Maternal undernutrition during lactation alters nicotine

International Journal of Developmental Neuroscience 65 (2018) 45–53

Contents lists available at ScienceDirect

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

Maternal undernutrition during lactation alters nicotine reward and DOPAC/dopamine ratio in cerebral cortex in adolescent mice, but does not affect nicotine-induced nAChRs upregulation

MARK

Ana C. Dutra-Tavaresb, Juliana O. Silvab, André L. Nunes-Freitasb, Vinícius M.S. Guimarãesb, Ulisses C. Araújob, Ellen P.S. Conceiçãob, Egberto G. Mourab, Patrícia C. Lisboab, ⁎ Cláudio C. Filgueirasb, Alex C. Manhãesb, Yael Abreu-Villaçab, Anderson Ribeiro-Carvalhoa, a Departamento de Ciências, Faculdade de Formação de Professores da Universidade do Estado do Rio de Janeiro, Rua Dr. Francisco Portela 1470 – Patronato, São Gonçalo, RJ, 24435-005, Brazil b 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

A R T I C L E I N F O

A B S T R A C T

Keywords: Drugs of abuse Conditioned place preference Development Cholinergic system Dopamine

Early undernutrition causes long lasting alterations that affect the response to psychoactive drugs. Particularly, undernutrition during lactation affects the acute locomotor response to nicotine during adolescence, but the reward effect of continued exposure to nicotine remains unknown. The goal of this study was to investigate the effects of undernutrition during lactation on the nicotine susceptibility indexed via conditioned place preference (CPP), on dopamine content and turnover and on nicotine-induced nicotinic cholinergic receptor (nAChR) upregulation in the cerebral cortex, midbrain and hippocampus of adolescent mice. The impact of undernutrition and nicotine exposure on stress-related hormones and leptin was also investigated. From postnatal day 2 (PN2) to weaning (PN21), dams were randomly assigned to one of the following groups: Control (C) – free access to standard laboratory diet (23% protein); Protein Restricted (PR) – free access to isoenergenetic diet (8% protein); Calorie Restricted (CR) – access to standard laboratory diet in restricted quantities (mean ingestion of PR). PR and CR groups showed less mass gain and less visceral fat mass. While C and CR were equally susceptible to nicotine-induced place preference conditioning, PR failed to show a conditioning pattern. In contrast, all groups presented a nicotine-evoked nAChR upregulation in the cerebral cortex. While dopamine and DOPAC levels did not differ between groups, the DOPAC/dopamine ratio was increased in CR animals. No differences in endocrine parameters were observed. Taken together, our results indicate that undernutrition during lactation programs for brain alterations later in life. Our data also suggest that early undernutrition does not affect the rewarding associative properties of nicotine at adolescence.

1. Introduction Undernutrition during development remains an important public health problem in developing countries (FAO, 2013). This insult has negative health effects later in life, affecting among other features the brain response to psychoactive drugs (Almeida et al., 1996; DutraTavares et al., 2015; FAO, 2013; Valdomero et al., 2006; Valdomero et al., 2007). For instance, a case-control study on prenatal famine during the Dutch hunger winter of 1944-45 indicated a positive association between early malnutrition and propensity to addiction later in life (Franzek et al., 2008). Corroborating this idea, preclinical studies demonstrate an increased response of the mesocorticolimbic

⁎

Corresponding author. E-mail address: [email protected] (A. Ribeiro-Carvalho).

http://dx.doi.org/10.1016/j.ijdevneu.2017.10.007 Received 5 July 2017; Received in revised form 15 October 2017; Accepted 16 October 2017 Available online 18 October 2017 0736-5748/ © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

dopaminergic pathway to the rewarding effects of cocaine (Valdomero et al., 2006) and morphine (Valdomero et al., 2007) in early malnourished rats. In addition, perinatal undernutrition seems to facilitate morphine cross-sensitization to cocaine (Velazquez et al., 2010). The existence of animal models has been useful to understand the later consequences of early malnutrition. An interesting model of early malnutrition is the protein and/or caloric deprivation of the rodent dam during lactation. The first weeks of postnatal life encompass a transient period of intense brain growth characterized by neurogenesis, dendritic arborization, synaptogenesis and migration of numerous neuronal populations (Bandeira et al., 2009; Dobbing and Sands, 1979). Interestingly, it is also a period of vulnerability to nutrition insults, which


A.C. Dutra-Tavares et al.

generate several neurobiological disturbances that result in physiological consequences throughout life (Fraga et al., 2011; Hernandes and Almeida, 2003; Reyes-Castro et al., 2012). Animal models of undernutrition during lactation that investigated food intake and the hypothalamic leptin-signaling pathway later in life, further suggest that nutritional insults in this period promote changes in mechanisms of reward control (Lisboa et al., 2012; Passos et al., 2004). Nicotine, the most important psychoactive component of tobacco smoke, promotes synaptic alterations that affect the responsiveness of reward systems and generate addictive behavior (Mansvelder et al., 2002; Ribeiro-Carvalho et al., 2008; Zhao-Shea et al., 2011). Besides, the central cholinergic system, the primary target of nicotine, is susceptible to early undernutrition, which evokes, in rodents, long-lasting cholinergic alterations and inappropriate responses to cholinergic pharmacological treatments (Almeida et al., 1996; Fukuda et al., 2007; Nakagawasai et al., 2006). These findings raise the possibility that early undernutrition increases the reward properties of nicotine. However, to date, only one study investigated the influence of this insult on nicotine susceptibility. In this study, undernutrition during lactation failed to increase the locomotor response to a nicotine challenge during adolescence (Dutra-Tavares et al., 2015). Considering that continued exposure to drugs of abuse is known to evoke neuroplastic adaptations that play a role in mechanisms of reward and that are not identified after acute exposure (Ortells and Arias, 2010), in the current study, we investigated whether undernutrition during lactation increases the susceptibility of adolescent mice to reinforcing effects of repeated exposure to nicotine indexed via conditioned place preference (CPP). Nicotine exposure evokes a robust upregulation of nicotinic acetylcholine receptors (nAChRs) in rodents during adolescence (AbreuVillaca et al., 2003; Ribeiro-Carvalho et al., 2008), which is the period when humans usually begin to smoke (Prevention, 2010), and this effect is hypothesized to play a role in addiction mechanisms (Ortells and Arias, 2010). In addition, chronic nicotine generates neuroplastic alterations in dopamine reward systems that are fundamental to nicotine conditioned reward (Brunzell et al., 2009). In this sense, to further investigate the influence of early undernutrition on nicotine susceptibility, immediately after the CPP, we evaluated nAChRs binding, dopamine content and turnover. Finally, maternal food restriction affects the response to stress during the weaning period (Vieau et al., 2007) and it has been described that an altered response to stress influences the stimulatory response of nicotine in the mesolimbic dopaminergic system (Enrico et al., 2013). In fact, in a previous study we demonstrated that undernutrition during lactation of mice evokes adrenocorticotropic hormone (ACTH) and catecholamine hypersecretion during adolescence (DutraTavares et al., 2015). Considering that stress-related endocrine responses affect reward, we evaluated total catecholamine (adrenaline and noradrenaline) content in the adrenal medulla, ACTH and corticosterone serum levels. Leptin signaling has also been implicated in altered responses to drugs of abuse (Opland et al., 2010). Since early undernutrition decreases serum leptin levels during adolescence (DutraTavares et al., 2015), we assessed this hormone levels to investigate whether leptin is associated with alterations on nicotine reward processing.

