Neonatal tactile stimulation changes anxiety-like behavior and improves responsiveness of rats to diazepam

brain research 1474 (2012) 50–59

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Research Report

Neonatal tactile stimulation changes anxiety-like behavior and improves responsiveness of rats to diazepam Nardeli Boufleura, Caren T.D. Antoniazzia, Camila S. Pasea, Dalila M. Benvegnu´a, Raquel C.S. Barcelosa, Geisa S. Dolcia, Veroˆnica T. Diasb, Katiane Roversib, Karine Roversib, ~ G. Rosaa, Leonardo J.G. Barcellosa,c, Marilise E. Bu¨rgera,n Gessi Koakoskiaa, Joao a

~ em Farmacologia, Universidade Federal de Santa Maria, RS, Brazil Programa de po´s Graduac- ao Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, RS, Brazil c Faculdade de Agronomia e Medicina Veterina´ria, Universidade de Passo Fundo, RS, Brazil b

ar t ic l e in f o

abs tra ct

Article history:

In this study we evaluated the influence of neonatal tactile stimulation (TS) on behavioral

Accepted 1 August 2012

and biochemical effects related to a low dose of diazepam (DZP) in adult rats. Male pups of

Available online 8 August 2012

Wistar rats were handled (TS) daily from PND1 to PND21 for 10 min, while unhandled (UH)

Keywords:

rats were not touched. In adulthood, half the animals of each group received a single

Neonatal handling

administration of diazepam (0.25 mg/kg body weight i.p.) or vehicle and then were

Tactile stimulation

submitted to behavioral and biochemical evaluations. In the TS group, DZP administration

Anxiety

reduced anxiety-like symptoms in different behavioral paradigms (elevated plus maze,

Diazepam

EPM; staircase and open-field and defensive burying) and increased exploratory behavior.

Antioxidant defense

These findings show that neonatal TS increased DZP pharmacological responses in adulthood compared to neonatally UH animals, as observed by reduced anxiety-like symptoms and lower levels of plasma cortisol. TS also changed plasma levels of antioxidant defenses such as vitamin C and glutathione peroxidase, whose increase may be involved in lower oxidative damages to proteins in cortex, subthalamic region and hippocampus of these animals. Here we are showing for the first time that neonatal TS is able to change responsiveness to benzodiazepine drugs in adulthood and provides better pharmacological responses in novel situations of stress. & 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Environmental changes originate individual adaptations, whose responses may generate a dysfunctional state of high stress and anxiety and result in neuropsychiatric disorders Abbreviations: BZ, benzodiazepines; DBT,

(Salomons et al., 2010). These pathologies include insomnia, mood disorders, panic and others, often developed in highly anxious persons who tend to present impaired adaptive capacity (Beck et al., 1985) and slow recovery after stressful situations (Hoehn-Saric and McLeod, 1988).

defensive burying test; DZP,

diazepam; EPM,

elevated plus maze; GSH,

reduced

gluthatione; OF, open field; PND, postnatal day; PC, protein carbonyl; SAP, stretched-attend postures n Correspondence to: Departamento de Fisiologia e Farmacologia, Centro de Cio´ncias da Sau´de-CCS, Universidade Federal de Santa Maria (UFSM) 97105-900, Santa Maria, RS- BRAZIL. Fax: þ55 55 3220 7686. E-mail address: [email protected] (M.E. Bu¨rger). 0006-8993/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2012.08.002

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brain research 1474 (2012) 50–59

Clinical and preclinical evidence link GABAergic system dysfunctions to the development of anxiety symptoms (Millan, 2003). Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the adult mammalian central nervous system (CNS), and is involved in regulation of physiological, emotional, cognitive and behavioral functions (Vekovischeva et al., 1999). Benzodiazepines (BZs) are a prototypical class of drugs widely prescribed to relieve anxiety symptoms (Carlini, 2003; Gallager and Primus, 1993; Woods et al., 1992), whose action mechanism consist in modulating the functionality of the GABAergic system by enhancing chloride ion flux through GABA-A receptors at a number of limbic areas (Caldji et al., 2003; Gonzalez et al. 1996; Gray, 1987; Pesold and Treit 1995; Thomas et al. 1985). Despite showing higher selectivity than other CNS depressant drugs, continued use of higher doses of benzodiazepines has been related to sedation, memory loss and amnesia (Woods et al., 1992). It is widely known that during early development, the CNS presents great plasticity and can be very sensitive to even moderate environmental interventions (Gschanes et al., 1998; Inazusta et al., 1999; Sternberg and Ridgway, 2003; Zhang and Cai, 2008). Studies showed that behavioral and physiological processes in adult rodents can be a result from exposure to distinct stimuli during the first weeks of life (Casolini et al., 1997; Pham et al., 1999). Neonatal tactile stimulation (TS), as one kind of external sensory stimuli, influences physiological and behavioral processes, like acceleration of cortical neuron maturation (Schapiro and Vukovich, 1970), improvement of passive avoidance response in adulthood (Zhang and Cai, 2006) and increase in pups weight gain (Levine and Otis, 1958). Moreover, this neonatal handling is related to reduced hypothalamic–pituitary–adrenal (HPA) response to stressful situations, since handled animals have lower plasma corticosterone levels after novel situations (Levine et al., 1967; Meaney et al., 1991, 1992). Moreover, neonatal manipulations have been used to study neurobiological changes associated with psychiatric disorders (Cirulli et al., 2003). Besides the physiological, neuroendocrine and morphological effects of neonatal handling, some studies have shown that such stimulations during infancy cause significant changes on fear-related behavior in adult animals (Ferna´ndez-Teruel et al., 1990; Hilakivi-Clarke et al., 1991; Nu´n˜ez et al., 1996; Vallee et al., 1997). Different studies have reported lesser anxiety in the

elevated plus-maze (Ferna´ndez-Teruel et al., 1990; Nu´n˜ez et al., 1995; Vallee et al., 1997; Wakshlak and Weinstock, 1990), the hyponeophagia (Bodnoff et al., 1987; Hilakivi-Clarke et al., 1991) or the Vogel conflict tests (Nu´n˜ez et al., 1996). Furthermore, it has been shown that early life experiences exert influence on the development of the GABA complex in brain regions that mediate stress reactivity and change the expression of fearfulness in rats (Caldji et al., 2000; Giachino et al., 2007). So, it is possible to hypothesize that alterations in GABA-A receptors density may affect the threshold for responsiveness to agonist drugs which act in the BZ site (Cirulli et al., 2010). As some studies demonstrated changes in the GABAergic system after neonatal handling and little is known about the possible influence of these manipulations on positive modulators of this system, more studies about this are necessary. So, we decided to investigate the influence of neonatal TS on behavioral parameters associated with diazepam (DZP) administration, a GABA-A receptor agonist, in rats. We also investigated the effects of this TS-DZP association on the development of anxiety-like symptoms, which were assessed in different behavioral paradigms, as well the oxidant/antioxidant status in blood and brain regions.

