Behavioural Processes 103 (2014) 297–305

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Tactile stimulation and neonatal isolation affect behavior and oxidative status linked to cocaine administration in young rats Caren T.D. Antoniazzi a , Nardeli Boufleur a , Camila S. Pase a , Fábio T. Kuhn a , Verônica T. Dias b , Hecson J. Segat b , Karine Roversi b , Katiane Roversi b , Dalila M. Benvegnú a , Marilise E. Bürger a,b,∗ a Programa de Pós Graduac¸ão em Farmacologia, Universidade Federal de Santa Maria, Av. Roraima, 1000, Prédio 21, Cidade Universitária, CEP 97105-900 Santa Maria, RS, Brazil b Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, Av. Roraima, 1000, Prédio 21, Cidade Universitária, CEP 97105-900 Santa Maria, RS, Brazil

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Article history: Received 1 August 2013 Received in revised form 15 January 2014 Accepted 15 January 2014 Available online 24 January 2014 Keywords: Tactile stimulation Conditioned place preference Anxiety-like behavior Oxidative status Cocaine

a b s t r a c t We investigated the influence of neonatal handling on cocaine-induced conditioned place preference (CPP), anxiety-like symptoms and oxidative status related to drug abstinence in young rats. Pups were submitted to tactile stimulation (TS) or neonatal isolation (NI10 or NI60 ) after birth, and then were submitted to CPP performed with cocaine. TS group did not show place preference, while unhandled (UH), NI10 and NI60 rats did. Handling was related to anxiety-like symptoms per se in UH and NI60 groups and this behavior was also observed in the cocaine-conditioned rats exposed to the same handlings. Both TS and NI10 pups treated or not with cocaine showed less anxiety-like behavior than animals submitted to other handlings. TS reduced protein carbonyl (PC) in cortex and NI60 increased PC in both striatum and hippocampus of cocaine-treated rats. Among cocaine-treated rats, both times of NI increased plasma lipoperoxidation levels, which was reduced by TS in erythrocytes. TS increased the catalase activity in brain areas, while other handlings did not change this. Both TS and NI10 increased plasma vitamin C levels. These findings indicate that neonatal handling can modify anxiety-like symptoms related to cocaine preference and abstinence, and its protective influence, especially TS, on the antioxidant system. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Cocaine is a psychostimulant drug commonly related to abuse, and its mechanism of action involves inhibition of neuronal monoamines re-uptake, especially dopamine (DA) (Hadfield and Nuggent, 1983; Izenwasser, 2004). Repeated use of cocaine is related to compulsion, despite adverse consequences, and a high prevalence of relapse following its withdrawal (A.P. Association, 1994).

∗ Corresponding author at: Departamento de Fisiologia e Farmacologia-CCS, Programa de Pós-Graduac¸ão em Farmacologia-CCS, Programa de Pós-Graduac¸ão em Bioquímica Toxicológica-CCNE, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. Tel.: +55 55 3220 8676; fax: +55 55 3220 8676. E-mail addresses: [email protected] (C.T.D. Antoniazzi), [email protected] (N. Boufleur), [email protected] (C.S. Pase), [email protected] (F.T. Kuhn), vel [email protected] (V.T. Dias), hecson [email protected] (H.J. Segat), karineroversi @hotmail.com (K. Roversi), [email protected] (K. Roversi), [email protected] (D.M. Benvegnú), [email protected], [email protected] (M.E. Bürger). http://dx.doi.org/10.1016/j.beproc.2014.01.011 0376-6357/© 2014 Elsevier B.V. All rights reserved.

Cocaine rewarding effects have been evaluated through conditioned place preference (CPP) (Bardo et al., 1995; Hoffman, 1989), an experimental model used for cues-elicited conditioning. This paradigm is based in the ability to find cues associated with drug to elicit place preference, which has important implications for drug “craving” and relapses in humans (Hand et al., 1989; Neisewander et al., 1990). Human studies have shown a relationship between vulnerability to drug abuse and adverse life events and/or stress exposure (Dube et al., 2003; Sinha, 2001). Early life experiences exert longlasting effects on behavior and stress reactivity (Kabbaj et al., 2002; Padoin et al., 2001) and may increase propensity to use drugs such as cocaine (Schenk et al., 1987), especially during adolescence (Casey et al., 2008; Izenwasser, 2005, for review; Spear, 2000). Because of the severity of this problem, CPP and others animal models have been used to study many of the psychobiological and social factors of the transition from infancy into adulthood, which in animal models is defined as the developmental period after weaning and before adulthood (Laviola et al., 2003).

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Adolescent rats present different neurochemical responsiveness to stress as compared to adults (Kellogg et al., 1998) and much greater behavioral effects following psychostimulant consumption than infant rats (Cirulli and Laviola, 2000). Animal models of early life stress have been developed to elucidate the consequent neurochemical and behavioral changes resulting from these stressors (Pryce and Feldon, 2003), including maternal separation (MS) and neonatal isolation (NI). In this sense, longer periods of MS and/or NI have been related to interruption of dam-pup interaction, thus affecting the development of the central nervous system (CNS) (Kalinichev et al., 2002; Knuth and Etgen, 2007; Kosten et al., 2004). Fundamentally, in the NI protocol, pups are separated individually from their dam, whereas in MS, the litter as a whole is separated from the dam. Indeed, prolonged separation from the dam during the neonatal period is considered one of the most powerful stressors to which rat pups can be exposed (Rosenfeld et al., 1992; Wigger and Neumann, 1999). These neonatal handlings were related to disruption of the adrenal response maturation to stress (Kuhn and Schanberg, 1998), showing the importance of early life social relationships on neurobiological systems. Of particular importance, neonatal stress is able to affect brain neurotransmitter systems such as dopamine and serotonin (Brake et al., 2004; Vicentic et al., 2006) and to promote dysfunctions in endogenous opioid system, suggesting its involvement in brain reward patterns in drug abuse in animals. Contrarily to stress caused by MS and/or NI, tactile stimulation (TS) has been related to accelerated maturation of cortical neurons with favorable consequences to behavioral and physiological changes that persist into adulthood (Pham et al., 1999; Silveira et al., 2005). Neonatal TS consists of external sensory stimuli that exerts influence on neural pathways from the skin to the CNS (Montagu, 1953) associated to accelerated maturation of cortical pyramidal neurons (Schapiro and Vukovich, 1970). Its experimental application has been linked to increased neurogenesis, prevention of hippocampal neuronal loss induced by stress or aging (Lemaire et al., 2006), suggesting its effectiveness to influence brain functions. Neonatal TS was related to prevention of neural stress markers alteration induced by maternal deprivation (van Oers et al., 1998), demonstrating also less emotionality in stressful situations (Levine and Otis, 1958). In fact, neonatal TS is used in animals to test the effects of external stimuli on the newborn’s life in a variety of behaviors and neuroendocrine systems (Padoin et al., 2001; Raineki et al., 2009; Todeschin et al., 2009) and is very similar to therapy through massage, used in humans (Field, 1998). In fact, the neuroendocrine system can be activated by repeated exposure to stress, causing an increase in levels of circulating hormones. When the trigger of this activation remains for a long time, the damage begins to occur mainly on the nervous system. Such deleterious effects have been associated with increased generation of reactive oxygen species (ROS), which when in excess can cause oxidative damage to various structures, affecting their function (Cochrane, 1991; McIntosh and Sapolsky, 1996). In the same oxidative scenario, cocaine is able to inhibit the activity of the presynaptic transporter, thereby reducing the reuptake of dopamine, which is vulnerable to autoxidation and consequently to generation of pro-oxidant metabolites (Boess et al., 2000). In this sense, oxidative stress occurs when there is an imbalance between the enzymatic antioxidant defenses and oxidative species (Gutteridge and Halliwell, 2000; Halliwell and Gutteridge, 2007), whose influence is able to compromise the physiologic functions of the different organs and tissues. Considering that NI is an animal model of early life stress related to neurochemical and behavioral changes, which may facilitate preference for drugs such as cocaine (Kosten et al., 2004; Marquardt et al., 2004), and that TS has been related to behavioral benefits and less emotionality in coping response to traumatic situations,