TABLE 1 Comparisons between Control and Protein Restricted diets. Controla

Protein Restrictedb

Ingredients (g/kg) Soybean + wheat Cornstarch Soybean oil Vitamin mixc Mineral mixc

230.0 676.0 50.0 4.0 40.0

80.0 826.0 50.0 4.0 40.0

Macronutrient composition (%) Protein Carbohydrate Fat

23.0 66.0 11.0

8.0 81.0 11.0

Total energy (kJ/kg)

17138.7

17138.7

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.

2.1. Maternal undernutrition during lactation At the second postnatal day (PN2), a total of twenty seven dams were assigned to one of the following groups: 1) Control (C, 10 litters) – which had free access to a standard laboratory diet containing 23% protein; 2) Protein restricted (PR, 10 litters) – which had free access to an isoenergenetic protein restricted diet, containing only 8% protein (components description in Table 1); 3) Calorie restricted (CR, 7 litters) – which received standard laboratory diet in restricted quantities that corresponded to the mean ingestion of the PR dams in the previous day (pair feeding group). This last group was used due to the fact that mice dams submitted to protein restriction present a reduction in food intake when compared to control dams (Dutra-Tavares et al., 2015). Undernutrition extended from PN2 to weaning (PN21). The PR diet was prepared in our laboratory by replacing part of the protein in the standard rat chow with cornstarch. Thus, PR diet consisted of 8% protein, 81% carbohydrate and 11% fat, and it was isoenergetic compared with the control diet, containing 17138.7 kj/kg. The amount of starch was calculated in order to compensate the lower energy content resulting from the protein reduction. Diets included recommended amounts of vitamins and minerals for rodent (Reeves et al., 1993). The food in each batch was stored in freezer (–20 °C) until use. We only used litters that, at birth, had 8–12 pups. Litters with 11 or 12 pups were culled to 10 at PN1. Pups’ body mass and food intake data of the same litter were averaged within each group to minimize litter effects and avoid over-sampling. After weaning, mice were separated by sex and housed in groups of two or three animals. During the lactation period, the body mass of the dams and pups and food intake of the dams were monitored. After weaning, pups had free access to standard laboratory diet and their food intake and body mass were evaluated every third day until PN30. Pups’ body masses were also evaluated after the end of CPP period (PN39). From PN28 onwards, nicotine reward properties were assessed using the CPP method. Immediately after completing the CPP testing procedure, animals were decapitated and the brain, adrenal medulla, and blood were collected.

2. Materials and methods 2.2. Conditioned place preference (CPP) test

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. All Swiss mice were bred and maintained in our animal facility with controlled temperature (around 21 °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 adjoining the animal facility.

In this test, the animals are conditioned to associate the effects of the drug with the environment where the drug is administered. The CPP apparatus (Insight, SP, Brazil) consists of a box with three adjoining chambers: the “start box” is centrally located and painted gray; two doors can be used by the animals to access the laterally located chambers. One of the lateral chambers is painted with alternating black 46



and white horizontal stripes on the wall and a steel grid floor, and the other is painted with alternating black and white vertical stripes on the wall and a parallel steel bars floor. The nicotine CPP was conducted using a biased design. Briefly, starting at PN28, the animals received a once-daily i.p. injection of saline for two consecutive days (habituation period). At PN30, the animals received the i.p. injection of saline and were immediately placed in the neutral chamber to freely explore the apparatus for 15 min (pre-test). This pre-test was video-recorded and the time spent in each lateral chamber was used to determine the preferred and non-preferred ones. The conditioning period, which started at PN31, lasted 8 days. During this period, animals were submitted to 2 CPP sessions per day: the first between 8:00 a.m. and 12:00 p.m. and the second between 2:00 and 6:00 p.m. (separated by at least a 6-h interval). After each session, the apparatus was cleaned with paper towels soaked in 70% ethanol and dried. No more than one male and one female from each litter and each diet group (C, PR or CR) were randomly assigned into either Saline (SAL) or Nicotine (NIC) exposure subgroups. The nicotine-exposed animals (C-NIC, n = 19, PR-NIC, n = 14 and CR-NIC, n = 12) were administered nicotine (0.5 mg/kg, free base) once a day: in one session the animals received nicotine paired with the non-preferred side (biased design) and, in the other session, they received saline paired with the preferred side. The sessions sequence was alternated along the conditioning period. To avoid the interference of the time of the day, the schedule of injections was counterbalanced within the conditioning period so that half of the mice received nicotine and the other half received saline in the morning of the first conditioning trial. This sequence of events was reversed every day. The saline groups (C-SAL, n = 16; PR-SAL, n = 15 and CR-SAL, n = 11) received saline in both sessions/chambers. Immediately after each injection, mice were confined to the appropriate chamber for 15 min. On the ninth day (test day, PN39), no injections were administered. The animals were placed in the starting box and were allowed to freely explore the entire CPP apparatus for 15 min. This test was video-recorded and the time spent in each lateral chamber was quantified. In the CPP, a higher preference for the nicotine-associated chamber is considered to reflect the rewarding effects of the context associated with the drug (Natarajan et al., 2011).

