Withdrawal from methylphenidate increases neural reactivity of dorsal midbrain

Neuroscience Research 68 (2010) 290–300

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Withdrawal from methylphenidate increases neural reactivity of dorsal midbrain R. Ferreira a,b , G.S. Bassi a,b , A. Cabral a,b , M.J. Nobre a,b,∗ a b

Instituto de Neurociências & Comportamento – INeC, Campus USP, Ribeirão Preto, SP 14040-901, Brazil Laboratório de Neuropsicofarmacologia, Departamento de Psicologia e Educac¸ão, FFCLRP, Campus USP, Ribeirão Preto, SP 14040-901, Brazil

a r t i c l e

i n f o

Article history: Received 30 June 2010 Received in revised form 25 August 2010 Accepted 31 August 2010 Available online 9 September 2010 Keywords: Methylphenidate Withdrawal Anxiety Brainstem

a b s t r a c t Ritalin (methylphenidate hydrochloride, MP) is a non-amphetamine psychostimulant and is the drug of choice to treat children and adults diagnosed with the attention deficit hyperactivity disorder (ADHD). Several studies have demonstrated that rats treated with MP during early developmental stage exhibit alterations in anxiety-related processes such as an increased response to stressful stimuli and elevated plasma levels of corticosterone. Accordingly, the present study was designed to further characterize the neural and behavioral consequences of withdrawal from MP in adult rats and its influence on the neural reactivity of the dorsal midbrain. After initial exposure to an elevated plus-maze (EPM), brainstem neural activation, elicited by exposure to EPM aversive cues, was analyzed using a Fos-protein immunolabeling technique. Additional independent groups of animals were submitted to electrical stimulation of the dorsal column (DPAG) or the startle response procedure, in order to verify the influence of withdrawal from MP on the expression of unconditioned fear induced by DPAG activation and the effects of or withdrawal from MP on motor response, respectively. Our results provide new findings about the influence of MP treatment in adult rats, showing that, after a sudden MP treatment-break, increased anxiety, associated with the neural sensitization of anxiety-related regions, ensues. © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

1. Introduction Ritalin (methylphenidate hydrochloride, MP) is a nonamphetamine psychostimulant and is the drug of choice to treat children and adults diagnosed with the attention deficit hyperactivity disorder (ADHD) (Solanto, 1998; Leonard et al., 2004) and narcolepsy (Leonard et al., 2004). For the most part, the stimulants used to treat ADHD (MP and dextroamphetamine) have been found to be safe and effective (Spencer et al., 1996). However, concerns about the possible overuse of MP in young children have been disseminated both in the media and in scientific publications (Zito et al., 2000; Accardo and Blondis, 2001; Kollins et al., 2001; Kollins, 2003), and there is controversy over whether MP treatment has the potential to elicit drug dependence in the same way as other psychostimulants, such as cocaine and amphetamine (Jaffe, 1991; Kollins, 2003). In addition, clinical studies showed that even therapeutic doses of MP can induce severe manic-like or psychotic-like symptoms in children for which MP is used as a treatment for ADHD (Ross, 2006). This is attested by retrospective data showing high prevalence rates of schizophrenia or bipolar

∗ Corresponding author at: Laboratório de Neuropsicofarmacologia, Departamento de Psicologia e Educac¸ão, FFCLRP, Campus USP, Ribeirão Preto, SP 14040-901, Brazil. Tel.: +55 1636023788; fax: +55 1636024830. E-mail address: [email protected] (M.J. Nobre).

disorder in children under MP treatment (Schaeffer and Ross, 2002). MP has a neuropharmacological profile similar to that of cocaine (Volkow et al., 1999), releasing catecholamines and also inhibiting their reuptake, primarily that of dopamine (DA), through its inhibitory action on the DA transporter (Izenwasser et al., 1999). This causes an increase in extracellular DA levels (Hurd and Ungerstdet, 1989; Challman and Lipsky, 2000; Leonard et al., 2004), a phenomenon that has been linked to its reinforcing properties (Ritz et al., 1987; Sonders et al., 1997; Volkow et al., 1999). Despite the large number of children being treated with MP it is surprising that few studies have addressed the consequences of chronic MP exposure or withdrawal after long-or even short-term use. Behavioral studies have demonstrated that rats treated with MP during early developmental stage exhibit alterations in anxietyrelated processes such as an increased response to stressful stimuli, elevated plasma levels of corticosterone and depression-like symp˜ et al., 2003; Carlezon et al., 2003). For toms in adulthood (Bolanos ˜ example, Bolanos et al. (2003) investigated the long-term effects of chronic administration of MP in adolescent rats, and showed that MP-treated animals were significantly more sensitive to stressful situations, when tested at later stages of development (adult phase). Therapeutic doses of stimulants, as MP, can cause manic- or psychotic-like symptoms in children under MP treatment, among then increased irritability and aggression. Anxiety is also a frequent