the present study was designed to evaluate the possible influence of distinct forms of neonatal handling such as tactile stimulation (TS) and brief and longer times of neonatal isolation on cocaine preference behavioral responses. We also investigated the influence of these neonatal handlings on stress and anxiety-like behaviors during the drug abstinence period (Sarnyai et al., 1995; Bannerman et al., 2003; Kuhn et al., 2013), as well as the oxidant/antioxidant status in blood and brain areas. 2. Material and methods 2.1. Animals and experimental procedure Fourteen pregnant female Wistar rats from the breeding facility of Universidade Federal de Santa Maria (UFSM), RS, Brazil, were kept in plexiglass cages with free access to food and water in a room with controlled temperature (22–23 ◦ C) and on a 12 h-light/dark cycle with lights on at 7:00 a.m. All procedures were in accordance with the rules of the Committee on Care and Use of Experimental Animals of the UFSM, which follows international rules (NIH Publication No. 80-23; revised 1978). The birth day (postnatal day 0 – PND0) was monitored and litters were culled to 8 pups (5 males and 3 females) to ensure adequate nutritional status. At postnatal day one (PND1) male pups were randomly assigned to one of the four experimental groups (n = 14): unhandled (UH), 10 min of tactile stimulation (TS), 10 min of neonatal isolation (NI10 ) (which was considered a time control of TS) and 60 min of neonatal isolation (NI60 ). Daily, after each neonatal procedure, all pups were returned to their mothers at the original nests, in order to maintain breastfeeding. At PND22, pups of the same condition (only one pup per litter for each handling procedure) were weaned and housed in groups (3 or 4 rats) and left undisturbed up to 40 days of age. TS, NI10 and NI60 were applied daily from PND1 to PND21, between 8:00 and 10:00 a.m. TS consisted in gently stimulating the dorsal surface of the pups with the index finger in rostral caudal direction for 10 min out of the nest (Rodrigues et al., 2004), and NI10 consisted in removing the pups from the nest and keeping them warm (32◦ C) in individual plastic boxes lined with soft paper for 10 min. For NI60 , pups were submitted to the same procedures for 60 min. The UH group remained in their nest without any touch by human hand. Animals were submitted to conditioned place preference (CPP) paradigm, and two days after this training/test, anxiety-like symptoms were sequentially evaluated in order to observe abstinence behaviors in the animals exposed to cocaine. One day after the behavioral evaluations, all the animals were anesthetized with pentobarbital (80 mg/kg body weight i.p.) and euthanized by exsanguination (blood was collected by cardiac puncture in heparinized tubes). The collected blood was centrifuged at 1300 × g for 15 min, erythrocytes were used for lipid peroxidation evaluation (TBARS) and plasma was used for vitamin (VIT C) and TBARS determination. Brains were immediately removed and cut coronally at the caudal border of the olfactory tubercle. Cortex, striatum and hippocampus were dissected according to Paxinos and Watson (2007), and homogenized in 10 volumes (w/v) of 10 mM Tris–HCl buffer (pH7.4) for the determination of protein carbonyl (PC) levels and catalase (CAT) activity. 2.2. Conditioned place preference (CPP) paradigm CPP is an animal model used to evaluate hedonic effects of addictive drugs. It involves repeated pairing in a compartment with a specific stimulus (drug), while pairing in the opposite compartment occurs with a neutral stimulus. On the test day (after the conditioning and in the absence of the stimuli), the animal is allowed free

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access to both compartments. Longer time spent in the drug-paired compartment indicates a preference for drug whereas shorter time spent in it indicates aversion (Tzschentke, 1998, 2007). The CPP apparatus consisted of a box divided into two compartments (either with black walls/white floor or with white walls/grid floor) of equal size (45 × 45 × 50) accessible through guillotine doors actuated manually from an outer common rectangular area (18 × 36 × 50) with gray walls and floor. At 40 days of age (PND40), animals were placed for 15 min in each compartment of the box for habituation. At the next day (PND41), the time spent by the animals in each compartment was monitored for 15 min, which was considered the preconditioning test. The conditioning of place preference (PND42–51) was performed by daily injection of cocaine (Merck, Germany) (20 mg/kg-ip; n = 7) for 10 days in the non preferred compartment, which was paired with saline administration on alternate turns in the opposite compartment (Martin and Itzhak, 2000). Control group (n = 7) was injected with saline in both non preferred and opposite compartments, alternatively. After each drug injection, the animals were immediately placed and maintained inside the compartment for 30 min. At PND52 all animals were submitted to 15 min post-conditioning test, which was performed similarly to the preconditioning test. 2.3. Elevated plus maze (EPM) In order to evaluate the influence of neonatal handling on cocaine-abstinence, anxiety-like symptoms were quantified in the EPM on PND 54, which is based on the innate fear rodents have for open and elevated spaces (Montgomery, 1955). The apparatus was made of wood and consisted of a plus-shaped platform elevated 50 cm from the floor. Two opposite arms (50 cm × 10 cm) were enclosed by 40 cm high walls whereas the other two arms had no walls. The four arms had at their intersection a central platform (10 cm × 10 cm), which gave access to any of the four arms. At the beginning of each test the rat was placed in the central platform facing an open arm. Time spent and entries number in the open and closed arms were registered during five-minute test. Time spent (expressed as a percentage of the total test duration) and entries number in the open arms of the maze were used as measures of the anxiety-like behavior (Hlavacova et al., 2010). The apparatus was cleaned with a 20% alcohol solution using wet sponge and paper towel before the introduction of each animal. Observers were blind to treatment during all behavioral observations. 2.4. Biochemical evaluations 2.4.1. Protein carbonyl (PC) quantification PC was quantified according Yan et al. (1995), with some modifications. Soluble protein was mixed with 2,4dinitrophenylhydrazine (DNPH; 10 mM in 2 M HCl) or HCl (2 M) and incubated at room temperature for 1 h. Denaturing buffer (150 mM sodium phosphate buffer, pH 6.8, with 3% sodium dodecyl sulfate), ethanol (99.8%) and hexane (99.5%) were added, mixed by shaking and centrifuged. The protein isolated from the interface was washed twice with ethyl acetate/ethanol 1:1 (v/v) and suspended in denaturing buffer. Each DNPH sample was read at 370 nm in a spectrophotometer against the corresponding HCl sample (blank). Results were expressed as nmol carbonyl/g tissue. 2.4.2. Catalase (CAT) activity CAT activity was spectrophotometrically quantified by the method of Aebi (1984), which involves monitoring the disappearance of H2 O2 in the presence of cell homogenate (pH 7 at 25 ◦ C) at 240 nm for 120 s. The enzymatic activity was expressed in ␮mol H2 O2 /min/g tissue.