(Bicinchoninic Acid kit). [3H]cytisine is a selective ligand that binds to the α4β2 nAChR, the predominant receptor subtype in the mammalian brain, and that is upregulated by nicotine (Abreu-Villaca et al., 2003; Nunes-Freitas et al., 2011; Ribeiro-Carvalho et al., 2008). Binding was determined using a final ligand concentration of 2 nM; specific binding was displaced with 100 μM nicotine. Values were expressed as binding (fmol) per mg of membrane protein. 2.5. DA and DOPAC evaluations Dopamine (DA) and 3,4 dihydroxyphenylacetic acid (DOPAC) content were analyzed in the right cerebral cortex by High-Performance Liquid Chromatography (HPLC) using a fluorescence detector (Shimadzu Prominence LC-20AT, RF-20A detector). Our methodology was adapted from Nohota et al. In summary, each cortex was thawed and homogenized with 200 μL of HClO4 (0.1 M) plus sodium ascorbate (20 μM) (Precellys, BERTIN Technologies, Montigny-le-Bretonneux, France) and centrifuged at 5200 × g for 30 min (4 °C). The supernatant was filtrated in a 0.22 μm PVDF filter (Millipore) and diluted in 4 vols of milli-Q water. DA and DOPAC derivatization reaction was accomplished using 10 μL standard solution or tissue sample + 20 μL of Glycine (2.5 M) + 10 μL NaIO4 (5 mM) + 20 μL of acetonitrile + 50 μL of derivatization solution (0.1 M 1,2-Diphenyl-ethylenediamine/0.1 M Glycine/62 mM sodium methoxide/3.75 mM potassium hexacyanoferrate III). After 3 min in ambient temperature, a 20 μL portion of the final reaction mixture was injected onto the chromatograph. The detector wavelengths of excitation/emission were 345 nm/ 480 nm. The mobile phase was a gradient formed by acetonitrile and acetate buffer (20 mM, pH = 4.5) with EDTA (0.5 mM). We used a 0.1 mL/min flow rate and ambient temperature (20–23 °C). A reversephase column (150 mm × 2.6 mm i.d., packed with C 18 silica, 3 μm) was used for separation. 2.6. Endocrine measurements Trunk blood was collected for the evaluation of total corticosterone, leptin and ACTH concentrations in serum and the adrenal medullas were dissected for total catecholamine (adrenaline and noradrenaline) content. The endocrine measurements have been described in details in a previous paper (Dutra-Tavares et al., 2015). Briefly, for serum hormone evaluations, trunk blood was centrifuged (1000 × g, 4 °C for 20 min) and the serum was stored at −20 °C. Total corticosterone and leptin levels were measured using a radioimmunoassay kit (for total corticosterone: ICN Biomedicals Inc., Cleveland, OH, USA; for Leptin: Linco Research Inc., St Charles, MO, USA) with a detection range from 25 to 1000 ng/mL or 0.5–50 ng/mL, respectively. 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. For the adrenal medulla catecholamine evaluation, we used the trihydroxyindole fluorescence method for total adrenaline and noradrenaline content (Trevenzoli et al., 2010). Right adrenal glands were homogenized in 200 μL 10% acetic acid and centrifuged (1120 × g, 5 min). Supernatant fraction (50 μL) was mixed with 250 μL 0.5 M buffer phosphate (pH 7.0) and 25 μL 0.5% potassium ferricyanate and incubated for 20 min in ice bath. Subsequently, 500 μL ascorbic acid 5 M NaOH (1:19) was used to stop the reaction. We used 420 nm to excitation and 510 nm to emission as parameters to the fluorometer (Plate Chameleon V, Hidex, Turku, Finland). Results were obtained by plotting the values into a linear regression of the standard adrenaline curve (expressed as μM of total catecholamines).

2.3. Brain dissection Mice were decapitated between 2:00 and 6:00 p.m., immediately after the behavioral test (at PN39). The brain regions were dissected by making blunt cuts through the cerebellar peduncles whereupon the cerebellum was lifted from the underlying tissue. The cerebral cortex was separated from the midbrain/brain stem by a cut made caudal to the thalamus. The midbrain was dissected from the hindbrain by making a cut caudal to the inferior colliculus. The hippocampus was removed from both left and right cerebral cortices. The brain regions were weighted, frozen in liquid nitrogen, and stored at −45 °C until assayed. 2.4. nAChRs binding We evaluated nAChRs binding in left cerebral cortex, midbrain and hippocampus. The nAChRs binding method has been described in detail in previous papers (Abreu-Villaca et al., 2016; Lima et al., 2013; NunesFreitas et al., 2011; Ribeiro-Carvalho et al., 2008; Ribeiro-Carvalho et al., 2009). Briefly, tissues were thawed and homogenized (UltraTurrax T10 basic, IKA, São Paulo, SP) in 40 vols of ice-cold 50 mM Tris–HCl (pH 7.4), the homogenates were sedimented at 40,000 × g for 10 min and the supernatant solution was discarded. The membrane pellet was resuspended (Ultra-Turrax) in the previous volume of buffer, resedimented, and the pellet was resuspended in 10 vols (based on the mass of the tissue) of the same buffer using a smooth glass homogenizer fitted with a Teflon pestle. Aliquots were withdrawn for measurements of [3H]cytisine binding and for membrane protein evaluation