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R. Ferreira et al. / Neuroscience Research 68 (2010) 290–300

complaint among patients with bipolar disorders. Patients with bipolar disorder and high levels of anxiety symptoms are more likely to exhibit suicidal behavior. In addition, the strong association between anxiety and alcohol abuse suggests that patients with bipolar disorder and high levels of anxiety may be at risk of alcohol-related problems. In this regard, we have consistently demonstrated that withdrawal from other drugs of abuse as benzodiazepines, opiates, and alcohol promotes similar emotional disturbances, increasing the neural sensitivity of brainstem structures that are mainly involved in the modulation/expression of unconditioned anxiety- and fear-related behaviors (Avila et al., 2008; Castilho et al., 2008; Fontanesi et al., 2007; Souza-Pinto et al., 2007). Accordingly, the present study was designed to further characterize the consequences of withdrawal from MP on the anxiety-like behavior, motor response and neural reactivity of the dorsal periaqueductal gray neurons in rats, using behavioral, electrophysiological and immunohistochemical techniques. 2. Materials and methods 2.1. Animals Eighty-nine naive male Wistar rats, weighing 200–210 g at the beginning of the treatment, were obtained from the animal house on the campus of Ribeirão Preto, University of São Paulo. They were housed in groups of four in Plexiglas-walled cages, lined with wood shavings that were changed every 3 days, and were maintained in a 12:12 dark/light cycle (lights on 07:00 h) at 24 ± 1◦ C. The rats were given free access to food and water. Before the beginning of treatment, the animals had a 3-day habituation period to the lodging conditions. 2.2. Ethical statements The authors declare that all experiments received formal approval from the Committee on Animal Research and Ethics (CEUA) of the University of São Paulo. In addition, the experiments were performed in compliance with the recommendations of the Brazilian Society for Neuroscience and Behavior, which are in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals, in an effort to minimize the number of animals used and their suffering. 2.3. Administration of methylphenidate Rats were given 20 MP (Ritalin, Novartis) subcutaneous (s.c.) injections (days 1–3 = 5 mg, days 4–6 = 10 mg, days 7–11 = 20 mg/kg) twice a day (09:00 and 19:00 h), for 11 days, in a procedure adapted from the study by Chase et al. (2005). The first injection was given in the evening of the first day (19:00 h) and the last s.c. injection was administered on the morning of the last day of treatment (09:00 h). Analysis of the chronic effects and withdrawal of the drug was conducted 30 min or 48 h after the last injection, respectively. The use of this procedure was mainly to: (1) avoid the commonly sensitization to locomotor behavior, frequently noted when MP was administered at more than 1 mg/kg but less than or equal to 4 mg/kg, and not seen when MP doses exceeded 5 mg/kg (Gaytan et al., 1997, 2000, 2002; Sripadab et al., 1998; Eckermann et al., 2001; Kuczenski and Segal, 2002; Yang et al., 2001), and (2) counterbalance the tolerance developed to repeated administration of higher doses (9–25 mg/kg) of MP (Browne and Segal, 1977; McNamaraa et al., 1993; Crawford et al., 1998; Izenwasser et al., 1999).