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2.4.3. Vitamin C (VIT C) level Plasmatic VIT C was estimated as described by Galley et al. (1996) with some modifications (Jacques-Silva et al., 2001). Fresh isolated plasma was precipitated with 5% trichloroacetic acid solution and centrifuged. Supernatants were mixed with 2,4dinitrophenylhydrazine (4.5 mg/mL) and 13.3% trichloroacetic acid, and incubated (3 h at 37 ◦ C). Sulfuric acid solution (65%) was added and samples were measured at 520 nm. Results are expressed as ␮g VIT C/mL plasma. 2.4.4. Thiobarbituric acid reactive species (TBARS) levels TBARS assay measures lipid peroxidation (LP) which occurs by excessive ROS generation. LP was estimated through the pink chromogen produced by the reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA) at 100 ◦ C, measured spectrophotometrically at 535 nm. In plasma and erythrocytes, TBARS was estimated by the method described by Lapenna et al. (2001). Results were expressed as nmol MDA/mL plasma and nmol MDA/mL erythrocytes. 2.5. Statistical analysis Data were analyzed by two-way ANOVA followed by Duncan’s Post Hoc tests when appropriate. (Software package Statistica 8.0 for Windows was used). Values of P < 0.05 were considered statistically significant for all comparisons made. 3. Results 3.1. Development of preference for cocaine evaluated in CPP is shown in Fig. 1 Two-way ANOVA revealed a significant main effect of handling and drug on CPP [F(3,48) = 3.94, P < 0.05 and F(1,48) = 43.39, P < 0.001], respectively. Duncan’s test showed that TS significantly decreased cocaine preference in comparison with other groups (UH, NI10 and NI60 ), which showed similar preference for drug compartment (Fig. 1). 3.2. Anxiety-like symptoms evaluated in elevated plus maze (EPM) are shown in Fig. 2 Two-way ANOVA revealed a significant main effect of handling on time spent in open arms [F(3,48) = 13.56, P < 0.0001] and a significant main effect of handling and handling × drug interaction on time spent in closed arms [F(3,48) = 12.54, P < 0.0001 and F(3,48) = 4.51, P < 0.05, respectively] and on the ratio between open/total entries number of EPM [F(3,48) = 12.38, P < 0.0001 and F(3,48) = 2.91, P < 0.05, respectively]. Additionally, two way ANOVA revealed a significant main effect of handling, drug and a significant main effect of handling × drug interaction on the open arms entries number [F(3,48) = 15.17, P < 0.0001; F(1,48) = 4.45, P < 0.05 and F(3,48) = 3,82, P < 0.05, respectively] of EPM. Duncan’s test showed that both saline/cocaine-treated groups exposed to TS and to NI10 spent more time in the open arms as compared to both UH and NI60 groups, and these last showed similar values to each other (Fig. 2A). In addition, NI60 group spent more time in closed arms of EPM than all other experimental groups treated with saline. In cocaine-conditioned groups, time spent in closed arms was shorter in TS and NI10 than in UH and NI60 , which had similar values to each other. In fact, TS and NI10 also spent similar time in this compartment of EPM (Fig. 2B). Post hoc test showed that in saline-treated groups, TS increased entries number in the open arms (Fig. 2C) as well as the ratio between open/total arm entries of the EPM (Fig. 2D). In cocaine-treated animals, TS and

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Fig. 1. Influence of neonatal handling on time spent in the saline- and cocaine-associated compartment in CPP during post-conditioning test in young rats. Data are expressed as mean ± S.E.M. (n = 7). *Significant difference from control at the same neonatal handling (P < 0.05). Different lowercase indicates significant difference between neonatal handling in the same treatment (P < 0.05). Abbreviations: UH, unhandled; TS, tactile stimulation; NI10 , neonatal isolation 10 min; NI60 , neonatal isolation 60 min.

Fig. 2. Influence of neonatal handling on EPM during abstinence of cocaine in young rats. Anxiety-like symptoms were evaluated by % of time in open (A) and closed (B) arms, open arms entries number (C), and open/total arm entries ratio (D). Data are expressed as mean ± S.E.M. (n = 7). *Significant difference from control at the same neonatal handling (P < 0.05). Different lowercase indicates significant difference between neonatal handling in the same treatment (P < 0.05). Abbreviations: UH, unhandled; TS, tactile stimulation; NI10 , neonatal isolation 10 min; NI60 , neonatal isolation 60 min.

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Fig. 3. Influence of neonatal handling on protein carbonyl (PC) levels in cortex (A), striatum (B) and hippocampus (C) and on catalase (CAT) activity in the same brain areas (D, E, F, respectively) of young rats treated or not with cocaine. Data are expressed as mean ± S.E.M. (n = 7). *Significant difference from control at the same neonatal handling (P < 0.05). Different lowercase indicates significant difference between neonatal handling in the same treatment (P < 0.05). Abbreviations: UH, unhandled; TS, tactile stimulation; NI10 , neonatal isolation 10 min; NI60 , neonatal isolation 60 min.

NI10 increased the open arms entries number (Fig. 2C), while the open/total arm entries ratio was lower in the NI60 group (Fig. 2D).