2.7. Statistical analysis Repeated measures analyses of variance (rANOVA) were performed for body mass and food intake. Treatment (C, PR and CR) and Sex were used as between-subjects factors. Day was considered the within47



subjects factor. These data are shown as Supplementary material 1. Regarding the CPP, a univariate analysis of variance (uANOVA) was used to evaluate the Conditioning score (time spent in the nicotinepaired side minus time spent in the saline-paired side). A positive score indicates a preference for the drug-paired side. In addition, the time spent in the nicotine-paired side was taken as the variable in the rANOVA and Day (pre-test and test) was considered the within-subjects factor. Treatment, Exposure (nicotine or saline) and Sex were used as between-subjects factors. Separate uANOVA were performed for body mass at the last day of CPP (PN39), endocrine measures, nAChRs binding, DA and DOPAC evaluations, brain region mass and visceral fat mass. Treatment, Exposure 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 significant sex effects or interactions involving Sex were observed, thus figures represent the averaged values of males and females. 3. Results 3.1. Body mass, visceral fat mass and brain region mass As detailed in the Supplementary material 1, undernutrition reduced the body mass of both dams and pups during lactation. This effect persisted long after the end of the period of undernutrition, since PR and CR offspring still had less body mass when compared with the controls at PN39 (Treatment: F(2.82) = 14.7, P < 0.001, Supplementary 1, Table 1). Exposure to nicotine did not influence body mass. Undernutrition affected visceral fat mass (Treatment: F(2.82) = 6.3, P = 0.003, Table 2): Both PR and CR mice presented less visceral fat mass than controls (PR and CR < C, P < 0.05). In addition, undernutrition reduced total brain mass (F(2,120) = 10.8, P < 0.001), as well cerebral cortex (F(2,120) = 12.9, P < 0.001) and hippocampus (F(2,120) = 7.7, P < 0.001), but not midbrain mass (Table 2).

Fig. 1. Conditioning place preference (CPP): Effects of nicotine exposure during adolescence in mice malnourished during lactation. A, time spent in nicotine-paired side before and after conditioning; B, conditioning score (time spent on the nicotine-paired side minus time spent on the saline-paired side). C, Control; PR, Protein restricted; CR, calorie restricted. C, n = 19, PR, n = 14 and CR, n = 12. Values are means ± S.E.M. Statistical differences between groups were assessed by FPLSD; *P < 0.05; **P < 0.01.

3.2. CPP test

CR groups. There was no difference in the time spent in the non-preferred chamber between the pre-test and test of saline treated animals (Supplementary Fig. 1).

The time spent in the nicotine-paired side was affected by undernutrition during lactation (Treatment × day: F (2.42) = 3.2, df = 2, P = 0.05). This result was confirmed by the fact that only C and CR offspring spent more time in the nicotine-paired chamber after the conditioning sessions (P = 0.01 and P = 0.007 respectively, Fig. 1A). The analysis of the conditioning score corroborates this effect of undernutrition (Treatment: F2,42 = 4.4, P = 0.02). While both C and CR offspring presented a positive score, indicating a preference for the nicotine-paired side, the PR group did not present a conditioning pattern (Fig. 1B). No significant differences were observed between C and

3.3. nAChRs binding Nicotine-elicited nAChR upregulation was identified in the cerebral cortex (Exposure: F(1.98) = 9.76; P = 0.002, Fig. 2A), but this effect was not affected by early undernutrition. In the hippocampus and midbrain, nicotine exposure did not evoke nAChR upregulation and no differences among groups in nAChR binding were observed (Fig. 2B and C).

Table 2 Mean body mass, visceral fat mass, brain region mass of adolescent mice submitted to protein and/or calorie deprivation during lactation.

Body Massa Visceral fat massb Brain massc Cerebral cortex Hipoccampus Midbrain

C

PR

CR

27.2 ± 0.6 0.43 ± 0.02 635.4 ± 18.4 199.9 ± 2.0 36.5 ± 3.7 63.2 ± 1.3

24.0 ± 0.6*** 0.30 ± 0.02** 569.8 ± 5.7*** 185.8 ± 2.0*** 26.6 ± 0.6*** 62.1 ± 1.2

23.0 ± 0.9*** 0.32 ± 0.03* 576.4 ± 6.1*** 189.4 ± 2.0*** 26.1 ± 0.4*** 61.0 ± 1.1

3.4. Dopamine and DOPAC levels There were no significant differences among groups or effect of nicotine exposure for dopamine and DOPAC content in cerebral cortex. However, DOPAC:DA was greater in CR mice, suggesting that dams caloric restriction during lactation promotes an increase in dopamine turnover in CR offspring (Treatment: F2,58 = 3.0, P = 0.05; CR > C and CR > PR, P < 0.05; Fig. 3).

Data presented as means ± S.E.M. Body mass and visceral fat mass measurements at PN39. Measurements from the weaning (PN21) to PN30 were used to calculate mean values of food intake. *P < 0.05, **P < 0.01 and *P < 0.001 vs C; C, control group; PR, Protein restricted; CR, Calorie restricted. a Grams. b (g/100 g body mass). c Milligrams.

3.5. Endocrine evaluations As depicted in Fig. 4, no differences among groups were observed in the endocrine parameters: Neither diet during lactation nor nicotine exposure at early adolescence affected these measures. 48



Fig. 2. nAChRs binding: Effects of nicotine exposure during adolescence in cerebral cortex (A), hippocampus (B) and midbrain (C) in mice malnourished during lactation. In the insert, data are separated by Treatment (C, PR and CR) and Exposure (SAL and NIC). C, Control; PR, Protein restricted; CR, calorie restricted; SAL, saline; NIC, nicotine. C-SAL: n = 13–16; C-NIC: n = 14–17; PR-SAL: n = 14–17; PR-NIC: n = 13–15; CR-SAL: n = 14–17; CR-NIC: n = 15–16. Values are means ± S.E.M.

4. Discussion

evaluated here were observed in any experimental groups. Overall, these results suggest that maternal undernutrition during lactation does not increase reward properties of nicotine in adolescent mice. The ensuing paragraphs elaborate this essential conclusion.

Increased responsiveness to psychoactive substances has been observed in experimental models of deficient nutritional status during early life (Valdomero et al., 2006; Velazquez et al., 2010). Here, while C and CR offspring was equally susceptible to nicotine-induced place preference conditioning, PR offspring was not affected by nicotine. Early undernutrition failed to alter nicotine-evoked nAChR upregulation in cerebral cortex. Regarding DA and DOPAC levels, there were no differences between groups, however, the DOPAC/DA ratio was higher in CR animals. In addition, no differences in endocrine parameters