2.

Results

2.1. Anxiety-like symptoms evaluated in elevated plus maze (EPM) are shown in Table 1 Two-way ANOVA revealed a significant main effect of handling, DZP and a significant handling  DZP interaction on time spent in the open arms [F(1,22)¼6.11; 5.53; and F(2,22)¼6.15; Po0.05, respectively], and a significant main effect of handling and DZP on the number of entries in the open arms [F(1,22)¼18.37 and 6.21; Po0.05, respectively]. Duncan’s test showed that neonatal TS increased the number of entries in the open arms in relation to UH animals. DZP administration in adulthood was able to increase both entries number and time spent in the open arms of animals exposed to TS, but this was not observed in UH animals. Two-way ANOVA revealed a significant main effect of handling on time spent in the closed arms [F(1,22)¼6.11; Po0.05].

Table 1 – Effects of neonatal handlings on elevated plus maze task performed in adult rats. Behavior

UHþV

UHþDZP

TSþV

TSþDZP

% Time in OA % Time in CA OA entries number CA entries number Rearing number Head dipping number SAP

5.372.4 78.673.7 1.270.4 7.271.0 14.470.3 2.670.7 9.070.8

5.071.4 81.172.1 2.770.4 6.870.4 14.871.0 2.170.8 10.070.9

5.371.5 69.875.8 4.270.4b 6.770.6 13.870.8 6.071.4b 10.070.4

18.874.6a,b 65.372.3b 6.871.5a,b 6.770.9 18.071.0a,b 8.371.6b 5.470.7a,b

UH, unhandled; TS, tactile stimulation; V, vehicle; DZP, diazepam, OA, open arms; CA, closed arms; SAP, stretched-attend postures, Data are expressed as mean7SEM, (n ¼ 7). a Different from vehicle at the same neonatal handling (Po0.05). b Different from UH at the same treatment (Po0.05).

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Rats submitted to neonatal TS and treated with DPZ in adulthood also spent less time in the closed arms in relation to UHþDZP group. This result cannot be considered as a false-positive, mainly because the animals of both experimental groups showed similar number of entries in the closed arms, which is indicative of locomotor activity in the EPM. DZP and TS modified not only the classical spatiotemporal measures of anxiety, but also the ethological parameters related to exploration and risk assessment behavior in a manner consistent with an anxiolytic outcome. Two-way ANOVA revealed a significant main effect of handling on head dipping number [F(1,22)¼17.88; Po0.001]. Neonatal TS group per se showed higher head dipping number compared to UH group. Regarding DZP treatment in adulthood, TSþDZP group presented increased head dipping number compared to UHþDZP group. DZP reduced frequency of stretched-attend postures (SAP) in TS group and TSþDZP showed reduced SAP compared to UHþDZP group. Moreover, DZP increased rearing number in handled animals, and this experimental group presented higher rearing number than unhandled DZP-treated rats.

2.2. Anxiety-like symptoms evaluated in staircase test are shown in Fig. 1 Two-way ANOVA revealed a significant main effect of handling and DZP on the number of stairs climbed [F(1,22)¼21.7, Po0.001 and 5.6; Po0.05, respectively] and a significant main effect of DZP on rearing number [F(1,22)¼ 9.9; Po0.05]. Duncan’s test showed that TS group presented higher number of steps climbed in relation to UH group. DZP was able to increase this parameter in UH animals, but this value was lower than in TSþDZP treated group (Fig. 1A). TSþDZP group also showed increased rearing number in relation to UHþDZP (Fig. 1B).

Fig. 1 – Effects of tactile stimulation in staircase test. UH, unhandled; TS, tactile stimulation. Data are expressed as mean7SEM. , Different from vehicle at the same neonatal handling (Po0.05); þ, different from UH at the same treatment (Po0.05).

2.3. Locomotor and exploratory performance evaluated in open field (OP) are shown in Fig. 2 Two-way ANOVA revealed a significant main effect of handling on the number of crossings [F(1,22)¼ 20.41; Po0.001]), and a significant main effect of handling, DZP and a significant handling  DZP interaction on the number of rearings [F(1,22)¼ 19.3, Po0.001; 6.35, Po0.05 and F(2,22)¼8.93; Po0.05, respectively] and central squares crossed [F(1,22)¼17.64; 17.63; and F(2,22)¼ 14.34; Po0.001, respectively]. Post hoc test showed that neonatal TS increased the number of crossings in relation to UH animals (Fig. 2A). DZP treatment in adulthood increased the number of rearings (Fig. 2B) and central squares crossing (Fig. 2C) by TS-submitted animals. TSþDZP group presented higher numbers of crossing, rearing and central squares compared to UHþDZP group.

2.4. Anxiety-like symptoms observed in defensive burying test (DBT) are shown in Fig. 3 Two-way ANOVA revealed a significant main effect of handling on duration of immobility behavior [F(1,22)¼ 23.64; Po0.001]). Duncan’s test showed no differences in burying time, indicating that the handling did not change this behavioral parameter (Fig. 3A). In relation to immobility or freezing time, neonatal TS was able to reduce this behavior when compared to UH animals (Fig. 3B). Furthermore, TS-submitted animals treated with DZP in adulthood showed lower immobility time in relation to both UH and TS treated with vehicle.

2.5.

Biochemical assays

2.5.1. Cortisol, VIT C and GSH plasma levels are shown in Table 2 Two-way ANOVA revealed a significant main effect of handling and DZP on cortisol plasma levels [F(1,22)¼ 5.25 and 7.80; Po0.05, respectively], a significant main effect of handling and a significant handling  DZP interaction on VIT plasma level [F(1,22)¼17.19; and F(2,22)¼5.76; Po0.05, respectively] and a significant main effect of handling on GSH plasma levels [F(1,22)¼10.22; Po0.05]). Post hoc test showed that neonatal TS did not change plasma cortisol levels of the control rats in relation to UH animals, but reduced the plasma hormone levels in the adult rats treated with DZP. In fact, DZP did not change this biochemical parameter in UH animals but it decreased plasma cortisol levels in animals submitted to neonatal TS. Neonatal TS increased VIT C plasma levels of control adult animals, but this effect was not observed in DZP-treated rats. Similar levels of plasma GSH were observed in both UH and TS groups injected with saline, but between animals injected with DZP, TS increased the plasma levels of this antioxidant compound.