3.3. Estimation of oxidative damage and antioxidant defense measured by protein carbonyl (PC) levels and catalase (CAT) activity in cortex, striatum and hippocampus are shown in Fig. 3 In cortex, two-way ANOVA revealed a significant main effect of handling and drug on PC levels [F(3,48) = 8.18 and F(1,48) = 14.74, P < 0.001, respectively]; a significant main effect of handling and drug, and a significant handling × drug interaction on CAT activity [F(3,48) = 3.33, P < 0.05; F(1,48) = 22.04, P < 0.001 and F(3,48) = 7.94, P < 0.001, respectively]. Duncan’s test showed that among saline-treated rats, both NI times (NI10 and NI60 ) increased PC levels in cortex in relation to UH and TS, which had comparable PC levels between each other. Cocaine administration increased PC levels in cortex of UH and NI60 , but did not alter this oxidative parameter in both NI10 and TS groups. In fact, in cocaine-treated animals, PC level was lower in TS than in all other experimental groups (Fig. 3A). In saline injected animals, TS and NI10 decreased the CAT activity in cortex as compared to UH and NI60 groups, which showed similar enzyme activity to each other. Cocaine administration significantly decreased the CAT activity in cortex of UH and NI60 groups, whose values were similar to NI10 and to each other. In fact,

CAT activity was higher in TS group than in all other experimental groups (Fig. 3D). In striatum, two-way ANOVA revealed a significant main effect of handling and drug on PC levels [F(3,48) = 4.54 and F(1,48) = 10.14, P < 0.05, respectively]; a significant main effect of handling and drug, and a significant handling × drug interaction on CAT activity [F(3,48) = 6.59, F(1,48) = 6.24, P < 0.05 for both and F(3,48) = 13.27, P < 0.001, respectively]. Post hoc test showed that the different handling caused no differences on PC levels in striatum of animals injected with saline, but cocaine administration increased PC levels in striatum of UH and NI60 , and did not change this oxidative parameter significantly in TS and NI10 groups. In cocaine-treated animals, the highest level of PC was observed in NI60 as compared to all experimental groups, whose values were similar to one another (Fig. 3B). In saline-injected animals, NI60 showed increased striatal CAT activity in relation to UH, TS and NI10 . Cocaine administration significantly increased the activity of this enzyme in striatum of TS group, while it was decreased in NI60 . In rats injected with cocaine, TS showed increased CAT activity, whose value was higher than in other experimental groups (Fig. 3E). In hippocampus, two-way ANOVA revealed a significant main effect of handling and drug on PC levels [F(3,48) = 8.59 and F(1,48) = 12.85, P < 0.001, respectively]; a significant main effect of handling and a significant handling × drug interaction on CAT activity [F(3,48) = 4.75 and 5.24, P < 0.05, respectively].

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Table 1 Effects of neonatal handling on biochemical evaluations performed in blood of adolescent rats treated or not with cocaine. VIT C plasma (␮g/mL)

TBARS erythrocytes (nmol/mL)

TBARS plasma (nmol/mL)

UH

Vehicle Cocaine

15.6 ± 1.2b 13.9 ± 1.3b

36.0 ± 3.3b 49.1 ± 4.8a

17.1 ± 2.0b 16.1 ± 4.4b

TS

Vehicle Cocaine

23.0 ± 1.1a 19.2 ± 1.1* ,a

31.8 ± 4.7b 25.3 ± 1.1b

14.6 ± 1.9b 13.6 ± 3.1b

NI10

Vehicle Cocaine

14.5 ± 1.1b 17.9 ± 0.6* ,a

35.5 ± 5.0b 52.9 ± 6.6* ,a

32.6 ± 3.5a 32.1 ± 5.3a

NI60

Vehicle Cocaine

17.5 ± 1.0b 13.2 ± 1.0* ,b

54.3 ± 6.5a 52.5 ± 6.9a

28.7 ± 5.6a 37.8 ± 1.5a

Data are expressed as mean ± S.E.M. (n = 7). Different lowercase indicates significant difference between neonatal handling in the same treatment (P < 0.05). Abbreviations: UH, unhandled; TS, tactile stimulation; NI10 , neonatal isolation 10 min; NI60 , neonatal isolation 60 min. * Significant difference from control at the same neonatal handling (P < 0.05).

Post hoc test showed increased PC level in hippocampus of animals submitted to NI60 , in comparison to both UH and TS groups. In fact, in saline-injected, UH, TS and NI10 groups, PC levels in hippocampus were comparable to each other. Cocaine injection increased PC levels in hippocampus of UH and NI60 , but not in TS and NI10 groups. In cocaine-treated rats, while PC levels were higher in NI60 than in the other groups, hippocampal PC levels were lower in TS than in UH and NI10 , which had similar levels to each other (Fig. 3C). In saline-injected animals, NI60 showed increased CAT activity in relation to all other groups, which showed similar CAT activity to each other. Cocaine administration increased CAT activity in TS, while such activity was decreased in NI60 . In cocaineinjected animals, CAT activity was higher in TS than in all other groups, which showed similar CAT activity to each other (Fig. 3F). 3.4. Vitamin C (VIT C) levels in plasma and generation of thiobarbituric acid reactive species (TBARS) in erythrocytes and plasma are shown in Table 1 Two-way ANOVA revealed a significant main effect of handling, drug [F(3,48) = 14.33, P < 0.0001 and F(1,48) = 4.26, P < 0.05, respectively], and a significant handling × drug interaction [F(3,48) = 5.50, P < 0.05) on plasma VIT C levels. In saline-injected groups, Duncan’s test showed that TS increased VIT C levels in relation to UH, NI10 and NI60 groups, which showed similar values to each other. In cocaine-treated animals, TS and NI10 groups presented similar levels of VIT C, which were higher than those observed in UH and NI60 groups. In fact, UH and NI60 showed comparable plasma VIT C levels to each other (Table 1). Two-way ANOVA revealed a significant main effect of handling on TBARS levels in plasma [F(3,48) = 13.86, P < 0.0001] and erythrocytes [F(3,48) = 7.36, P < 0.0001]. In saline- and cocaine-injected rats, post hoc test showed that generation of plasma TBARS in NI60 and NI10 was higher than in UH and TS groups, which showed comparable values to each other. In fact, cocaine administration did not change plasma TBARS level in relation to saline-treated groups, whose values remained comparable to each other (Table 1). In saline injected groups, TBARS generation in erythrocytes was higher in NI60 than in UH, TS and NI10 groups, whose values were similar to each other. Cocaine injection increased TBARS generation in erythrocytes of NI10 , but did not change this oxidative parameter in the other experimental groups. In fact, in cocaine-treated groups, TBARS generation in erythrocytes was lower in TS than in the other experimental groups, which had comparable values to each other (Table 1).