4.1. Effects of undernutrition during lactation on body mass, visceral fat mass, food intake and region brain mass Both PR and CR offspring presented a reduction in body mass gain, resulting in lighter animals when compared to controls during adolescence. In addition, the reduced visceral fat mass in PR and CR mice 49



initially described by Morgan and Naismith (1982) and Katz and Davies (1982) (Katz and Davies, 1982; Morgan and Naismith, 1982). Poor formation of neuronal circuits and reduction on neurogenesis promoted by undernutrition could explain the reduction in brain mass observed here (for review about the effects of early undernutrition on brain development see, Alamy and Bengelloun, 2012). For example, undernutrition during gestation and lactation increase the number of apoptotic cells (Jordan and Clark, 1983), decrease neuronal cell density in the hippocampus and reduces hippocampal volume (Ranade et al., 2008). Cordero et al. (2003) also demonstrated that undernutrition during lactation alters neuronal circuits, affecting dendritic morphology and orientation of neocortical pyramidal cells (Cordero et al., 2003). The midbrain mass was not affected by early undernutrition in the present study, which could be due to the fact that the cellular formation in the brain stem is established early during development. In this regard, neurons of midbrain nuclei are generated in two waves, on gestational days l2 and 17 in rodents (Altman and Bayer, 1981). 4.2. Effects of early undernutrition on nicotine susceptibility Both C and CR offspring displayed a similar increase in the time spent in the nicotine-paired side of the CPP apparatus, thus showing that nicotine was capable of inducing a reward-seeking behavior. Distinctively, PR animals displayed no such preference indicating that a diet with adequate protein content during lactation is critical to the conditioning effects of nicotine in the CPP during adolescence. In a previous study, we observed that acute administration of nicotine elicited locomotor hyperactivity in PR, but not in CR adolescent mice (Dutra-Tavares et al., 2015). This discrepancy is intriguing since nicotine-induced dopamine release in the mesocorticolimbic pathway mediates both reward learning that generates CPP and locomotor hyperactivity (Museo and Wise, 1994a, 1994b). Despite that, several mechanisms could modulate this dopaminergic activity. For example, Mineur et al. (2009) demonstrated that β2 subunit-containing nAChRs in the ventral tegmental area (VTA) neurons mediate nicotine-induced locomotor activation but do not support nicotine reward in CPP test (Mineur et al., 2009). Here, we failed to observe significant effects of protein or caloric undernutrition on α4β2 nAChRs binding, however, we cannot exclude the possibility that other *β2 subunit-containing nAChRs might explain the discrepancy between nicotine induced CPP and locomotor hyperactivity. It is also possible that the lack of conditioning in PR animals is not restricted to impairments in the rewarding system, but could reflect a cognitive deficit caused by early protein restriction. Accordingly, several studies show that early postnatal undernutrition impairs learning and memory (for review (Laus et al., 2011)). However, a set of studies on the influence of protein undernutrition in the rewarding properties of cocaine and morphine demonstrated that a more prolonged deprivation schedule (from the 14th gestational day to 40 days old) is capable of inducing place conditioning in adult rats, suggesting that protein restriction per se was not able to block the place conditioning effect caused by these drugs (Valdomero et al., 2006; Valdomero et al., 2007). In addition, these experiments indicated a higher rewarding effect of cocaine and morphine in PR animals. It is possible to speculate that developmental malnutrition sensitizes catecholamine (cocaine) and opiate (morphine) pathways, but does not increase the cholinergic response to nicotine in brain reward systems. However, another possible explanation is based on the fact that, in cocaine and morphine studies, the malnutrition period initiated during gestation. The lack of effects in the present study could indicate that prenatal life in rodents is a critical period to program for increased susceptibility to drugs of abuse. Nicotine addiction is a complex behavioral phenomenon in which various factors are involved. Among these, the nicotine-induced upregulation of nAChRs described to occur in both smokers and animal models of nicotine and tobacco smoke exposure (Abreu-Villaca et al., 2016; Abreu-Villaca et al., 2003; Ribeiro-Carvalho et al., 2008; Ribeiro-

Fig. 3. Dopamine and DOPAC levels: Effects of undernutrition during lactation in cerebral cortex of adolescent mice. A, dopamine levels; B, DOPAC levels; C, dopamine turnover (DOPAC:dopamine). C, Control; PR, Protein restricted; CR, calorie restricted. C: n = 22; PR: n = 22; CR: n = 22. Values are means ± S.E.M. Statistical differences between groups were assessed by FPLSD; *P < 0.05.

demonstrate that our procedure was efficient in eliciting undernutrition and corroborates previous findings that show reduced body mass and body fat mass in mice pups whose dams were submitted to protein or caloric restriction during lactation (Dutra-Tavares et al., 2015). Both PR and CR did not present an increase in food intake after the undernutrition period, suggesting no compensatory increase on food consumption. Our data shows that undernutrition during lactation was able to reduce the brain mass. In fact, the brain seems to be vulnerable to insults during the lactation period of rodents, mostly during the first 14 days of post-natal life (Filgueiras et al., 2013; Filgueiras et al., 2009). During this period, the brain rapidly increases in mass, a called “brain growth spurt” phase, during which intense neurogenesis and synaptogenesis takes place (Bandeira et al., 2009; Dobbing and Sands, 1979). It was expected that caloric/protein deprivation during this period would affect cell proliferation and disturb synaptic structure and function, as 50



Fig. 4. Endocrine parameters after conditioning place preference (CPP): Effects of undernutrition during lactation on adolescent mice. A, serum ACTH levels; B, serum corticosterone levels; C, catecholamine total content in the adrenal gland; D, serum leptin levels. C, Control; PR, Protein restricted; CR, calorie restricted. C: n = 12; PR: n = 12; CR: n = 12. Values are means ± S.E.M.

undernutrition are not persistent. This discrepancy may also be associated with the fact that while Dutra-Tavares et al. (2015) evaluated the endocrine response to stress 10-min after a single open field session, in the present study; it was evaluated after 18 sessions in the CPP chamber. Some studies have demonstrated that the intensity of hormonal, physiological, and behavioral responses to a repeated stressful stimulus often wanes over time (Cyr and Romero, 2009; Grissom and Bhatnagar, 2009). This waning is generally associated with a phenomenon of habituation, which is characterized by a reduction in the response to stress as a novel stressor becomes familiar with repetition (Cyr and Romero, 2009). Accordingly, the absence of differences between undernourished groups and controls regarding hormonal stress mediators may reflect the fact that mice no longer perceived the stimulus to be stressful. Alternatively, chronic stress may lead to a desensitization of stress-related physiological responses or even the saturation of the neuro-endocrine system (Cyr and Romero, 2009). It has been recognized that leptin modulates the response to food and non-food rewards (Fulton et al., 2006; Opland et al., 2010). In our previous study, we observed that protein restriction during lactation reduces serum leptin concentrations at PN30, which was associated with a reduction in body fat mass (Dutra-Tavares et al., 2015). Since the CPP protocol began on PN28, this effect could have contributed for PR animals failure to present a nicotine-induced place conditioning. However, the reduction in serum leptin levels was not observed after CPP test at PN39, suggesting that this effect was attenuated during subsequent development.