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2.4. Experiment I: Effects of withdrawal from MP on the anxiety-like behavior of rats tested in the elevated plus-maze This experiment was carried out in order to analyze the chronic effects of and withdrawal from MP on the expression of anxietylike behaviors elicited by EPM exposure in 37 adults male Wistar rats. 2.4.1. Experimental apparatus The EPM was made of wood and consisted of four arms of equal dimensions (50 cm × 12 cm). Two of the arms were enclosed by 40cm high walls and were arranged perpendicularly to two opposite open arms. The apparatus was elevated 50 cm above the floor, with a 1-cm Plexiglas rim surrounding the open arms to prevent falls (Pellow et al., 1985). The apparatus was located inside an isolated room with 20 lx of luminosity at the end of the open arms. The behavior of the animals in the plus-maze was recorded via a camera (Everfocus, USA) linked to a monitor and videocassette, external to the experimental room. 2.4.2. Variables recorded The performance of rats in the EPM was measured by recording the traditional (percentage of time spent and entries into open arms, and frequency of closed arm entries) and naturalistic behaviors (number of times the animals reached the end of the open arms, number of times the animals performed an unprotected head-dipping, number of stretching-attend postures and rearing behavior, and time spent self-grooming). Three groups were formed: saline (n = 13), MP chronic effects (n = 12) and MP withdrawal effects (n = 12). Six animals in the saline group were exposed to the plus-maze 30 min after the last saline injection. The other seven animals were tested 48 h after this injection. Since no statistical significant difference was obtained between the groups the data were collapsed and presented together. 2.4.3. Experimental procedure The tests were conducted 30 min (under the chronic effects of the drug) or 48 h (under withdrawal, a condition in which the animals were tested free from the drug effects) after the last s.c. injection. The animals were placed in the center of the plus-maze facing one of the closed arms and were allowed to explore the environment for 5 min. Each animal was tested only once. The apparatus was cleaned with 20% ethanol and water before each test. 2.4.4. Statistical analysis The data are presented as mean ± SEM. Data were transformed through the use of the square root of raw data and were analyzed by a one-way ANOVA for each measure in the study. The post hoc Newman–Keuls test was employed when p < 0.05. 2.4.5. Results With regard to the traditional measures, the one-way ANOVA revealed significant differences in the percentage of time spent in the open arms of the maze [F(2,34) = 6.31; p < 0.005] (Fig. 1(B)). On the other hand, there was no significant difference between the frequency of closed- [F(2,34) = 1.14; p > 0.05] (Fig. 1(A)) and open-arm entries [F(2,34) = 1.76; p > 0.05] (Fig. 1(B)). With regard to the naturalistic measures (Fig. 2), ANOVA showed significant differences in the frequency of end arm exploration [F(2,34) = 8.98; p < 0.005], rearing [F(2,34) = 9.04; p < 0.001], unprotected head-dipping [F(2,34) = 8.10; p < 0.005] and the frequency of stretching-attend postures [F(2,34) = 3.25; p < 0.05]. There was no significant difference in the time spent self-grooming between the groups tested [F(2,34) = 0.45; p > 0.05]. The Newman–Keuls post hoc test revealed that chronic treatment with MP, per se, did not have any effect on the behavior of rats tested after the interruption

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Fig. 1. Mean ± SEM of the percentage of entries and time spent in the open arms (B), and number of closed arm entries (A) of rats pretreated with MP and tested under chronic or withdrawal (Withd) effects. For statistical comparisons one-way ANOVA was used followed by the Newman–Keuls post hoc test, in the case of p < 0.05. *Significant difference from saline group.

of long-term treatment, as there were no significant differences in any of the measures, when compared with those obtained in control animals. On the other hand, 48 h of withdrawal significantly enhanced the expression of anxiety-like behaviors since the

percentage of open arm time (Fig. 1(B)), the number of times MPwithdrawn rats visited the end of open arms, and the frequency of rearing and unprotected head-dipping decreased, whereas the stretching-attend postures increased (Fig. 2).

Fig. 2. Mean ± SEM of the number of end arm explorations, rearings, head-dippings, stretching-attend postures (SAP) and time spent self-grooming during 5 min EPM exposure of saline and MP pretreated rats tested under chronic effects or withdrawal (Withd). Data were analyzed by a one-way ANOVA, followed by the Newman–Keuls post hoc test. *Significant difference from saline group at the level of p < 0.05.