4. Discussion In the present study, saline did not cause place preference across different neonatal handlings, while in cocaine-treated animals, UH, NI10 and NI60 increased the time spent in the drug-paired compartment, while this behavior was not observed in TS group. Indeed, NI has been linked to cocaine-seeking behavior (Lynch et al., 2005), which was reduced by less time of isolation (Matthews et al., 1999). In contrast, in our study, NI10 group was unable to prevent drug seeking behaviors. This inability was not observed in TS-exposed animals. While different authors have shown the influence of NI on stress development and its connections with higher susceptibility to addictive drugs preference (Kosten et al., 2000; Lynch et al., 2005), so far no study has shown the influence of neonatal TS on the search for addictive drugs like cocaine in rats. Some authors suggested that such handling is related to increased neonatal neurogenesis (Gibb et al., 2010; Rodrigues et al., 2004), but the exact mechanism involved in the beneficial effects of TS on cocaine seeking behavior is poorly known. A recent study showed a relation between synaptic plasticity and neonatal TS, whose influence was reflected on the recovery from cortical injury in adult rats (Kolb and Gibb, 2010). Different studies have shown that stress and anxiety are closely related to drug abstinence symptoms (Buffalari et al., 2012; Rudoy and Bockstaele, 2007), suggesting that these factors can also be predictive of addiction (Sinha et al., 2003). Our findings showed a relationship between different neonatal handlings and anxiety-like symptoms, which were related to the cocaine-abstinence period observed in the EPM: while saline-treated TS and NI10 spent more time in the open arms, saline-treated NI60 stayed longer in the closed arms, indicating anxiety-like symptoms per se. In contrast, TS and NI10 were associated with shorter time spent in closed arms during cocaine abstinence, indicating reduced emotionality and fear-symptoms. Additional data confirmed our findings, when TS per se increased open arms entries and open/total arm entries ratio, while during cocaine abstinence, this ratio was reduced only in NI60 . Moreover, the total number of entries in both arms of EPM was not affected by neonatal handling (data not shown), indicating that differences observed in time spent in open and/or closed arms is not an artifact from lower locomotor activity, but a result of modified emotionality elicited by the neonatal handling. Our results suggest that TS exerts a beneficial influence on drug seeking, while fewer anxiety-like symptoms observed in both TS and NI10 were comparable to each other. In fact, such behavioral responses can be explained by the following considerations: (i) TS allows an additional stimulus by the experimenter, which reduces stress and anxiety-like symptoms, as observed in this experimental group; (ii) NI10 consists of 10 min of maternal separation without

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additional stimulus, which may be considered as an incentive because pups are subject to more licking and grooming from their dams than UH and NI60 groups (Rodrigues et al., 2004); (iii) NI60 is a handling related to a disturbance of the dam-pup contact leading to enhanced susceptibility to stress in adulthood (Knuth and Etgen, 2007; Kosten et al., 2004). We emphasize that although these findings are preliminary, they are promising because the behavioral psychopathologies, particularly stress, anxiety and drug abuse, could be naturally prevented or attenuated by positive neonatal handling. Additionally, other consequences such as increased metabolic rates and ROS generation have been related to neurobehavioral disturbances (Zhang et al., 2009), such as neonatal stress. Neonatal TS, and in minor extent neonatal MS, have been reported to alter the HPA axis in an opposing way, thereby its activation is positively related to emotionality and stress resulting in increased monoamine release and ROS generation (Ader and Grota, 1969; Ladd et al., 1996). In this sense, a recent study of our group showed that rats exposed to neonatal TS presented fewer anxiety-like symptoms and lower content of protein carbonyl in different brain areas (Boufleur et al., 2013). Supportive of the beneficial influence of TS and/or the harmful influence of NI on stress and anxiety development, the oxidative status of different brain areas and blood of drug-free animals showed that (i) NI60 exposure per se was associated with increased PC generation in both cortex and hippocampus, increased LP in plasma and erythrocytes, as well as increased CAT activity in striatum and hippocampus; (ii) NI10 showed increased LP levels in plasma, increased PC levels in cortex but reduced CAT activity in cortex, and (iii) in TS group, higher levels of VIT C in plasma and decreased CAT activity in cortex were observed. In fact, along with blood, these brain areas are recognized to be linked with stress and emotionality, as observed by serious damage to proteins and lipids. However, in absence of cocaine, NI60 was primarily related to increase CAT activity in all brain areas evaluated, possibly as a compensatory response against the increased reactive species generation. Such findings reinforce the fundamental role of CAT as an important antioxidant defense tool, whose activity is related to detoxification of hydroperoxides in brain tissues (Teixeira et al., 2008, 2009). In this scenario, we can propose that while NI60 is able to stimulate the HPA axis and release monoamines, which are vulnerable to auto-oxidation and generation of pro-oxidant metabolites, TS is negatively related to these events, offering protection against oxidative damage in brain tissues. In line with this, cocaineconditioned animals show an additional source of ROS. Indeed, cocaine exerts its action by blocking dopamine transporter (DAT), leading to DA accumulation in the synaptic cleft (Kuhar et al., 1991), thus marking a high pro-oxidant potential of that psychoactive drug. This oxidant property is due to autoxidation of DA, which consequently generates DA-quinones and reactive species (Boess et al., 2000), allowing a relationship between chronic use of cocaine and development of brain oxidative stress, whose continuity can lead to neurodegeneration (Fantel and Person, 2002; Sharan et al., 2003). In the present study, oxidative damage markers were also observed in rats exposed to different handlings and treated with cocaine: (i) TS decreased PC levels in cortex, while NI60 increased this oxidative marker in striatum and hippocampus; (ii) CAT activity was increased in all evaluated brain areas by TS, suggesting that lower PC levels can be closely related to preservation of cysteineterminal groups, which are present in this enzyme; (iii) a higher level of plasma VIT C was observed in TS group, reinforcing its protective role in the antioxidant defenses mainly due to reduced generation of reactive species, which has been linked to this handling (Boufleur et al., 2012); (iv) while plasma LP was increased in

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both NI10 and NI60 , such oxidative marker was decreased in erythrocytes of TS-exposed rats. As previously described, cocaine inhibits the DA reuptake increasing its pro-oxidant metabolites. When this pro-oxidative event occurs along with neonatal stress such as NI60 , the ROS amount may exceed the capacity of the antioxidant defense system, generating oxidative damage. In contrast, animals submitted to TS and also exposed to cocaine present a reduced amount of oxidative metabolites in relation to NI60 , and levels can be controlled by the antioxidant defense system. In this regard, determination of the activity of antioxidant enzymes such as CAT is really complex, mainly because this enzyme is itself vulnerable to ROS damage, making its activity a consequence of its production and inactivation. In our study, CAT activity in the cocaine-exposed NI60 rats was reduced in relation to the TS group in all evaluated brain areas, which can be a result of its inactivation. Moreover, carbonylation of proteins is the result of ROS action (Stadtman et al., 1991), which reacts with amino acid residues and modify protein function (Stadtman and Levine, 2000). These findings suggest that TS was able to stimulate the antioxidant defense system and attenuate the cocaine-induced oxidative damage, confirming the protective role of TS through lower oxidative damage to proteins, even in cocaine-treated rats. A recent study showed that a brief time of NI may endanger some brain structures, leading to a state of imbalance in the antioxidant defense system (Marcolin et al., 2012), reinforcing the importance of previous life events when studying behavioral and psychological disorders. Such events were confirmed here, when rats exposed to NI60 showed more damage to cortical proteins, while cocaine-treated animals showed more damage to striatal and hippocampal proteins. Although NI10 caused some damage to cortical proteins in rats not treated with cocaine, this brief handling did not have harmful influence in striatum and hippocampus. In addition, our findings also showed that plasma antioxidant status was improved by TS, reinforcing previous studies which postulated this tissue as the first protective barrier of the organism against oxidative insults (Boufleur et al., 2012; Bernhard and Wick, 2006). In contrast, aversive experiences, like NI or social isolation, are able to change the brain synaptic organization (Ferdman et al., 2007), which can exert influence on the oxidative/antioxidant status, as proposed in the present study. In agreement with our hypothesis, Marcolin et al. (2012) recently suggested that neonatal stress is able to unbalance the antioxidant defense system in some brain areas.