2.5.2. Protein carbonyl (PC) levels in cortex, hippocampus and subthalamic region are shown in Table 3 Two-way ANOVA revealed a significant main effect of handling and DZP on PC levels in cortex [F(1,22)¼ 8.56 and 6.03; Po0.05, respectively], a significant main effect of handling on PC levels

brain research 1474 (2012) 50–59

53

Fig. 2 – Effects of tactile stimulation in open field test. UH, unhandled; TS, tactile stimulation. Data are expressed as mean7SEM. , Different from vehicle at the same neonatal handling (Po0.05); þ, different from UH at the same treatment (Po0.05).

3.

Fig. 3 – Effects of tactile stimulation in defensive burying test. UH, unhandled; TS, tactile stimulation. Data are expressed as mean7SEM. , Different from vehicle at the same neonatal handling (Po0.05); þ, different from UH at the same treatment (Po0.05).

in hippocampus and subthalamic region [F(1,22)¼ 9.42 and 4.28; Po0.05, respectively]. Neonatal handling did not change the cortical and subthalamic PC levels of control rats, but the level of this oxidative parameter was reduced by neonatal TS in DZP-treated adult rats, indicating that DZP injection reduced PC levels in cortex and subthalamic region of TS, but not in UH group. Neonatal TS reduced PC levels in hippocampus of adult control animals but not of those treated with DZP, whose levels of PC showed similar values. The DZP injection did not change hippocampal PC levels of both UH and TS groups.

Discussion

It is well known that early manipulations of the infant–mother interaction can induce neurochemical, physical and psychological changes in offspring (Cirulli et al., 2003; Imanaka et al., 2008; Pryce and Feldon, 2003; Weaver et al., 2004) and might result in prolonged behavioral effects in adulthood (Cameron et al., 2005; Giachino et al., 2007). Mild and brief (3–15 min) neonatal daily manipulations exert persistent effects on behavior of adults including decreased fearfulness to novelty and reduced emotionality (Bodnoff et al., 1987; Denenberg, 1964; Levine, 1957, 1962), and enhanced curiosity and exploratory behavior (Caldji et al., 2000; Denenberg and Smith, 1963; Levine, 1960). Animals handled during neonatal development have also shown beneficial changes in the functionality of the HPA axis (Levine, 1957), and so their adaptation to novel and/or stressful stimuli can be significantly increased (Meaney et al., 1991). These beneficial effects of neonatal handling may be explained by early maturation of neural pathways from skin to CNS (Montagu, 1953), and also by changes in maternal behavior patterns (Cirulli et al., 2000, Levine, 1994) since mothers of handled pups exhibit increased levels of licking/grooming and arched-back nursing behavior (Caldji et al., 1998; Lee and Williams, 1975; Liu et al., 1997; Pryce et al., 2001). Moreover, handled animals may also present important alterations in the neurotransmitter systems involved in the regulation of emotionality, such as the GABAergic one (Giachino et al., 2007). Most of the brain GABA-A receptor alterations in rat occur during early life, prior to 20 days of age (Laurie et al., 1992). Neonatal handling was shown to increase GABAergic interneuron densities in hypothalamus and amygdale, which are brain regions related to stress response and emotional behavior (Giachino et al.,

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Table 2 – Effects of neonatal tactile stimulation on biochemical evaluations performed in plasma of adult rats treated or not with diazepan. Measure

UHþV

UHþDZP

TSþV

TSþDZP

Cortisol (mg/dL) GSH (nmol/mL) VIT C (mg/mL)

5.370.63 342.1721.1 13.070.6

4.770.6 314.2730.5 15.3170.6a

4.570.5 400.7714.4 17.270.7þ

2.570.4a,b 414.2726.9b 16.570.7

UH, unhandled; TS, tactile stimulation; V, vehicle; DZP, diazepam. Data are expressed as mean7SEM (n ¼7). a Different from vehicle at the same neonatal handling (Po0.05). b Different from UH at the same treatment (Po0.05).

Table 3 – Effects of neonatal tactile stimulation on levels of protein carbonyl (PC) in brain regions of adult rats treated or not with diazepan. Brain area

UHþV

UHþDZP

TSþV

TSþDZP

Cortex Hippocampus Subthalamic region

1779.5793.3 970.27219.2 2753.6791.06

1547.57116.9 714.5737.63 2684.37162.3

1491.17156.8 524.0741.41þ 2676.3792.35

1133.17101.8a,b 414.5753.10 2250.27135.1a,b

UH, unhandled; TS, tactile stimulation; V, vehicle; DZP, diazepam. Data are expressed as mean7SEM (n ¼ 7). a Different from vehicle at the same neonatal handling (Po0.05). b Different from UH at the same treatment (Po0.05).

2007). In addition, high levels of maternal care were also related to increase of GABA-A/benzodiazepine receptor density in locus coeruleus of the offspring (Caldji et al., 2000, 2003). Here, we evaluated the influence of neonatal TS on anxiety and locomotion of adult rats treated with a low dose of DZP in comparison to UH animals. Exposure of rodents to a novel ambient can induce both exploratory and fear behaviors, thus creating an approach-avoidance conflict (Montgomery, 1955). The EPM test is based on the conflict between rodents’ innate aversion to exposed spaces and a tendency to explore new environments (Lister, 1987). In our study, this behavior was observed in the TS-DZP group, as evidenced by higher time spent and greater number of entries in open arms, as well as by lower time spent in closed arms. Thus, we can hypothesize that neonatal TS modified the pharmacological response to BZ drugs in adulthood, since animals subjected to TSþDZP showed reduced levels of anxiety and fear as compared to those exposed to either TS or DZP alone. In fact, DZP combined with neonatal TS reduced anxiety-like symptoms by modifying the classical spatiotemporal measures of anxiety and also the ethological parameters related to exploration and risk assessment behavior. Risk assessment, mainly observed by SAP movements in EPM (Lepicard et al., 2000; Rodgers and Johnson, 1995; Shepherd et al., 1994), is positively correlated with anxiety-like behavior and is considered one of the most responsive parameters for assessment of anxiety-like behavior (Rodgers and Dalvi, 1997; Setem et al., 1999). Our findings clearly showed lower SAP frequency in the TSþDZP group, confirming that risk assessment behaviors are sensitive to anxiolytic drugs (Cole and Rodgers, 1993; Handley, 1991). In line with this, full agonists of BZ receptors typically increase exploratory head dipping of rodents (Cole