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2.5. Experiment II: Effects of withdrawal from MP on the brainstem neural activation induced by aversive plus-maze cues We evaluated the Fos-protein expression in 16 rats that were randomly chosen from the groups tested in the EPM (n = 6 for each group). After immunohistochemical procedures two animals were removed from the experiments due to problems with histology. The groups were composed as follows: saline (n = 5), MP chronic (n = 6) and MP withdrawal (n = 5). 2.5.1. Analysis of Fos-protein immunoreactivity Two hours after the plus-maze test, the animals were deeply anaesthetized with urethane (1.25 g/kg, i.p., Sigma, USA) and intracardially perfused with 0.1 M phosphate-buffered saline followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were removed and immersed (4 ◦ C) for 2 h in paraformaldehyde and then stored for at least 48 h in 30% sucrose in 0.1 M PBS cryoprotectant. They were then quickly frozen in isopentane (−40 ◦ C) and sliced using a cryostat (−19 ◦ C). Two adjacent series of 40 ␮m thick brain slices were obtained. One series was Nissl stained and used for neuroanatomical comparison purposes, and the other series was collected for immunohistochemical studies. Tissue sections were collected in 0.1 M PBS and subsequently processed free-floating according to the avidin–biotin procedure, using the Vecstatin ABC Elite peroxidase rabbit IgG kit (Vector, USA, ref. PK 6101). All reactions were carried out under agitation at room temperature. The slices were first incubated with 1% H2 O2 for 10 min, washed four times with 0.1 M PBS (5 min each) and then incubated overnight at room temperature with the primary Fos rabbit polyclonal IgG (Santa Cruz, USA, SC-52) at a concentration of 1:2000 in PBS+ (0.1 M PBS enriched with 0.2% Triton-X and 0.1% bovine serum albumin, BSA). Sections were again washed three times (5 min each) with 0.1 M PBS and incubated for 1 h with secondary Fos biotinylated anti-rabbit IgG (H + L) (Vecstatin, Vector Laboratories) at a concentration of 1:400 in PBS. After another series of three 5-min washes in 0.1 M PBS the sections were incubated for 1 h with the avidin–biotin–peroxidase complex in 0.1 M PBS (A and B solution of the ABC kit, Vecstatin, Vector Laboratories) at a concentration of 1:250 in 0.1 M PBS, and then were again washed three times in 0.1 M PBS (5 min per wash). Fos immunoreactivity was revealed by the addition of the chromogen 3,3 -di-aminobenzidine (DAB, 0.02%, Sigma), to which hydrogen peroxide (0.04%) was added just prior to use. Finally, the tissue sections were washed twice with 0.1 M PBS. 2.5.2. Quantification of Fos-positive cells Tissue sections were mounted on gelatin-coated slides, and dehydrated for observation and cell counting under bright-field microscopy. The nomenclature and nuclear boundaries utilized were based on the atlas of Paxinos and Watson (2005). Cells containing a nuclear brown-black reaction product with areas between 10 ␮m2 and 80 ␮m2 were identified and automatically counted as Fos-positive neurons by a computerized image analysis system (Image Pro Plus 4.0, Media Cybernetics, USA). Sections of 11 different midbrain regions, at different levels, were collected according to a method used in previous studies (Lamprea et al., 2002; Vianna et al., 2003; Ferreira-Netto et al., 2005). Mounted sections of the tissue were observed using a light microscope (Olympus BX-50) equipped with a video-camera module (Hamatsu Photonics C2400) and coupled to the computerized image analysis system mentioned above. Counting of Fos-positive cells was performed under a 10× objective at a magnification of 100× in one field per area, encompassing the entire brain region included in quantification. An area of the same shape and size per brain region was used for each rat. The system was calibrated to ignore background stain-

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Table 1 Influence of the exposure to EPM aversive cues on the neural activation of midbrain regions of rats tested under the chronic effects of, or withdrawal from, MP, as revealed by the use of a Fos-immunolabeling technique. Mean (±SEM) number of Fos-positive neurons per 0.1 mm2 ) Structures

Nacc AcbC AcbSh Midbrain DMPAG DLPAG LPAG VLPAG DpSC CIC DR MnR LC

Saline

MP (Chronic)

MP (Withd)

Mean ± SEM

Mean ± SEM

Mean ± SEM

1.08 ± 0.45 1.21 ± 0.38

2.17 ± 0.35 2.12 ± 0.32

1.23 ± 0.27 1.91 ± 0.32

* *

± ± ± ± ± ± ± ± ±

* * * * * * * * *

1.0 0.9 1.1 1.1 0.3 0.4 1.8 2.3 1.9

± ± ± ± ± ± ± ± ±

0.2 0.2 0.1 0.1 0.01 0.1 0.2 0.1 0.1

0.8 1.0 1.1 0.7 0.8 1.2 1.4 1.0 1.3

± ± ± ± ± ± ± ± ±

0.2 0.2 0.3 0.1 0.2 0.1 0.1 0.2 0.1

2.6 2.4 2.4 2.1 1.3 1.8 3.2 2.2 2.0

0.4 0.4 0.1 0.1 0.2 0.2 0.3 0.4 0.1

p < 0.05

Data were transformed via the square root of the raw data and are presented as mean ± SEM (mean of right plus left brain side). Fos-positive cells were counted individually. Shaded areas represent a significant difference when compared with the saline control group.