5. Conclusions In conclusion, we are showing for the first time that TS is able to prevent oxidative damage induced or not by cocaine administration, as well as to preserve parts of the antioxidant defense system in brain areas involved in stress/anxiety development and drug seeking behaviors. However, NI was harmful as regards drug seeking and oxidative markers, especially when such exposure lasted 60 min, but not after only 10 min. Up to now, the majority of studies have been performed with neonatal handling, which consist in simply manipulating animals without the additional stimulus of TS. In fact, TS has been related to reduced corticosteroid production, as well as an increased arborization in the hippocampus, which may cause behavioral and morphological influences (Gilad et al., 2000; Zhang and Cai, 2008). Nevertheless, we believe that this innovative study contributes to the beginning of new preventative approaches, which may turn out to be helpful tools for novel therapeutic strategies, raising efforts by the scientific community to elucidate the molecular mechanisms possibly involved in the benefits of neonatal TS on neural

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development. This study is pioneer in the beneficial influence of TS on addictive drug seeking, and it should therefore be continued through molecular mechanisms investigations that shall be carried out in the near future. Declaration of interest The authors report no conflicts of interest. Acknowledgments This study was supported by grants from CNPq, CAPES, FAPERGS fellowships (C.T.D.A., N.B., C.S.P., D.M.B. and F.T.K.). References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Ader, R., Grota, L., 1969. Effects of early experience on adrenocortical reactivity. Physiol. Behav. 4, 303–305. A.P. Association, 1994. Diagnostic and Statistical Manual of Mental Disorders (DSMIV). American Psychiatric Press, Washington, DC. Bannerman, D.M., Grubb, M., Deacon, R.M.J., Yee, B.K., Feldon, J., Rawlins, J.N.P., 2003. Ventral hippocampal lesions affect anxiety but not spatial learning. Behav. Brain Res. 139 (1–2), 197–213. Bardo, M.T., Rowlett, J.K., Harris, M.J., 1995. Conditioned place preference using opiate and stimulant drugs: a meta-analysis. Neurosci. Biobehav. Rev. 19, 39–51. Bernhard, D., Wick, G., 2006. In vitro models for the analysis of cigarette smoke effects. In: Wang, X.L., Scott, D.A. (Eds.), Molecular Mechanisms of Tobacco Induced Diseases. Nova Science Publishers, New York. Boess, F., Ndikum-Moffor, F.M., Boelsterli, U.A., Roberts, S.M., 2000. Effects of cocaine and its oxidative metabolites on mitochondrial respiration and generation of reactive oxygen species. Biochem. Pharmacol. 60 (5), 615–623. Boufleur, N., Antoniazzi, C.T.D., Pase, C.S., Benvegnú, D.M., Barcelos, R.C.S., Dolci, G.S., Dias, V.T., Roversi, K., Roversi, Kr., Koakoski, G., Rosa, J.G., Barcellos, L.J.G., Bürger, M.E., 2012. Neonatal tactile stimulation changes anxietylike behavior and improves responsiveness of rats to diazepam. Brain Res. 1474, 50–59. Boufleur, N., Antoniazzi, C.T.D., Pase, C.S., Benvegnú, D.M., Dias, V.T., Segat, H.J., Roversi, K., Roversi, Kr., Dalla-Nora, M., Koakoski, G., Rosa, J.G., Barcellos, L.J.G., Bürger, M.E., 2013. Neonatal handling prevents anxiety-like symptoms in rats exposed to chronic mild stress: behavioral and oxidative parameters. Stress 16 (3), 321–330. Brake, W.G., Zhang, T.Y., Diorio, J., Meaney, M.J., Gratton, A., 2004. Influence of early postnatal rearing conditions on mesocorticolimbic dopamine and behavioral responses to psychostimulants and stressors in adult rats. Eur. J. Neurosci. 19, 1863–1874. Buffalari, D.M., Baldwin, C.K., Feltenstein, M.W., See, R.E., 2012. Corticotrophin releasing factor (CRF) induced reinstatement of cocaine seeking in male and female rats. Physiol. Behav. 105, 209–214. Casey, B.J., Jones, R.M., Hare, T.A., 2008. The adolescent brain. Ann. N.Y. Acad. Sci. 1124, 111–126. Cirulli, F., Laviola, G., 2000. Paradoxical effects of d-amphetamine in infant and adolescent mice: role of gender and environmental risk factors. Neurosci. Biobehav. Rev. 24, 73–84. Cochrane, C.G., 1991. Mechanisms of oxidant injury of cells. Mol. Aspects Med. 12, 137–147. Dube, S.R., Felitti, V.J., Dong, M., Chapman, D.P., Giles, W.H., Anda, R.F., 2003. Childhood abuse, neglect and household dysfunction and the risk of illicit drug use: the adverse childhood experiences study. Pediatrics 111, 564–572. Fantel, A.G., Person, R.E., 2002. Involvement of mitochondria and other free radical sources in normal and abnormal fetal development. Ann. N.Y. Acad. Sci. 959, 424–433. Ferdman, N., Murmu, M., Bock, J., Braun, K., Leshem, M., 2007. Weaning age, social isolation, and gender, interact to determine adult explorative and social behavior, and dendritic and spine morphology in prefrontal cortex of rats. Behav. Brain Res. 180, 174–182. Field, T., 1998. Massage therapy effects. Am. Psychol. 53, 1270–1281. Galley, H., Davies, M.J., Webster, N.R., 1996. Ascorbil radical formation in patients with sepsis: effects of ascorbate loading. Free Radic. Biol. Med. 20, 139–143. Gibb, R., Gonzalez, C., Wegenast, W., Kolb, B., 2010. Tactile stimulation promotes motor recovery following cortical injury in adults rats. Behav. Brain Res. 214, 102–107. Gilad, V.H., Rabey, J.M., Eliyayev, Y., Gilad, G.M., 2000. Different effects of acute neonatal stressors and long-term postnatal handling on stress-induced changes in behavior and in ornithine decarboxylase activity of adult rats. Dev. Brain Res. 120, 255–259. Gutteridge, J.M.C., Halliwell, B., 2000. Free radicals and antioxidants in the year 2000. A historical look to the future. Ann. N.Y. Acad. Sci. 899, 136–147. Hadfield, M.G., Nuggent, E.A., 1983. Cocaine: comparative effect on dopamine uptake in extrapyramidal and limbic systems. Biochem. Pharmacol. 32, 744–746.