Carvalho et al., 2009; Teaktong et al., 2004) could play a major role in nicotine addiction (Govind et al., 2009; Ortells and Arias, 2010; Picciotto et al., 2008). Here, we observed that daily nicotine injection for 8 days was able to induce α4β2 nAChR upregulation in the cerebral cortex, but not in the midbrain and hippocampus. This effect occurred not only in controls but also in undernourished nicotine-exposed groups, indicating that malnutrition during lactation did not affect the capability of nicotine to induce nAChR upregulation. In addition, the absence of conditioning in the PR group suggests that the upregulation per se is not sufficient to elicit nicotine CPP. While α4β2 nAChRs are fundamental for nicotine reward, self-administration and behavioral sensitization, it is now known that other nAChRs subtypes also play a role in nicotine addiction (Abreu-Villaça et al., 2017). Thus, the effects of early undernutrition on the expression and function of other subtypes is an important concern to be addressed in future studies. Early malnutrition affects dopamine function in the brain (Alamy et al., 2005; Manuel-Apolinar et al., 2014). Prenatal undernutrition significantly decreased basal extracellular dopamine in the medial prefrontal cortex of rats (Mokler et al., 2007). In the present study, undernutrition during lactation did not affect dopamine levels in cerebral cortex, suggesting that lactation is no longer a critical period to malnutrition-induced reduction in dopamine levels. Despite the absence of differences in dopamine levels, we observed an increase in dopamine turnover in CR animals that could indicate basal dopaminergic hyperactivity. This effect could represent a compensatory response to caloric restriction, which fails to occur due to protein deprivation and, in this sense, contributes to explain the differences between PR and CR groups in their capability to develop nicotine-induced CCP. Accordingly, dopaminergic hyperactivity seems to predispose the development of drug addiction (Marinelli et al., 2006; McCutcheon et al., 2012).

5. Conclusions Current findings support the idea that undernutrition during lactation generates alterations later in life and suggest that the diet type (caloric or protein restriction) determines differential phenotype profiles. Despite the fact that early undernutrition did not affect nicotineinduced upregulation in cerebral cortex, protein restriction during lactation impeded nicotine conditioning in the CPP. In addition, our results indicate that caloric restriction, but not protein restriction, during lactation results in an increase in dopamine turnover in the cerebral cortex. Even though early undernutrition enhances the reinforcing properties of other drugs (Valdomero et al., 2006, 2007), under the parameters of the present study, our results suggest that this is not true for nicotine during adolescence.

4.3. Endocrine measures associated with stress response and serum leptin concentration in malnourished animals Maternal food restriction reduces hypothalamic pituitary adrenal axis activity in response to stress during the weaning period in rats (Vieau et al., 2007). In mice, we observed that both protein and caloric restriction during lactation period promote a hypersecretion pattern of ACTH and catecholamine after exposure to a new environment at PN30 (Dutra-Tavares et al., 2015). However, in the present study no alterations were observed in endocrine measures associated with stress response at PN39, indicating that these stress-related effects of 51