and Rodgers, 1993, 1995), and this effect was also observed in handled rats treated with DZP. Regarding exploratory behavior, inhibition of this conduct is often related to high emotionality or anxiety (Archer, 1973). Furthermore, studies showed that some anxiolytic drugs at low doses increased the number of steps climbed and rearing in the staircase test (Jacobson and Cryan, 2008; Lepicard et al., 2000; Ste´ru et al., 1987). Our findings showed that handled animals treated with DZP presented higher exploratory behavior as observed by rearing number in EPM, open field and staircase tests, as well as greater number of stairs climbed. A recent study showed that increased locomotor activity in animals may reflect an anxiolytic status (Shoji and Mizoguchi, 2010), while a decrease in the central squares crossed in open field can demonstrate increased anxiety-like behavior (File, 1997). Since exploratory behavior is hard to dissociate from general and locomotor activity, both types of conduct had to be considered in the experimental studies (Lepicard et al., 2000). Here, neonatal TS was able to increase the number of crossings in the open-field, revealing an increase in locomotor activity. On the other hand, only the animals submitted to neonatal TS and treated with DZP in adulthood showed increased rearing and number of central squares crossed, revealing higher exploration and lower fear symptom, respectively. Regarding the defensive burying test (DBT), an increased anxiety-like behavior in this test can be observed through two types of strategies: the active behavior associated with burying the shock probe and the passive behavior associated with immobility or freezing (Matuszewich et al., 2007). Our results showed that neonatal TS decreased immobility behavior in both groups treated or not with DZP in adulthood,

brain research 1474 (2012) 50–59

whose animals explored more the apparatus and presented a quieter behavior during the test. Besides altering behavioral profiles, TS also changed the antioxidant status of the control animals, as evidenced by higher plasma VIT C levels. Moreover, when combined with DZP, TS increased plasma GSH levels in relation to the UH group. These findings allow us to hypothesize that TS is able to modulate and improve the antioxidant status of blood, which is considered as the first protective barrier of the organism against oxidative insults (Bernhard and Wick, 2006). Indeed, TS decreased the PC levels in cortex and subthalamic region of DZP-treated animals, as well as in hippocampus of control rats. Taken together, these findings indicate that this reduced oxidative damage may be related to the increased levels of antioxidant defense observed in plasma of handled animals. Contrarily, but confirming our findings, anxiety disorders have been related to depleted blood levels of such non-enzymatic antioxidants like vitamin E and GSH (Hovatta et al., 2010). In addition, some animal models have shown that antioxidants can decrease oxidative damages, thereby reducing anxiety-like behavior (Salim et al., 2010), which reinforces the relationship between oxidative stress and anxiety-like symptoms shown here. Overall, TS was able to reduce anxiety-like symptoms observed in different behavioral paradigms. In fact, this neonatal handling acted favorably on the anxiolytic effects of a low dose of DZP. In contrast, UH animals and treated with the same dose of DZP in adulthood showed higher anxiety grade, which may indicate an insufficient effect of the BZ drug. Our findings are in agreement with other studies that showed a close relationship between neonatal handling and the GABAergic system (Caldji et al., 2000; Giachino et al., 2007) and confirm that handling can affect the responsiveness to BZ agonists, as recently proposed by Cirulli et al. (2010). Bodnoff et al. (1987) showed influence of neonatal handling on the density of BZ receptors, which are fundamental components of the GABA-A complex (Caldji et al., 2000). When an agonist binds to the BZ receptor, the GABA-A complex enhances its affinity for GABA, causing hyperpolarization and inhibition of fear and anxiety symptoms (Caldji et al., 2000). This is the first study about effects of TS on higher responsiveness of a BZ agonist, also showing an increase in plasma antioxidant defenses and lower oxidative status in brain regions. At this time it is possible to hypothesize that neonatal TS is related to less emotionality of adult animals exposed to novel environments. The continuity of studies in animal models involving binding sites of BZ receptors, as well as its activation after stress exposure, are some future prospects and merit attention.

3.1.

Conclusion

In summary, the present study indicated that a low dose of DZP in adulthood was able to promote just a small anxiolytic effect in UH animals, but when combined with neonatal TS, it showed a clear and relevant anxiolytic effect, as demonstrated here in different behavioral assessments. Our findings suggest that adequate neonatal handling in early life provides better responses in novel situations and environments in

55

adulthood and also allows reduction in BZ doses, if such pharmacological intervention is required.

4.

Experimental procedures

4.1.

Animals

Fourteen pregnant female Wistar rats from the breeding facility of Universidade Federal de Santa Maria (UFSM), RS, Brazil were individually kept in plexiglas cages with free access to food and water in a room with controlled temperature (22–23 1C) 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 (EC Directive 86/ 609/EEC).

4.2.

Neonatal handling

The births were monitored and the litters were culled to eight pups (five males and three females) to ensure adequate nutritional status. At postnatal day one (PND1) two male pups of 14 litters were randomly assigned to one of two handlings: unhandled (UH, not touched; n¼ 14) and tactile stimulation (TS, n¼14). Neonatal TS was applied from PND1 to PND21, between 1 and 3 p.m. Pups submitted to TS were removed from the nest, held gently by experimenter and stroked with the index finger on the dorsal surface, in the rostral caudal direction, for 10 min (Rodrigues et al., 2004). The UH group remained in their nest without any touch by human hand. The entire litter remained together with their mother until weaning. At PND22, the litters were weaned, and one half of each handling group (TS and UH) was assigned to receive diazepam or vehicle at 70 days of age, thus forming four final experimental groups: TSþdiazepan (TSþDZP); TSþvehicle (TSþC); UHþdiazepan (UHþDZP) and UHþvehicle (UHþC). Only one male pup from each litter and each neonatal handling was assigned to each experimental group, i.e., pups of one experimental group were never from the same litter, thus representing an experimental unit.

4.3.

Drugs and experimental procedure

~ Paulo, SP, Brazil) was dissolved in physiological DZP (Deg, Sao saline with one drop of Tween 80 (Sigma Aldrich, Brazil); vehicle (V) consisted of physiological saline containing one drop of Tween 80. Thirty minutes before behavioral evaluation, animals received a single dose of DZP (0.25 mg/kg body weight i.p.), based on pilot studies conducted in our laboratory (data not shown) and on other studies (Loiseau et al., 2006; Saldı´var-Gonza´lez et al., 2000), or vehicle. All animals were then submitted to behavioral assessment.