ing. All brain regions were bilaterally counted for each rat. The analyzed encephalic regions and their respective AP coordinates from the bregma (Paxinos and Watson, 2005) were as follows: dorsomedial (DMPAG), dorsolateral (DLPAG), lateral (LPAG) and ventrolateral (VLPAG) parts of the intermediate and caudal portions of the periaqueductal gray (AP: −6.84 mm to −7.88 mm), the deep layers of the superior colliculus (DpSC) – gray and white layers (AP: −7.44 mm to −7.68 mm), the ventral part of the central nucleus of the caudal inferior colliculus (CIC) (AP: −8.16 mm to −8.52 mm), the dorsal (DR) and median raphe nucleus (MnR) – all parts (AP: −7.68 mm to −7.80 mm) and the locus coeruleus (LC) (−9.48 mm to −9.96 mm). Nuclei was counted individually and expressed as the number of Fos-positive cells per 0.1 mm2 (Lamprea et al., 2002; Vianna et al., 2003; Ferreira-Netto et al., 2005), as shown in Table 1. The selection of the areas sampled in this study considered the importance of these regions in the modulation of anxiety-like and fear-motivated behaviors (Brandão et al., 1999, 2003, 2005; Campeau et al., 1997; Charney et al., 1998; Graeff, 1990, 1994; Silveira et al., 1993, 1995), although we are aware that many other brain regions, not specified here, also contribute to the modulation/expression of behaviors elicited by fear stimuli. As an additional control Fos immunoreactivity induced in the core (AcbC) and shell (AcbSh) regions of the nucleus accumbens (NAcc) was also recorded. 2.5.3. Statistical analysis Data were transformed via the square root of the raw data. Fos-protein expression was analyzed by one-way ANOVA with the treatments and the structures in the study as the main levels. Statistical analysis was followed, when appropriate, by the Newman–Keuls post hoc test. The significance level was set at p < 0.05. 2.5.4. Results Statistical analysis of the chronic effects or withdrawal from MP on Fos-protein expression was conducted in brain areas of 16 animals randomly chosen from each of the groups tested in the plus-maze. The mean Fos-protein immunoreactivity in neuronal nuclei is shown in Fig. 3, for each brain region in study (see also Table 1). Fig. 4 shows a photomicrograph of the dorsal and lateral columns of the periaqueductal gray matter and the inferior colliculus, two of the main regions belonging to the well-known

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Fig. 3. Number of Fos-immunoreactive neurons in the core and shell regions of the NAcc and several mesencephalic regions of rats pretreated with saline or MP and tested under chronic MP effects or after 48 h of withdrawal (Withd) in the EPM. Data are expressed as mean ± SEM of Fos-positive cells in a 0.1 mm2 area of tissue. *Significant difference in the number of Fos-positive cells in each structure studied between MP and saline group. The analysis was performed using one-way ANOVA followed by the Newman–Keuls post hoc test. The level of significance was set at p < 0.05.

brain aversion system, which is involved in the modulation and expression of unconditioned anxiety and fear-like behaviors. Statistical analysis indicated significant differences in Fos expression in all areas studied (Fig. 3), as follows: AcbC [F(2,13) = 27.59; p < 0.0001], AcbSh [F(2,13) = 8.92; p < 0.005], DMPAG [F(2,13) = 11.36; p < 0.005], DLPAG [F(2,13) = 10.00; p < 0.005], LPAG [F(2,13) = 13.98; p < 0.005], VLPAG [F(2,13) = 25.87; p < 0.0001], DpSC [F(2,13) = 6.18; p < 0.05], CIC [F(2,13) = 30.58; p < 0.0001], DR [F(2,13) = 21.09; p < 0.0001], MnR [F(2,13) = 6.80; p < 0.05] and LC [F(2,13) = 9.15; p < 0.005]. Comparisons a posteriori showed that rats tested under chronic MP effects showed significant neural activation in the CIC (Fig. 4, bottom, middle). MP was effective at reducing Fos expression in the MnR and LC. On the other hand, 48 h of MP withdrawal resulted in significant increases in Fos-immunolabeling in almost all areas, including all periaqueductal gray columns (Fig. 4, top, right) and the CIC (Fig. 4, bottom, right), except for the MnR and LC. MP treatment resulted in significant Fos expression in both core (AcbC) and shell (AcbSh) regions of the NAcc (see Table 1). Post hoc comparisons showed