Halliwell, B., Gutteridge, F.A., 2007. Free Radicals in Biology and Medicine, fourth ed. Oxford University Press, Oxford. Hand, T.H., Stinus, L., Le Moal, M., 1989. Differential mechanisms in the acquisition and expression of heroin-induced place preference. Psychopharmacology 98, 61–67. Hlavacova, N., Bakos, J., Jezova, D., 2010. Eplerenone, a selective mineralocorticoid receptor blocker, exerts anxiolytic effects accompanied by changes in stress hormone release. J. Psychopharmacol. 24, 779–786. Hoffman, D.C., 1989. The use of place conditioning in studying the neuropharmacology of drug reinforcement. Brain Res. Bull. 23, 373–387. Izenwasser, S., 2004. The role of the dopamine transporter in cocaine abuse. Neurotox. Res. 6, 379–384. Izenwasser, S., 2005. Differential effects of psychoactive drugs in adolescents and adults. Crit. Rev. Neurobiol. 17, 51–68. Jacques-Silva, M.C., Nogueira, C.W., Broch, L.C., Flores, E.M., Rocha, J.B., 2001. Diphenyl diselenide and ascorbic acid changes deposition of selenium and ascorbic acid in liver and brain of mice. Toxicol. Appl. Pharmacol. 88, 119–125. Kabbaj, M., Isgor, C., Watson, S.J., Akil, H., 2002. Stress during adolescence alters behavioral sensitization to amphetamine. Neuroscience 113, 395–400. Kalinichev, M., Easterling, K.W., Plotsky, P.M., Holtzman, S.G., 2002. Long-lasting changes in stress-induced corticosterone response and anxiety-like behaviors as a consequence of neonatal maternal separation in Long-Evans rats. Pharmacol. Biochem. Behav. 73, 131–140. Kellogg, C.K., Awatramani, G.B., Piekut, D.T., 1998. Adolescent development alters stressor-induced fos immunoreactivity in rat brain. Neuroscience 83, 681–689. Knuth, E.D., Etgen, A.M., 2007. Long-term behavioral consequences of brief, repeated neonatal isolation. Brain Res. 1128, 139–147. Kolb, B., Gibb, R., 2010. Tactile stimulation after frontal or parietal cortical injury in infant rats facilitates functional recovery and produces synaptic changes in adjacent cortex. Behav. Brain Res. 214, 115–120. Kosten, T.A., Miserendino, M.J., Kehoe, P., 2000. Enhanced acquisition of cocaine selfadministration in adult rats with neonatal isolation stress experience. Brain Res. 875, 44–50. Kosten, T.A., Sanchez, H., Zhang, X.Y., Kehoe, P., 2004. Neonatal isolation enhances acquisition of cocaine self-administration and food responding in female rats. Behav. Brain Res. 151, 137–149. Kuhar, M.J., Ritz, M.C., Boja, J.W., 1991. The dopamine hypothesis of the reinforcing properties of cocaine. Trends Neurosci. 14, 299–302. Kuhn, C.M., Schanberg, S.M., 1998. Responses to maternal separation: mechanism and mediators. Int. J. Dev. Neurosci. 16, 261–270. Kuhn, F.T., Roversi, Kr., Antoniazzi, C.T.D., Pase, C.S., Trevizol, F., Barcelos, R.C.S., Dias, V.T., Roversi, K., Boufleur, N., Benvegnú, D.M., Piccolo, J., Emanuelli, T., Bürger, M.E., 2013. Influence of trans fat and omega-3 on the preference of psychostimulant drugs in the first generation of young rats. Pharmacol. Biochem. Behav. 110, 58–65. Ladd, C.O., Owens, M.J., Nemeroff, C.B., 1996. Persistent changes in corticotrophinreleasing factor neuronal system induced by maternal deprivation. Endocrinology 137, 1212–1218. Lapenna, D., Ciofani, G., Pierdomenico, S.D., Giamberardino, M.A., Cuccurullo, F., 2001. Reaction conditions affecting the relationship between thiobarbituric acid reactivity and lipid peroxides in human plasma. Free Radic. Biol. Med. 31, 331–335. Laviola, G., Macri, S., Morley-Fletcher, S., Adriani, W., 2003. Risk-taking behavior in adolescent mice: psychobiological determinants and early epigenetic influence. Neurosci. Biobehav. Rev. 27, 19–31. Lemaire, V., Lamarque, S., Le Moal, M., Piazza, P.V., Abrous, D.N., 2006. Postnatal stimulation of the pups counteracts prenatal stress-induced deficits in hippocampal neurogenesis. Biol. Psychiatry 59, 768–792. Levine, S., Otis, L.S., 1958. The effects of handling before and after weaning on the resistance of albino rat to later deprivation. Can. J. Psychol. 12, 103–108. Lynch, W.J., Mangini, L.D., Taylor, J.R., 2005. Neonatal isolation stress potentiates cocaine seeking behavior in adult male and female rats. Neuropsychopharmacology 30, 322–329. Marcolin, M.L., Noronha, A.S.B., Arcego, D.M., Noschang, C., Krolow, R., Dalmaz, C., 2012. Effects of early life interventions and palatable diet on anxiety and on oxidative stress in young rats. Physiol. Behav. 106, 491–498. Marquardt, A.R., Ortiz-Lemos, L., Lucion, A.B., Barros, H.M.T., 2004. Influence of handling or aversive stimulation during rats’ neonatal or adolescence periods on oral cocaine self-administration and cocaine withdrawal. Behav. Pharmacol. 15, 403–412. Martin, J.L., Itzhak, Y., 2000. 7-Nitroindazole blocks nicotine-induced conditioned place preference but not LiCl-induced conditioned place aversion. Neuroreport 11, 947–949. Matthews, K., Robbins, T.W., Everitt, B.J., Caine, S.B., 1999. Repeated neonatal maternal separation alters intravenous cocaine self-administration in adult rats. Psychopharmacology 141, 123–134. McIntosh, L.J., Sapolsky, R.M., 1996. Glucocorticoids increase the accumulation of reactive oxygen species and enhance adriamycin-induce toxicity in neuronal culture. Exp. Neurol. 141, 201–206. Montagu, A., 1953. The sensory influences of the skin. Tex. Rep. Biol. Med. 11, 292–301. Montgomery, K.C., 1955. The relation between fear induced by novel stimulation and exploratory behavior. J. Comp. Physiol. Psychol. 48, 254–260.