4.4.

Behavioral testing

4.4.1.

Elevated plus maze (EPM) test

This test 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

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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  13.5 cm), which gave access to any of the four arms (Pellow et al., 1985). At the beginning of each test, the rat was placed in central platform facing an open arm and evaluated for 5 min. Spatiotemporal measures were the number of entries and time spent in both arms of the maze (expressed as a percentage of total test duration), used as measures of the anxiety level. Ethological measures included the frequency of head dipping (exploratory movement of head/shoulders over sides of the open arms and down towards the floor), stretched-attend postures (SAP; when the animal stretches to its full length with the forepaws and turns back to its original position without moving forward) and rearing. These categories were defined according to previous studies (Albrechet-Souza et al., 2007; Griebel et al., 2002; Hlavacova et al., 2010; Lepicard et al., 2000; Rodgers and Dalvi, 1997). The apparatus was cleaned with alcoholic solution (20%) using a wet sponge and a paper towel before introduction of each animal.

4.4.2.

Staircase test

This is a simple and rapid test used to study some components of exploratory behavior in the rat. The procedure was first proposed by Molinengo and Ricci-Gamalero (1970) and the apparatus comprised an enclosed staircase made of wood with five steps. Each step was 2.5 cm in height, 7.5 cm in length, and 10 cm in width, so that the staircase rose to a height of 12.5 cm at the top step. The total length of the apparatus was 45 cm and it was surrounded by walls 12.5 cm in height at one end, rising to 25 cm at the other. Rats were individually placed on the floor of the box, facing away from the stairs. The scores of rearing and number of steps climbed in a 3-min period were recorded (Thie´bot et al., 1973). The apparatus was cleaned with alcoholic solution (20%) and paper towel between each test.

4.4.3.

Open field test

This behavioral measure is relevant for rodents because it involves the natural tendency of animals to explore new environments, despite having stress and conflict (Henderson et al., 2004). Each rat was individually placed for 5 min in the center of the open-field arena (40  40  30 cm) subdivided into nine equal squares as described elsewhere (Kerr et al., 2005). The number of crossings (horizontal squares crossed) and rearings (vertical movements) was used respectively as measures of locomotor activity and exploratory behavior, respectively, whereas the numbers of entries in central squares were used as measures of anxiety (Henderson et al., 2004).

4.4.4.

Defensive burying test (DBT)

Anxiety-like behavior following a single shock from a novel object, a shock probe, can be assessed in this test (Matuszewich et al., 2007). The apparatus was a modified home cage (40  30  50 cm) with 4 cm of wood chip bedding material evenly distributed throughout the cage (Matuszewich et al., 2007). One end of the cage contained a shock probe with a constant current of approximately 1.0 mA

(Treit et al., 1981). Each animal was placed individually into the testing apparatus facing away from the shock probe for 10 min (Gutie´rrez-Garcı´a et al., 2006). When an animal received a shock by making contact with the shock probe, the current was terminated so as not to provide additional shocks. Duration of burying behavior was measured and this conduct was defined as any spraying or throwing of the bedding with the head or forepaws towards the shock probe, which is often used as a measure of coping strategy. The immobility time (standing on four feet with body and head motionless) was assessed as well, which is typically recognized as the anxiety measure in this test (Matuszewich et al., 2007). After 10 min, the animal was removed and returned to his home cage, the apparatus was cleaned and new bedding was placed into the cage for the next rat.

4.5.

Biochemical assays

One day after behavioral testing, 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 for plasma and used for glutathione peroxidase (GSH), cortisol and vitamin C (VIT C) determination. Brains were immediately removed and cut coronally at the caudal border of the olfactory tubercle. Cortex, subthalamic region and hippocampus were dissected according to Paxinos and Watson (2007), and homogenized in 10 volumes (w/v) of 10 mM Tris–HCl buffer (pH 7.4) for the determination of protein carbonyl (PC) levels, which estimates oxidative damages to proteins.

4.5.1.

Cortisol measurement

Corticosterone and cortisol are regulated in the same way and released in parallel (Saito et al., 1992) allowing cortisol to stand as a general stress measure for adrenocortical function (Milanes et al., 1991). Different laboratories (Issa et al., 2010; Prasad et al., 2006; Radahmadi et al., 2006) have used cortisol to estimate stress development in rats, whose level was quantified in the present study using a commercially available EIA kit (EIAgenTM Cortisol, Adaltis Italy S.p.A).The specificity of the test was evaluated by comparing the parallelism between the standard curve and serial dilutions in PBS (pH 7.4) of plasma samples.

4.5.2.

Vitamin C levels

Plasmatic vitamin C (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 1 volume of a cold 5% trichloroacetic acid solution followed by centrifugation. An aliquot of the supernatants was mixed with 2,4-dinitrophenylhydrazine (4.5 mg/mL) and 13.3% trichloroacetic acid and incubated for 3 h at 37 1C. After the addition of 65% sulfuric acid an orange red color was measured at 520 nm. A standard curve using ascorbic acid was used to calculate the content of VIT C and expressed as mg VIT C/mL plasma.

brain research 1474 (2012) 50–59

4.5.3.

Reduced gluthatione (GSH) levels

Plasma GSH content was determined after reaction with 5,50 dithiobis-(2-nitrobenzoic acid); the yellow color developed was read at 412 nm, in accordance with Boyne and Ellman (1972) after modifications (Jacques-Silva et al., 2001). A standard curve using glutathione was constructed in order to calculate the content of GSH, expressed as nmol GSH/mL plasma.

4.5.4.

Protein carbonyl (PC) quantification

PC was measured according to Yan et al. (1995), with some modifications. Soluble protein was mixed with 2,4-dinitrophenylhydrazine (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). The results were expressed as nmol carbonyl/g tissue.

4.6.

Statistical analysis

Data were analyzed by two-way ANOVA followed by Duncan’s post hoc tests when appropriated. (Software package Statistica for Windows version 8.0 was used.) The values of Po0.05 were considered statistically significant for all comparisons made.