that MP increases neural activation in both nuclei. However, after 48 h of withdrawal this activation was observed only in the shell region. 2.6. Experiment III: Effects of MP withdrawal on the aversive thresholds determined by electrical stimulation of the DPAG In this experiment, we evaluated the effects of withdrawal from MP on the sensitization of the mesencephalic areas observed using Fos immunohistochemistry procedures. Since the DPAG is fundamentally involved in the expression of unconditioned anxiety and fear-related behaviors, we chose this area as the main site for electrical stimulation. Freezing and escape thresholds were the dependent variables. Independent groups of animals were used as follows: saline (n = 10) and MP (n = 10). 2.6.1. Surgery Surgery was performed 5 days before the end of the treatments (on day 5). The animals were anaesthetized with tribromoethanol

Fig. 4. Photomicrograph of Fos-like immunoreactive cells (dark dots) in coronal sections showing Fos expression throughout the regions of the dorsomedial (DMPAG), dorsolateral (DLPAG) and lateral (LPAG) columns of the periaqueductal gray and in the central nucleus of the inferior colliculus (CIC) of saline or MP pretreated rats tested under chronic or withdrawal effects in the EPM. SC, superior colliculus, Aq, aqueduct of Sylvius, ECIC, external cortex of the inferior colliculus.

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(250 mg/kg, i.p.) and fixed in a stereotaxic frame (Insight, São Paulo, Brazil). A bipolar electrode was implanted in the midbrain, aimed at the DPAG. The upper incisor bar was set at 2.5 mm below the interaural line so that the skull was horizontal between the bregma and lambda. The electrode was introduced using the following coordinates (Paxinos and Watson, 2005) with the bregma serving as the reference for each plane: anteroposterior = −7.5 mm, mediolateral = 1.9 mm, dorsoventral = 4.8 mm. The electrode was fixed to the skull by acrylic resin and two stainless steel screws. At the end of surgery each animal received an intramuscular injection (0.2 ml) of a veterinary pentabiotic (penicillin – 120,000 UI, plus 100 mg/ml of dihydrostreptomycin sulphate and streptomycin sulphate), followed by an injection of the anti-inflammatory and analgesic banamine (flunixin meglumine, 2.5 mg/kg). 2.6.2. Experimental apparatus The experimental setting in which brain stimulation was delivered consisted of a box (25 cm × 20 cm × 20 cm) with walls made of Plexiglas. The floor was made of stainless steel bars of 5 mm diameter, spaced 1.5 cm apart. This box was placed inside a sound attenuation chamber made of plywood, illuminated from within by a red 5 W lamp, and with 65 dB permanent background noise delivered by a fan. The behavior of the animals was recorded by a video-camera positioned beside the experimental box and monitored via a closed-circuit TV camera. The box was cleaned with a 5% ammonium solution immediately before each animal was placed within it. The brain was stimulated electrically, by means of a digital sine wave stimulator (Insight, São Paulo, Brazil), as previously described (Castilho et al., 1999). The stimulation current was monitored by measuring the voltage drop across a 1 k resistor with an oscilloscope (Philips, USA). 2.6.3. Experimental procedure Forty-eight hours following the interruption of the chronic treatment with saline or MP the animals were handled for 5 min and then placed in the experimental box, where they remained undisturbed for an additional 10-min period of habituation. After that, the electrical stimulation procedure started. Brain stimuli (AC, 60 Hz, 10 s) were presented at variable intervals (3 min ± 50%), with the current intensity increasing by steps of 10 ␮A for determination of the aversive thresholds. The freezing threshold was defined as the lowest intensity producing complete immobility except for respiratory movements. The escape threshold was defined as the lowest current intensity that produced running or jumping in two successive ascending series of electrical stimulation (as soon as the escape threshold was determined, electrical stimulation was stopped). Animals with an escape threshold above 200 ␮A (peakto-peak) were discarded from the experiment.