C.T.D. Antoniazzi et al. / Behavioural Processes 103 (2014) 297–305 Neisewander, J.L., Pierce, R.C., Bardo, M.T., 1990. Naloxone enhances the expression of morphine-induced conditioned place preference. Psychopharmacology 100, 201–205. Padoin, M.J., Cadore, L.P., Gomes, C.M., Barros, H.M.T., Lucion, A.B., 2001. Long-lasting effects of neonatal stimulation on the behavior of rats. Behav. Neurosci. 115, 1332–1340. Paxinos, G., Watson, C., 2007. The Rat Brain in Stereotaxic Coordinates, sixth ed. Academic Press, New York. Pham, T.M., Söderström, S., Winblad, B., Mohammed, A.H., 1999. Effects of environmental enrichment on cognitive function and hippocampal NGF in the non-handled rats. Behav. Brain Res. 103, 63–70. Pryce, C.R., Feldon, J., 2003. Long-term neurobehavioural impact of the postnatal environment in rats: manipulations, effects and mediating mechanism. Neurosci. Biobehav. Rev. 27 (1–2), 57–71. Raineki, C., De Souza, M.A., Szawka, R.E., Lutz, M.L., De Vasconcellos, L.F., Sanvitto, G.L., Izquierdo, I., Bevilaqua, L.R., Cammarota, M., Lucion, A.B., 2009. Neonatal handling and the maternal odor preference in rat pups: involvement of monoamines and cyclic AMP response element-binding protein pathway in the olfactory bulb. Neuroscience 159, 31–38. Rodrigues, A.L., Arteni, N.S., Abel, C., Zylbersztejn, D., Chazan, R., Viola, G., Xavier, L., Achaval, M., Netto, C.A., 2004. Tactile stimulation and maternal separation prevent hippocampal damage in rats submitted to neonatal hypoxia-ischemia. Brain Res. 1002, 94–99. Rosenfeld, P., Wetmore, J.B., Levine, S., 1992. Effects of repeated maternal separations on the adrenocortical response to stress of preweanling rats. Physiol. Behav. 52, 787–791. Rudoy, C.A., Bockstaele, E.J., 2007. Betaxolol, a selective ␤1 -adrenergic receptor antagonist, diminishes anxiety-like behavior during early withdrawal from chronic cocaine administration in rats. Prog. Neuropsychopharmacol. Biol. Psychiatry. 31, 1119–1129. Sarnyai, Z., Bíró, E., Gardi, J., Vecsernyés, M., Julesz, J., Telegdy, G., 1995. Brain corticotropin-releasing factor mediates ‘anxiety-like’ behavior induced by cocaine withdrawal in rats. Brain Res. 675, 89–97. Schapiro, S., Vukovich, K.R., 1970. Early experience effects upon cortical dendrites: a proposed model for development. Science 167, 292–294. Schenk, S., Lacelle, G., Gorman, K., Amit, Z., 1987. Cocaine self-administration in rats influenced by environmental conditions: implications for the etiology of drug abuse. Neurosci. Lett. 81, 227–231. Sharan, N., Chong, V.Z., Nair, V.D., Mishra, R.K., Hayes, R.J., Gardner, E.L., 2003. Cocaine treatment increases expression of a 40 kDa catecholamine-regulated protein in discrete brain regions. Synapse 47, 33–44. Silveira, P.P., Portella, A.K., Clemente, Z., Gamaro, G.D., Dalmaz, C., 2005. The effect of neonatal handling on adult feeding behavior is not an anxiety-like behavior. Int. J. Dev. Neurosci. 23, 93–99. Sinha, R., 2001. How does stress increase risk of drug abuse and relapse? Psychopharmacology 158, 343–359.

305

Sinha, A.R., Talih, M., Malison, R., Cooney, N., Anderson, G.M., Kreek, M.J., 2003. Hypothalamic-pituitary-adrenal axis and sympatho-adreno-medullary responses during stress-induced and drug cue-induced cocaine craving states. Psychopharmacology 170, 62–72. Spear, L.P., 2000. The adolescent brain and age-related behavioral manifestations. Neurosci. Biobehav. Rev. 24, 417–463. Stadtman, E.R., Oliver, C.N., Starke-Reed, P.E., 1991. Implication of metal catalyzed oxidation of enzymes in aging, protein turnover, and oxygen toxicity. Korean J. Biochem. 23, 49–54. Stadtman, E.R., Levine, R.L., 2000. Protein oxidation. Ann. N.Y. Acad. Sci. 899, 191–208. Teixeira, A.M., Trevizol, F., Colpo, G., Garcia, S.C., Charão, M., Pereira, R.P., Fachinetto, R., Rocha, J.B., Bürger, M.E., 2008. Influence of chronic exercise on reserpineinduced oxidative stress in rats: behavioral and antioxidant evaluations. Pharmacol. Biochem. Behav. 88, 465–472. Teixeira, A.M., Müller, L., Santos, A.A., Reckziegel, P., Emanuelli, T., Rocha, J.B.T., Bürger, M.E., 2009. Beneficial effects of gradual intense exercise in tissues of rats fed with a diet deficient in vitamins and minerals: a pilot study. Nutrition 25, 590–596. Todeschin, A.S., Winkelmann-Duarte, E.C., Jacob, M.H., Aranda, B.C., Jacobs, S., Fernandes, M.C., Ribeiro, M.F., Sanvitto, G.L., Lucion, A.B., 2009. Effects of neonatal handling on social memory, social interaction, and number of oxytocin and vasopressin neurons in rats. Horm. Behav. 56, 93–100. Tzschentke, T.M., 1998. Measuring reward with the conditioned place preference paradigm: a comprehensive review of drug effects, recent progress and new issues. Prog. Neurobiol. 56, 613–672. Tzschentke, T.M., 2007. Measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade. Addict. Biol. 12, 227–462. van Oers, H.J.J., de Kloet, E.R., Whelan, T., Levine, S., 1998. Maternal deprivation effect on the infant’s neural stress markers is reversed by tactile stimulation and feeding but not by suppressing corticosterone. J. Neurosci. 18, 10171–10179. Vicentic, A., Francis, D., Moffett, M., Lakatos, A., Rogge, G., Hubert, G.W., Harley, J., Kuhar, M.J., 2006. Maternal separation alters serotonergic transporter densities and serotonergic 1A receptors in rat brain. Neuroscience 140, 355–365. Wigger, A., Neumann, I.D., 1999. Periodic maternal deprivation induces genderdependent alterations in behavioral and neuroendocrine responses to emotional stress in adult rats. Physiol. Behav. 66, 93–302. Yan, L.Y., Traber, M.G., Packer, L., 1995. Spectrophotometric method for determination of carbonyls in oxidatively modified apolipoprotein B of human low-density lipoproteins. Anal. Biochem. 228, 349–351. Zhang, M., Cai, J.X., 2008. Neonatal tactile stimulation enhances spatial working memory, prefrontal long-term potentiation, and D1 receptor activation in adult rats. Neurobiol. Learn. Mem. 89, 397–406. Zhang, D., Wen, X.S., Wang, X.Y., Shi, M., Zhao, Y., 2009. Antidepressant effect of Shudihuang on mice exposed to unpredictable chronic mild stress. J. Ethnopharmacol. 123, 55–60.