compensatory increases in body mass in CR animals will occur only after the beginning of adolescence, while PR animals will remain lighter than normal throughout life. Here, we found that only PR animals showed a significantly reduced serum leptin concentration. The reduction of leptin levels could be associated with the reduction of body fat mass observed in the PR group. It must be pointed out, however, that PR and CR animals presented a similar reduction in body mass when compared to C ones, a pattern of results that indicates that protein undernutrition generates more intense alterations in leptin levels than calorie restriction. Previous studies have indicated that undernutrition during early postnatal life promotes an increase in plasma leptin during the malnutrition period in rats (particularly at PN10 and PN21), while no alteration is observed at PN60 (Vicente et al., 2004; Moura et al., 2002). Considering the aforementioned findings, it is conceivable to speculate that a brief period of hypoleptinemia is the result of the restoration of the standard laboratory diet to the protein malnourished animals. Fulton et al. (2006) showed that mice that lack the leptin receptor (ob/ob mice) have a reduced locomotor response to amphetamine that can be reversed by leptin

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infusion (Fulton et al., 2006). Here, the reduction of serum leptin concentration in PR animals did not seem to be associated with the nicotine-induced locomotor hyperactivity, suggesting that serum leptin concentration does not play a major role in the locomotor response to nicotine. 4.2. Effects of early undernutrition on nicotine susceptibility Our results indicate that undernutrition during lactation failed to program for an increased susceptibility to nicotine. Velazquez et al. (2010) demonstrated that perinatal undernutrition facilitates morphine sensitization and cross-sensitization to cocaine. It is possible to speculate that developmental undernutrition sensitizes catecholamine (cocaine) and opiate (morphine) pathways, but does not increase the cholinergic response to nicotine in brain reward systems. Another possible explanation is based on the fact that in cocaine and morphine studies, the undernutrition period initiated during gestation. The lack of effect in the present study could indicate that gestation period is more relevant than the lactation one to program an increase in drug susceptibility. Activation of the striatum and the nucleus accumbens is likely to be the initial step in drug reinforcement and development of addiction (Mineur et al., 2009). Nicotine, like other drugs of abuse, interacts with its receptors in the mesolimbic dopaminergic system (Dani and Bertrand, 2007). The nicotine-mediated modulation of dopamine release results in increased locomotor activity (Museo and Wise, 1994). Mineur et al. (2009) demonstrated that the ␤2 subunit-containing nAChRs in the ventral tegmental area (VTA) neurons mediates nicotine-induced locomotor activation (Mineur et al., 2009). Our results show that while PR-NIC offspring presented an increase in locomotor activity comparable to that observed in CNIC animals, CR animals did not respond to nicotine in the open field test. These results raise the possibility that the ␤2 subunitcontaining nAChRs in VTA pathways were affected by early calorie restriction. Interestingly, several alterations observed during lactation depend on the type of maternal malnutrition. For example, serum thyroid hormone levels during lactation and GH mRNA expression patterns at adulthood were shown to be differentially affected by PR and CR during lactation (De Moura et al., 2007; Passos et al., 2002). These differences could be the consequence of or underlie the disparity in brain function. 4.3. Alterations in endocrine measures associated with stress response and nicotine exposure in malnourished animals Maternal food restriction reduces hypothalamic pituitary adrenal axis activity in response to stress during the weaning period (Vieau et al., 2007). In this sense, it is possible that undernutrition during lactation affects stress response in pups, disturbing the behavioral response to nicotine. In the present study, acute nicotine was able to produce an increase in plasma ACTH only in control males. Distinctively, saline-injected PR and CR males that were tested in the OF showed a hypersecretion pattern of ACTH and, in these cases, nicotine did not cause alterations. The hypersecretion of ACTH was not accompanied by increases in circulating corticosterone levels, suggesting a decreased response of the adrenal gland to ACTH. In order to investigate this hypothesis, we analyzed the expression of MC2R in the adrenal glands of male animals. No differences among the groups were observed, indicating that the lack of increase in corticosterone levels could be due to alterations in MC2R signaling pathway. It is established that cigarette smoking increases circulating cortisol in humans (Pomerleau and Pomerleau, 1990), while nicotine administration has been shown to elevate plasma ACTH and corticosterone in animal models (Lutfy et al., 2012). Surprisingly, in the present study, nicotine did not promote an increase in serum corticosterone. In accordance, Cao et al.

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(2010) failed to demonstrate a nicotine-induced (0.6 mg/kg) release of corticosterone in adolescent males and females (Cao et al., 2010). The lack of effects on corticosterone levels suggests that this hormone is not relevant to the behavioral alterations identified in the present study. Malnourished animals (both PR and CR) showed a reduction in adrenal medulla catecholamine content after the OF test. However, adrenal TH and PNMT expressions were similar to control levels, suggesting that the reduced content in PR and CR animals was due to a greater catecholamine secretion. In fact, increased in vitro catecholamine secretion was observed in adult rats submitted to maternal protein restriction during lactation (Fagundes et al., 2007). In accordance, Petry et al. (2000) showed an increase in serum catecholamines in young adult rats whose dams were fed a PR diet during gestation and lactation (Petry et al., 2000). It is well known that the adrenal medulla has nicotinic receptors in which acetylcholine and its agonists, such as nicotine, directly bind to chromaffin cells, increasing catecholamine production and release (Hiremagalur et al., 1993; Slotkin and Seidler, 1975). Catecholamine synthesis is mainly regulated by the tyrosine hydroxylase (TH) enzyme. Studies have shown higher TH expression and activity in the adrenal medulla of rats that were exposed either acutely (Sterling and Tank, 2001) or chronically to nicotine (Sun et al., 2003). On the other hand, Cheng et al. (2005) found no changes in TH mRNA levels in rats chronically exposed to nicotine. Here, we did not find any effect of nicotine on adrenal medulla and serum corticosterone (Cheng et al., 2005). It is possible that, at this age, the adrenal medulla is resistant to nicotine effect. In addition, we suggest that the reduced catecholamine content observed in malnourished groups is not relevant to the behavioral response to nicotine, however, a more direct evaluation of catecholamines secretion, metabolism and the sensitivity of the central nervous system turns out to be an interesting issue to be investigated. 5. Conclusions The present study evaluated the consequences of two types of undernutrition during the lactation period on locomotor activity after acute nicotine exposure during early adolescence in mice. Despite the fact that the type of undernutrition was determinant to the observed behavioral effects, and reflected on a particular pattern of interaction with nicotine, neither PR nor CR groups presented increased susceptibility to nicotine in the OF. In addition, our results suggest that alterations in endocrine parameters associated with stress response in malnourished animals did not influence the behavioral response to nicotine. Considering that chronic exposure to nicotine promotes synaptic alterations in the VTA, which has a fundamental role in the acquisition of addictive behaviors (Feduccia et al. 2012), future studies focusing on the neurochemical responsiveness of reward systems will be useful to better understand the effects of early undernutrition on nicotine susceptibility. Acknowlegments This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundac¸ão de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and fellowships by Sub-reitoria de Pós-graduac¸ão e Pesquisa da Universidade do Estado do Rio de Janeiro (SR2-UERJ), Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior and FAPERJ. The authors are thankful to Ulisses Risso for animal care. 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.2015. 10.002.

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