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Fig. 5. The sites of the microinjections in the dorsal aspects of the periaqueductal gray (DPAG) in agreement with the coordinates of the Paxinos and Watson’s atlas (2005). A representative photomicrograph of the site of DPAG electrical stimulation in rats tested under withdrawal from MP is shown in the upper panel. (䊉) Saline group; () MP withdrawal group.

results obtained in each experiment were subjected to one-way ANOVA for comparison between the MP and saline control groups. A probability level of p < 0.05 was considered significant.

2.6.6. Results As the intensity of the current applied to the DPAG was increased, animals in both groups suddenly stopped, became immobile and often urinated and defecated. At higher intensities, freezing behavior was followed by vigorous running and jumping. Representative sites of DPAG stimulation as well as the location of the tips of the electrodes inside these regions are shown in Fig. 5. Fig. 6 shows the effects of 48 h of MP withdrawal on the aversive thresholds of animals submitted to electrical stimulation of the DPAG. One-way ANOVA showed a significant statistical decrease in freezing [F(1,18) = 4.60; p < 0.05] and a consequent increase in escape [F(1,18) = 8.14; p < 0.05] thresholds in the MP withdrawal group compared to control rats.

2.6.4. Histology Upon completion of the experiments, the animals were deeply anaesthetized with urethane and perfused intracardially with saline 0.9% followed by buffered formalin solution (4%). The brains were removed, immersed in formalin solution for 2 h and then kept in a sucrose solution (30%). Three days later the brains were frozen. Serial 60-␮m brain sections were cut using a cryostat (Leica, Wetzlar, Germany) and stained with Nissl in order to localize the positions of the electrode tips according to Paxinos and Watson’s atlas (2005). 2.6.5. Statistical analysis The results obtained in the present study are shown as mean ± SEM. In order to achieve normal variance and distribution data were transformed via the square root of the raw data. The

Fig. 6. Effects of 48 h of MP withdrawal on the freezing and escape thresholds determined by the procedure of electrical stimulation of the DPAG. Values represent mean ± SEM. *p < 0.05 in relation to control group, according to one-way ANOVA.

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2.7. Experiment IV: Influence of chronic effects of and withdrawal from MP on motor response of rats as determined by the unconditioned startle response procedure

drawal conditions as the dependent repeated factors. All statistical analyzes were followed, when appropriate, by the Newman–Keuls test. p < 0.05 was considered to be significant.

Since MP is a psychostimulant with the ability to induce increased motor behavior in rats tested during acute MP treatment, this experiment attempted to analyze its influence on the motor response of rats tested under chronic effects of the drug or during withdrawal. For this purpose, two independent groups of rats were used (n = 16 for each group).

2.7.4. Results Fig. 7 shows the influence of the chronic effects of and withdrawal from MP on the startle response amplitude. Twoway RM ANOVA showed no significant effects for the treatments (saline × MP) [F 1,30 = 1.90; p > 0.05], a significant difference between conditions (baseline × chronic × withdrawal) [F 2,60 = 16.85; p < 0.0001] and a significant interaction between condition × treatments [F 2,60 = 5.87; p < 0.005]. The post hoc Newman–Keuls test showed that rats tested under MP chronic effects had an increased startle amplitude when compared with saline pretreated animals. On the other hand, there were no significant differences in the startle amplitude of MP-withdrawn rats when compared with those in the saline group.

2.7.1. Experimental apparatus Two sets of startle response apparatus were used. Individually, they consisted of a wire-mesh cage (16.5 cm × 5.1 cm × 7.6 cm) that was attached to a stabilimeter (response platform – 36.5 cm × 11.5 cm × 4.5 cm) with four thumbnail-screws, suspended within a ventilated plywood sound attenuation box (96 cm × 48 cm × 45 cm), and divided by two chambers (48 cm × 48 cm × 45 cm), each one of which contained one startle hardware. The floor of the wire-mesh cage consisted of six stainless steel bars of 3.0 mm in diameter and spaced 1.5 cm apart. The startle amplitude was recorded within a time window of 200 ms after the onset of the startle stimulus. A loudspeaker located 15 cm from the rear of each of the chambers was used to deliver the startle stimuli, in addition to continuous background noise (white noise, 55 dB) delivered throughout the session. The startle reaction of the rats generated pressure on the stabilimeter and analog signals were amplified, digitized and analyzed by the software of the startle measurement system (Insight, São Paulo, Brazil), which also controlled all parameters of the session (intensity of the acoustic stimulus, inter-stimulus interval, etc.). In order to ensure equivalent sensitivities of the response platforms over the test period, calibration procedures were conducted prior to the experiment. Animal behavior was recorded via an infra-red camera (Safety View, São Paulo, Brazil) located behind the stabilimeter, allowing the discrimination of all possible behaviors, with the signal being relayed to a video and a monitor in another room via a closed-circuit system.