Acknowledgments The authors wish to thank CAPES, CNPq and PRPGP-UFSM for their fellowships and to FAPERGS for the financial support.

r e f e r e n c e s

~ M.L., Albrechet-Souza, L., de Carvalho, M.C., Franci, C.R., Brandao, 2007. Increases in plasma corticosterone and stretched-attend postures in rats naive and previously exposed to the elevated plus-maze are sensitive to the anxiolytic-like effects of midazolam. Horm. Behav. 52, 267–273. Archer, J., 1973. Tests for emotionality in rats and mice: a review. Anim. Behav. 21, 205–235. Beck, A.T., Emery, G., Greenberg, R.L., 1985. Anxiety Disorders and Phobias: A Cognitive Perspective. Basic Books, New York. 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. Bodnoff, S.R., Suranyi-Cadotte, B., Quirion, R., Meaney, M.J., 1987. Postnatal handling reduces novelty-induced fear and increases 3H-flunitrazepam binding in rat brain. Eur. J. Pharmacol. 144, 105–107. Boyne, A.F., Ellman, G.L., 1972. A methodology for analysis of tissue sulfhydryl components. Anal. Biochem. 46, 639. Caldji, C., Francis, F., Tannenbaum, B., Sharma, S., Meaney, M.J., 1998. Maternal care in infancy influences the development of neural systems mediating fearfulness in the rat. Proc. Natl. Acad. Sci. U.S.A. 95, 5335–5340.

57

Caldji, C., Francis, D., Sharma, S., Plotsky, P.M., Meaney, M., 2000. The effects of early rearing environment on the development of GABAA and central benzodiazepine receptor levels and novelty-induced fearfulness in the rat. Neuropsychopharmacology 22, 219–229. Caldji, C., Diorio, J., Meaney, M., 2003. Variations in maternal care alter GABAA receptor subunit expression in brain regions associated with fear. Neuropsychopharmacology 28, 1950–1959. Cameron, N.M., Champagne, F.A., Parent, C., Fish, E.W., OzakiKuroda, K., Meaney, M.J., 2005. The programming of individual differences in defensive responses and reproductive strategies in the rat through variations in maternal care. Neurosci. Biobehav. Rev. 29, 843–865. Carlini, E.A., 2003. Plants and the central nervous system. Pharmacol. Biochem. Behav. 75, 501–512.  G.S., Ruggieri, V., Angelucci, L., Casolini, P., Cigliana, G., Alema, Catalani, A., 1997. Effect of increased maternal corticosterone during lactation on hippocampal corticosteroid receptors, stress response and learning offspring in the early stages of life. Neuroscience 79, 1005–1012. Cirulli, F., Alleva, E., Antonelli, A., Aloe, L., 2000. NGF expression in the developing rat brain: effects of maternal separation. Dev. Brain. Res. 123, 129–134. Cirulli, F., Berry, A., Alleva, E., 2003. Early disruption of the mother–infant relationship: effects on brain plasticity and implications for psychopathology. Neurosci. Biobehav. 27, 73–82. Cirulli, F., Berry, A., Bonsignore, L.T., Caponea, F., D’Andrea, I., Aloe, L., Branchi, I., Alleva, E., 2010. Early life influences on emotional reactivity: evidence that social enrichment has greater effects than handling on anxiety-like behaviors, neuroendocrine responses to stress and central BDNF levels. Neurosci. Biobehav. Rev. 34, 808–820. Cole, J.C., Rodgers, R.J., 1993. An ethological analysis of the effects of chlordiazepoxide and bretazenil (Ro 16–6028) in the murine elevated plus-maze. Behav. Pharmacol. 4, 573–580. Cole, J.C., Rodgers, R.J., 1995. Ethological comparison of the effects of diazepam and acute/chronic imipramine on the behaviour of mice in the elevated plus-maze. Pharmacol. Biochem. Behav. 52, 473–478. Denenberg, V.H., Smith, A.S., 1963. Effects of infantile stimulation and age upon behavior. J. Comp. Physiol. Psychol. 56, 307–312. Denenberg, V.H., 1964. Critical periods, stimulus input, and emotional reactivity: a theory of infantile stimulation. Psychol. Rev. 71, 335–351. File, S.E., 1997. Animal models of anxiety. In: Crawley, J.N., Gerfen, C.R., McKay, R., Rogawski, M.A., Sibley, D.R., Skolnik, P. (Eds.), Current Protocols in Neuroscience. John Wiley and Sons, New York, pp. 8.3.1–8.3.15. Ferna´ndez-Teruel, A., Escorihuela, R.M., Jime´nez, P., Toben˜a, A., 1990. Infantile stimulation and perinatal administration of Ro 15–1788: additive anxiety-reducing effects in rats. Eur. J. Pharmacol. 191, 111–114. Gallager, D.W., Primus, R.J., 1993. Benzodiazepine tolerance and dependence: GABAA receptor complex locus of change. In: Biochemical Society Symposium, pp. 135–151. Galley, H., Davies, M.J., Webster, N.R., 1996. Ascorbil radical formation in patients with sepsis: effects of ascorbate loading. Free Radical Biol. Med. 20, 139–143. Giachino, C., Canalia, N., Capone, F., Fasolo, A., Alleva, E., Riva, M.A., Cirulli, F., Peretto, P., 2007. Maternal deprivation and early handling affect density of calcium binding proteincontaining neurons in selected brain regions and emotional behavior in periadolescent rats. Neuroscience 145, 568–578. Gonzalez, L.E., Andrews, N., File, S.E., 1996. 5-HT1A and benzodiazepine receptors in the basolateral amygdala modulate anxiety in the social interaction test, but not in the elevated plus-maze. Brain Res. 732, 145–153.