Neuron 51, 811–822. Govind, A.P., Vezina, P., Green, W.N., 2009. Nicotine-induced upregulation of nicotinic receptors: underlying mechanisms and relevance to nicotine addiction. Biochem. Pharmacol. 78, 756–765. Grissom, N., Bhatnagar, S., 2009. Habituation to repeated stress: get used to it. Neurobiol. Learn. Mem. 92, 215–224. Hernandes, A.S., Almeida, S.S., 2003. Postnatal protein malnutrition affects inhibitory avoidance and risk assessment behaviors in two models of anxiety in rats. Nutr. Neurosci. 6, 213–219. Jordan, T.C., Clark, G.A., 1983. Early undernutrition impairs hippocampal long-term potentiation in adult rats. Behav. Neurosci. 97, 319–322. Katz, H.B., Davies, C.A., 1982. The effects of early-life undernutrition and subsequent environment on morphological parameters of the rat brain. Behav. Brain Res. 5, 53–64. Laus, M.F., Vales, L.D., Costa, T.M., Almeida, S.S., 2011. Early postnatal protein-calorie malnutrition and cognition: a review of human and animal studies. Int. J. Environ. Res. Public Health 8, 590–612. Lima, C.S., Dutra-Tavares, A.C., Nunes, F., Nunes-Freitas, A.L., Ribeiro-Carvalho, A., Filgueiras, C.C., Manhães, A.C., Meyer, A., Abreu-Villaça, Y., 2013. Methamidophos exposure during the early postnatal period of mice: immediate and late-emergent effects on the cholinergic and serotonergic systems and behavior. Toxicol. Sci. 134, 125–139. Lisboa, P.C., Oliveira, E., Fagundes, A.T., Santos-Silva, A.P., Conceicao, E.P., Passos, M.C., Moura, E.G., 2012. Postnatal low protein diet programs leptin signaling in the hypothalamic-pituitary-thyroid axis and pituitary TSH response to leptin in adult male rats. Horm. Metab. Res. 44, 114–122. Mansvelder, H.D., Keath, J.R., McGehee, D.S., 2002. Synaptic mechanisms underlie nicotine-induced excitability of brain reward areas. Neuron 33, 905–919. Manuel-Apolinar, L., Rocha, L., Damasio, L., Tesoro-Cruz, E., Zarate, A., 2014. Role of prenatal undernutrition in the expression of serotonin, dopamine and leptin receptors in adult mice: implications of food intake. Mol. Med. Rep. 9, 407–412. Marinelli, M., Rudick, C.N., Hu, X.T., White, F.J., 2006. Excitability of dopamine neurons: modulation and physiological consequences. CNS Neurol. Disord. Drug Targets 5, 79–97. McCutcheon, J.E., Conrad, K.L., Carr, S.B., Ford, K.A., McGehee, D.S., Marinelli, M., 2012. Dopamine neurons in the ventral tegmental area fire faster in adolescent rats than in adults. J. Neurophysiol. 108, 1620–1630. Mineur, Y.S., Brunzell, D.H., Grady, S.R., Lindstrom, J.M., McIntosh, J.M., Marks, M.J., King, S.L., Picciotto, M.R., 2009. Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning. Genes Brain Behav. 8, 257–266. Mokler, D.J., Torres, O.I., Galler, J.R., Morgane, P.J., 2007. Stress-induced changes in extracellular dopamine and serotonin in the medial prefrontal cortex and dorsal hippocampus of prenatally malnourished rats. Brain Res. 1148, 226–233. Morgan, B.L., Naismith, D.J., 1982. The effect of early postnatal undernutrition on the growth and development of the rat brain. Br. J. Nutr. 48, 15–23. Museo, E., Wise, R.A., 1994a. Place preference conditioning with ventral tegmental injections of cytisine. Life Sci. 55, 1179–1186. Museo, E., Wise, R.A., 1994b. Sensitization of locomotion following repeated ventral tegmental injections of cytisine. Pharmacol. Biochem. Behav. 48, 521–524. Nakagawasai, O., Yamadera, F., Sato, S., Taniguchi, R., Hiraga, H., Arai, Y., Murakami, H., Mawatari, K., Niijima, F., Tan-no, K., Tadano, T., 2006. Alterations in cognitive function in prepubertal mice with protein malnutrition: relationship to changes in choline acetyltransferase. Behav. Brain Res. 167, 111–117. Natarajan, R., Wright, J.W., Harding, J.W., 2011. Nicotine-induced conditioned place preference in adolescent rats. Pharmacol. Biochem. Behav. 99, 519–523. Nunes-Freitas, A.L., Ribeiro-Carvalho, A., Lima, C.S., Dutra-Tavares, A.C., Manhães, A.C., Lisboa, P.C., Oliveira, E., Gaspar de, M.E., Filgueiras, C.C., Abreu-Villaça, Y., 2011. Nicotine exposure during the third trimester equivalent of human gestation: time course of effects on the central cholinergic system of rats. Toxicol. Sci. 123, 144–154. Opland, D.M., Leinninger, G.M., Myers Jr., M.G., 2010. Modulation of the mesolimbic dopamine system by leptin. Brain Res. 1350, 65–70. Ortells, M.O., Arias, H.R., 2010. Neuronal networks of nicotine addiction. Int. J. Biochem. Cell Biol. 42, 1931–1935. Passos, M.C., Vicente, L.L., Lisboa, P.C., de Moura, E.G., 2004. Absence of anorectic effect to acute peripheral leptin treatment in adult rats whose mothers were malnourished during lactation. Horm. Metab. Res. 36, 625–629. Picciotto, M.R., Addy, N.A., Mineur, Y.S., Brunzell, D.H., 2008. It is not either/or: activation and desensitization of nicotinic acetylcholine receptors both contribute to behaviors related to nicotine addiction and mood. Prog. Neurobiol. 84, 329–342. Prevention, C.f.D.C., 2010. Tobacco use among middle and high school students: United States 2000–2009. Morbidity and Mortality Weekly Report, vol. 59. pp. 1063–1068. Ranade, S.C., Rose, A., Rao, M., Gallego, J., Gressens, P., Mani, S., 2008. Different types of nutritional deficiencies affect different domains of spatial memory function checked in a radial arm maze. Neuroscience 152, 859–866. Reeves, P.G., Nielsen, F.H., Fahey Jr., G.C., 1993. AIN-93 purified diets for laboratory rodents: final report of the American institute of nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939–1951. Reyes-Castro, L.A., Rodriguez, J.S., Rodriguez-Gonzalez, G.L., Chavira, R., Bautista, C.J., McDonald, T.J., Nathanielsz, P.W., Zambrano, E., 2012. Pre- and/or postnatal protein restriction developmentally programs affect and risk assessment behaviors in adult male rats. Behav. Brain Res. 227, 324–329. Ribeiro-Carvalho, A., Lima, C.S., Filgueiras, C.C., Manhães, A.C., Abreu-Villaça, Y., 2008. Nicotine and ethanol interact during adolescence: effects on the central cholinergic systems. Brain Res. 1232, 48–60. Ribeiro-Carvalho, A., Lima, C.S., Medeiros, A.H., Siqueira, N.R., Filgueiras, C.C.,