58

brain research 1474 (2012) 50–59

Gray, J.A., 1987. The Psychology of Fear and Stress. Cambridge University Press, Cambridge, UK 16–21. Griebel, G., Simiand, J., Steinberg, R., Jung, M., Gully, D., Roger, P., Geslin, M., Scatton, B., Maffrand, JP., Soubrie´, P., 2002. 4(2-Chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)1,3-thiazol-2-amine hydrochloride (SSR125543A), a potent and selective corticotrophin-releasing factor(1) receptor antagonist II. Characterization in rodent models of stress-related disorders. Pharmacol. Exp. Ther. 30, 333–345. Gschanes, A., Eggenreich, U., Windisch, M., Crailsheim, K., 1998. Early postnatal stimulation influences passive avoidance behaviour of adult rats. Behav. Brain Res. 93, 91–98. Gutie´rrez-Garcı´a, A.G., Contreras, C.M., Mendoza-Lo´pez, M.R., Cruz-Sa´nchez, S., Garcı´a-Barradas, O., Rodrı´guez-Landa, J.F., Bernal-Morales, B., 2006. A single session of emotional stress produces anxiety in Wistar rats. Behav. Brain Res. 167, 30–35. Handley, S.L., 1991. Serotonin in animal models of anxiety: the importance of stimulus and response. In: Idzikowski, C., Cowen, P.J. (Eds.), Serotonin, Sleep and Mental Disorder, Wrightson, London, pp. 89–115. Henderson, N.D., Turri, M.G., DeFries, J.C., Flint, J., 2004. QTL analyses of multiple behavioral measures of anxiety in mice. Behav. Genet. 34, 267–293. Hilakivi-Clarke, L.A., Turkka, J., Lister, R.G., Linnoila, M., 1991. Effects of early postnatal handling on brain b-adrenoceptors and behavior in tests related to stress. Brain Res. 542, 286–292. 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. Hoehn-Saric, R., McLeod, D.R., 1988. The peripheral sympathetic nervous system. Its role in normal and pathologic anxiety. Psychiatr. Clin. North Am. 11, 375–386. Hovatta, I., Juhila, J., Donner, J., 2010. Oxidative stress in anxiety and comorbid disorders. Neurosci. Res. 68, 261–275. Imanaka, A., Morinobu, S., Toki, S., Yamamoto, S., Matsuki, A., Kozuru, T., et al., 2008. Neonatal tactile stimulation reverses the effect of neonatal isolation on open-field and anxiety-like behavior, and pain sensitivity in male and female adult Sprague-Dawley rats. Behav. Brain Res. 186, 91–97. Inazusta, J., Tejedor-Real, P., Varona, A., Costela, C., Gibert-Rahola, J., Casis, L., 1999. Effect of neonatal handling on brain enkephalindegrading peptidase activities. Neurochem. Int. 35, 357–361. Issa, G., Wilson, C., Terry Jr., A.V., Pillai, A., 2010. An inverse relationship between cortisol and BDNF levels in schizophrenia: data from human postmortem and animal studies. Neurobiol. Dis. 39, 327–333. Jacobson, L.H., Cryan, J.F., 2008. Evaluation of the anxiolytic-like profile of the GABAB receptor positive modulator CGP7930 in rodents. Neuropharmacology 54, 854–862. 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. Kerr, D.S., Bevilaqua, L.R., Bonini, J.S., Rossato, J.I., Ko¨hler, C.A., Medina, J.H., 2005. Angiotensin II blocks memory consolidation through an AT2 receptordependent mechanism. Psychopharmacology 179, 529–535. Laurie, D.J., Wisden, W., Seeburg, P.H., 1992. The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development. J. Neurosci. 12, 4151–4172. Lee, M.H.S., Williams, D.I., 1975. Long term changes in nest condition and pup grooming following handling of rat litters. Dev. Psychobiol. 8, 91–95.

Lepicard, E.M., Joubert, C., Hagneau, I., Perez-Diaz, F., Chapouthier, G., 2000. Differences in anxiety-related behavior and response to diazepam in BALB/cByJ and C57BL/6J strains of mice. Pharmacol. Biochem. Behav. 67, 739–748. Levine, S., 1957. Infantile experience and resistance to physiological stress. Science 126, 405. 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. Levine, S., 1960. Stimulation in infancy. Sci. Am. 202, 81–86. Levine, S., 1962. Plasma-free corticosteroid response to electric shock in rats stimulated in infancy. Science 135, 795–796. Levine, S., Haltmeyer, G.C., Karas, G.G., Denenberg, V.H., 1967. Physiological and behavioral effects of infantile stimulation. Physiol. Behav. 2, 55–59. Levine, S., 1994. Maternal behaviour as a mediator of pup adrenocortical function. Ann. N.Y. Acad. Sci 746, 260–275. Lister, R.G., 1987. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology 92, 180–185. Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S., Pearson, D., Plotsky, P.M., Meaney, M.J., 1997. Maternal care, hippocampal glucocorticoid receptor gene expression and hypothalamic–pituitary–adrenal responses to stress. Science 277, 1659–1662. Loiseau, F., Le Bihan, C., Hamon, M., Thie´bot, M.H., 2006. Effects of melatonin and agomelatine in anxiety-related procedures in rats: interaction with diazepam. Eur. Neuropsychopharmacol. 16, 417–428. Matuszewich, L., Karney, J.J., Carter, S.R., Janasik, S.P., O’Brien, J.L., Friedman, R,D., 2007. The delayed effects of chronic unpredictable stress on anxiety measures. Physiol. Behav. 90, 674–681. Meaney, M.J., Mitchel, J.B., Aitken, D.H., Bhatnagar, S., Bodnoff, S.R., Iny, L.J., Sarrieau, A., 1991. The effects of neonatal handling on the development of the adrenocortical response to stress: implications for neuropathology and cognitive deficits in later life. Psychoneuroendocrinology 16, 85–103. Meaney, M.J., Aitken, D.H., Sharma, S., Viau, V., 1992. Basal ACTH, corticosterone, and corticosteroid-binding globulin levels over the diurnal cycle, and hippocampal type I and type II corticosteroid receptors in young and old, handled and nonhandled rats. Neuroendocrinology 55, 204–213. Milanes, M.V., Gonzalvez, M.L., Fuente, T., Vargas, M.L., 1991. Pituitary-adrenocortical response to acute and chronic administration of U-50,488H in the rat. Neuropeptides 20, 95–102. Millan, M.J., 2003. The neurobiology and control of anxious states. Prog. Neurobiol. 70, 83–244. Molinengo, L., Ricci-Gamalero, S., 1970. The ‘‘staircase maze’’ and the ‘‘simple staircase’’ in the analysis of the psychopharmacologial action of CNS depressants. Pharmacology 4, 169–178. Montagu, A., 1953. The sensory influences of the skin. Tex. Rep. Biol. Med. 11, 292–301. Montgomery, K.C., 1955. The relationship between fear induced by novel stimulation and exploratory behavior. J. Comp. Physiol. Psychol. 48, 254–260. Nu´n˜ez, J.F., Ferre´, P., Garcia, E., Escorihuela, R.M., Ferna´ndezTeruel, A., Toben˜a, A., 1995. Postnatal handling reduces emotionality ratings and accelerates two-way active avoidance in female rats. Physiol. Behav. 57, 831–835. Nu´n˜ez, J.F., Ferre´, P., Escorihuela, R.M., Toben˜a, A., Ferna´ndezTeruel, A., 1996. Effects of postnatal handling of rats on emotional, HPA-axis, and prolactin reactivity to novelty and conflict. Physiol. Behav. 60, 1355–1359. Paxinos, G., Watson, C., 2007. The Rat Brain in Stereotaxic Coordinates, sixth ed. Academic Press, New York. Pellow, S., Chopin, P., File, S.E., Briley, M., 1985. Validation of open:closed arm entries in an elevated pluz mase as a measure of anxiety in the rat. J. Neurosci. Meth. 14, 149–167.

brain research 1474 (2012) 50–59

Pesold, C., Treit, D., 1995. The central and basolateral amygdala differentially mediate the anxiolytic effects of benzodiazepines. Brain Res. 671, 213–221. Pham, T.M., So¨derstro¨m, 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. Prasad, A., Naskar, R., Dubey, R., Raha, D., Ahmed, M.F., 2006. Modulation of serum cortisol by substance P in albino rats: evidence of a direct effect on adrenal gland. Indian J. Exp. Biol. 44, 163–164. Pryce, C.R., Bettschen, D., Feldon, J., 2001. Comparison of the effects of early handling and early deprivation on maternal care in the rat. Dev. Psychobiol. 38, 239–251. Pryce, C.R., Feldon, J., 2003. Long-term neurobehavioural impact of the postnatal environment in rats: manipulations, effects and mediating mechanisms. Neurosci. Biobehav. Rev. 27, 57–71. Radahmadi, M., Shadan, F., Karimian, S.M., Sadr, S.S., Nasimi, A., 2006. Effects of stress on exacerbation of diabetes mellitus, serum glucose and cortisol levels and body weight in rats. Pathophysiology 13, 51–55. Rodgers, R.J., Johnson, N.J., 1995. Factor analysis of spatiotemporal and ethological measures in the murine elevated plusmaze test of anxiety. Pharmacol. Biochem. Behav. 52, 297–303. Rodgers, R.J., Dalvi, A., 1997. Anxiety, defence and the elevated plus maze. Neurosci. Biobehav. Rev. 21, 801–810. 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. Saito, H., Sato, T., Kaba, H., Tadokoro, M., Edashige, N., Seto, K., Kimura, F., Kawakami, M., 1992. Influence of the electrical stimulation of the hypothalamus on adrenocortical steroidogenesis in hypophysectomized rats. Exp. Clin. Endocrinol. 99, 110–112. Saldı´var-Gonza´lez, A., Go´mez, C., Martı´nez-Lomelı´, I., Arias, C., 2000. Effect of flumazenil and diazepam on transient actions in defensive burying elicited by the social interaction experience in rats. Pharmacol. Biochem. Behav. 66, 265–273. Salim, S., Sarraj, N., Taneja, M., Saha, K., Tejada-Simon, M.V., Chugh, G., 2010. Moderate treadmill exercise prevents oxidative stress-induced anxiety-like behavior in rats. Behav. Brain Res. 208, 545–552. Salomons, A.R., Kortleve, T., Reinders, N.R., Kirchhoff, S., Arndt, S.S., Ohl, F., 2010. Susceptibility of a potential animal model for pathological anxiety to chronic mild stress. Behav. Brain Res. 209, 241–248. Schapiro, S., Vukovich, K.R., 1970. Early experience effects upon cortical dendrites: a proposed model for development. Science 167, 292–294. Setem, J., Pinheiro, A.P., Motta, V.A., Morato, S., Cruz, A.P.M., 1999. Ethopharmacological analysis of 5-HT ligands on the rat

59

elevated plus maze. Pharmacol. Biochem. Behav. 62, 515–521. Shepherd, J.K., Grewal, S.S., Fletcher, A., Bill, D.J., Dourish, C.T., 1994. Behavioural and pharmacological characterisation of the elevated zero-maze as an animal model of anxiety. Psychopharmacology 116, 56–64. Shoji, H., Mizoguchi, K., 2010. Acute and repeated stress differentially regulates behavioral, endocrine, neural parameters relevant to emotional and stress response in young and aged rats. Behav. Brain Res. 211, 169–177. Sternberg, W.F., Ridgway, C.G., 2003. Effects of gestational stress and neonatal handling on pain, analgesia, and stress behavior of adult mice. Physiol. Behav. 78, 375–383. Ste´ru, L., Thierry, B., Chermat, R., Millet, B., Simon, P., Porsolt, R.D., 1987. Comparing benzodiazepines using the staircase test in mice. Psychopharmacology 92, 106–109. Thie´bot, M.H., Soubrie´, P., Simon, P., Boissier, J.R., 1973. Dissociation of two components of rat behaviour by psychotropic drugs. Utilization for studying anxiolytic drugs. Psychopharmacology 31, 77–90. Thomas, S.R., Lewis, M.E., Iversen, S.D., 1985. Correlation of [3H]diazepam binding density with anxiolytic locus in the amygdaloid complex of the rat. Brain Res. 342, 85–90. Treit, D., Pinel, J.P., Fibiger, H.C., 1981. Conditioned defensive burying: a new paradigm for the study of anxiolytic agents. Pharmacol. Biochem. Behav. 15, 619–626. Vallee, M., Mayo, W., Dellu, F., Le Moal, M., Simon, H., Maccari, S., 1997. Prenatal stress induces high anxiety and postnatal handling induces low anxiety in adult offspring: correlation with stress induced corticosterone secretion. J. Neurosci. 17, 2626–2636. Vekovischeva, O.U., Haapalinna, A., Sarviharju, M., Honkanen, A., Korpi, E.R., 1999. Cerebellar GABA receptors and anxiolytic action of diazepam. Brain Res. 837, 184–187. Wakshlak, A., Weinstock, M., 1990. Neonatal handling reverses behavioral abnormalities induced in rats by prenatal stress. Physiol. Behav. 48, 289–292. Weaver, I.C.G., Cervoni, N., Champagne, F.A., D’Alessio, A.C., Sharma, S., Seckl, J.R., 2004. Epigenetic programming by maternal behavior. Nat. Neurosci. 7, 847–854. Woods, J.H., Katz, J.L., Winger, G., 1992. Benzodiazepines: use, abuse, and consequences. Pharmacol. Rev. 44, 151–347. Yan, L.Y., Traber, M.G., Packer, L., 1995. Spectrophotometric method for determination of carbonyls in oxydatively modified apolipoprotein B of human low-density lipoproteins. Anal. Biochem. 228, 349–351. Zhang, M., Cai, J.X., 2006. Effects of neonatal tactile stimulation and maternal separation on the anxiety and the emotional memory in adult female rats. Zool. Res. 27, 7. 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.