Acknowlegments This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and fellowships by Sub-reitoria de Pós-graduação e Pesquisa da Universidade do Estado do Rio de Janeiro (SR2-UERJ), Coordenação de Aperfeiçoamento 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.2017.10.007. References Abreu-Villaça, Y., Manhães, A.C., Krahe, T.E., Filgueiras, C.C., Ribeiro-Carvalho, A., 2017. Tobacco and alcohol use during adolescence: interactive mechanisms in animal models. Biochem. Pharmacol. 144, 1–17. Abreu-Villaca, Y., Seidler, F.J., Qiao, D., Tate, C.A., Cousins, M.M., Thillai, I., Slotkin, T.A., 2003. Short-term adolescent nicotine exposure has immediate and persistent effects on cholinergic systems: critical periods, patterns of exposure, dose thresholds. Neuropsychopharmacology 28, 1935–1949. Abreu-Villaca, Y., Correa-Santos, M., Dutra-Tavares, A.C., Paes-Branco, D., Nunes-Freitas, A., Manhaes, A.C., Filgueiras, C.C., Ribeiro-Carvalho, A., 2016. A ten fold reduction of nicotine yield in tobacco smoke does not spare the central cholinergic system in adolescent mice. Int. J. Dev. Neurosci. 52, 93–103. Alamy, M., Bengelloun, W.A., 2012. Malnutrition and brain development: an analysis of the effects of inadequate diet during different stages of life in rat. Neurosci. Biobehav. Rev. 36, 1463–1480. Alamy, M., Errami, M., Taghzouti, K., Saddiki-Traki, F., Bengelloun, W.A., 2005. Effects of postweaning undernutrition on exploratory behavior, memory and sensory reactivity in rats: implication of the dopaminergic system. Physiol. Behav. 86, 195–202. Almeida, S.S., Tonkiss, J., Galler, J.R., 1996. Malnutrition and reactivity to drugs acting in the central nervous system. Neurosci. Biobehav. Rev. 20, 389–402. Altman, J., Bayer, S.A., 1981. Development of the brain stem in the rat: v. thymidineradiographic study of the time of origin of neurons in the midbrain tegmentum. J. Comp. Neurol. 198, 677–716. Bandeira, F., Lent, R., Herculano-Houzel, S., 2009. Changing numbers of neuronal and non-neuronal cells underlie postnatal brain growth in the rat. Proc. Natl. Acad. Sci. U. S. A. 106, 14108–14113. Brunzell, D.H., Mineur, Y.S., Neve, R.L., Picciotto, M.R., 2009. Nucleus accumbens CREB activity is necessary for nicotine conditioned place preference. Neuropsychopharmacology 34, 1993–2001. Cordero, M.E., Valenzuela, C.Y., Rodriguez, A., Aboitiz, F., 2003. Dendritic morphology and orientation of pyramidal cells of the neocortex in two groups of early postnatal undernourished-rehabilitated rats. Brain Res. Dev. Brain Res. 142, 37–45. Cyr, N.E., Romero, L.M., 2009. Identifying hormonal habituation in field studies of stress. Gen. Comp. Endocrinol. 161, 295–303. Dobbing, J., Sands, J., 1979. Comparative aspects of the brain growth spurt. Early Hum. Dev. 3, 79–83. Dutra-Tavares, A.C., Manhaes, A.C., Silva, J.O., Nunes-Freitas, A.L., Conceicao, E.P., Moura, E.G., Lisboa, P.C., Filgueiras, C.C., Abreu-Villaca, Y., Ribeiro-Carvalho, A., 2015. Locomotor response to acute nicotine in adolescent mice is altered by maternal undernutrition during lactation. Int. J. Dev. Neurosci. 47, 278–285. Enrico, P., Sirca, D., Mereu, M., Peana, A.T., Mercante, B., Diana, M., 2013. Acute restraint stress prevents nicotine-induced mesolimbic dopaminergic activation via a corticosterone-mediated mechanism: a microdialysis study in the rat. Drug Alcohol Depend. 127, 8–14. FAO IFAD WFP, 2013. The state of food insecurity in the word. The Mutiple Dimensions of Food Security. FAO, IFAD, WFP, Rome. Filgueiras, C.C., Ribeiro-Carvalho, A., Nunes, F., Abreu-Villaça, Y., Manhães, A.C., 2009. Early ethanol exposure in mice increases laterality of rotational side preference in the free-swimming test. Pharmacol. Biochem. Behav. 93, 148–154. Filgueiras, C.C., Pohl-Guimaraes, F., Krahe, T.E., Medina, A.E., 2013. Sodium valproate exposure during the brain growth spurt transiently impairs spatial learning in prepubertal rats. Pharmacol. Biochem. Behav. 103, 684–691. Fraga, M.C., Moura, E.G., Silva, J.O., Bonomo, I.T., Filgueiras, C.C., Abreu-Villaca, Y., Passos, M.C., Lisboa, P.C., Manhaes, A.C., 2011. Maternal prolactin inhibition at the end of lactation affects learning/memory and anxiety-like behaviors but not noveltyseeking in adult rat progeny. Pharmacol. Biochem. Behav. 100, 165–173. Franzek, E.J., Sprangers, N., Janssens, A.C., Van Duijn, C.M., Van De Wetering, B.J., 2008. Prenatal exposure to the 1944–45 Dutch ‘hunger winter’ and addiction later in life. Addiction 103, 433–438. Fukuda, M.T., Francolin-Silva, A.L., Hernandes, A.S., Valadares, C.T., Almeida, S.S., 2007. Effects of early protein malnutrition and scopolamine on learning and memory in the Morris water maze. Nutr. Neurosci. 10, 251–259. Fulton, S., Pissios, P., Manchon, R.P., Stiles, L., Frank, L., Pothos, E.N., Maratos-Flier, E., Flier, J.S., 2006. Leptin regulation of the mesoaccumbens dopamine pathway.

52



2007. Increased rewarding properties of morphine in perinatally protein-malnourished rats. Neuroscience 150, 449–458. Velazquez, E.E., Valdomero, A., Orsingher, O.A., Cuadra, G.R., 2010. Perinatal undernutrition facilitates morphine sensitization and cross-sensitization to cocaine in adult rats: a behavioral and neurochemical study. Neuroscience 165, 475–484. Vieau, D., Sebaai, N., Leonhardt, M., Dutriez-Casteloot, I., Molendi-Coste, O., Laborie, C., Breton, C., Deloof, S., Lesage, J., 2007. HPA axis programming by maternal undernutrition in the male rat offspring. Psychoneuroendocrinology 32 (Suppl. 1), S16–S20. Zhao-Shea, R., Liu, L., Soll, L.G., Improgo, M.R., Meyers, E.E., McIntosh, J.M., Grady, S.R., Marks, M.J., Gardner, P.D., Tapper, A.R., 2011. Nicotine-mediated activation of dopaminergic neurons in distinct regions of the ventral tegmental area. Neuropsychopharmacology 36, 1021–1032.

Manhães, A.C., Abreu-Villaça, Y., 2009. Combined exposure to nicotine and ethanol in adolescent mice: effects on the central cholinergic systems during short and long term withdrawal. Neuroscience 162, 1174–1186. Teaktong, T., Graham, A.J., Johnson, M., Court, J.A., Perry, E.K., 2004. Selective changes in nicotinic acetylcholine receptor subtypes related to tobacco smoking: an immunohistochemical study. Neuropathol. Appl. Neurobiol. 30, 243–254. Trevenzoli, I.H., Pinheiro, C.R., Conceicao, E.P., Oliveira, E., Passos, M.C., Lisboa, P.C., Moura, E.G., 2010. Programming of rat adrenal medulla by neonatal hyperleptinemia: adrenal morphology, catecholamine secretion, and leptin signaling pathway. Am. J. Physiol. Endocrinol. Metab. 298, E941–E949. Valdomero, A., Bussolino, D.F., Orsingher, O.A., Cuadra, G.R., 2006. Perinatal protein malnutrition enhances rewarding cocaine properties in adult rats. Neuroscience 137, 221–229. Valdomero, A., Velazquez, E.E., de, O.S., de Olmos, J.S., Orsingher, O.A., Cuadra, G.R.,

53