3. Discussion Psychostimulants aimed at children with ADHD have been shown to be effective. In fact, the use of this class of drugs continues to be the main treatment method for the symptoms of this disorder (Leonard et al., 2004). However, exposure to MP during early periods of development has been shown to produce transient and long-term changes in some brain systems linked to rewarding effects as well as aversive stimuli, mainly the central dopaminergic systems (Moll et al., 2001; Brandon et al., 2003). In fact, MP treatment during premature developmental stages promotes increases in the levels of anxiety-related processes such as an elevated response to stressful stimuli, elevated plasma cor˜ ticosterone and depression-like states in adulthood (Bolanos et al., 2003; Carlezon et al., 2003). Our results revealed that MP can induce similar changes in the anxiety levels of adult MP-withdrawn rats, even after short-term treatment. The results obtained from the EPM test showed that following 48 h of withdrawal from MP, rats decreased the time spent in the open arms. In addition, the naturalistic variables that were recorded corroborated the

2.7.2. Experimental procedure The first startle baseline session was conducted before the beginning of each experiment. The second startle session was performed 30 min after the last saline or MP injection. The third session was performed 48 h after the last injection. These experiments were conducted in a simple paradigm that is routinely used in our laboratory (Nunes Mamede Rosa et al., 2005; Cabral et al., 2009). The acoustic startle test session consisted of two parts. The first was a 5-min period of acclimatization to the startle test chamber. Except for the background noise, no acoustic startle stimuli were presented during this period. The second part consisted of 40 presentations of an acoustic startle stimulus (pulse, 110-dB, 50-ms bursts of white noise having a rise-decay time of 5 ms). The inter-stimulus interval of 30 s used between trials was based on a previous study by our laboratory (Nunes Mamede Rosa et al., 2005; Cabral et al., 2009). The testing session lasted 20 min. Data obtained were initially stored on a hard disc and then transferred to tables in a spreadsheet program for off-line analysis. 2.7.3. Statistical analysis The results from the startle test were normalized via the square root of the raw data and are reported as means ± SEM. Startle responses were averaged for each animal across the entire session and used as the data for statistical analysis. A two-way repeated measures (RM) ANOVA was conducted on the startle test with the groups (saline × MP) as the independent factors and the amplitude of startle responses obtained during baseline, chronic or with-

Fig. 7. Mean ± SEM of startle response amplitude of rats tested in baseline condition or under chronic effects of, or withdrawal from, MP. *Significant difference in the startle response amplitude of the MP group when compared with the control group (saline). Data were analyzed by a two-way RM ANOVA, followed by the Newman–Keuls post hoc test. Significant differences were maintained at the level of p < 0.05.

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effects on traditional measures in such a way that withdrawn-rats visited the end of open arms less often, reduced the frequency of rearing and unprotected head-dipping and increased the frequency of stretching-attend postures, all of these measures being linked with anxiety and fear-related behaviors (Pellow et al., 1985; Cruz et al., 1994; Anseloni and Brandão, 1997). No differences in the number of closed arm entries were observed between the chronic MP and saline groups. This result is quite different from the increases in the locomotor behavior usually observed after short or long-term MP administration (for a review see the study by Askenasy et al., 2007). Nonetheless, in the majority of these studies, locomotor activation was observed following systemic (but not oral) administration of more than 1 mg/kg but less than or equal to 4 mg/kg (Eckermann et al., 2001; Gaytan et al., 2002). Since we used doses of MP that ranged between 5 mg/kg and 20 mg/kg in the present study, this could account for the absence of locomotor activation noted in MP pretreated rats, tested under MP effects. In addition, other studies found that tolerance developed after repeated administration of higher doses of MP (Wood et al., 1997; Crawford et al., 1998; Segal and Kuczenski, 1999). It is well established that MP binds to the dopamine transporter and inhibits dopamine reuptake in a similar manner to cocaine and amphetamine (Wise and Bozarth, 1987; Gatley et al., 1996; Koob et al., 1998). Thus it appears that MP presents the same rewarding properties as other stimulants, leading to increased dopamine levels in brain reward pathways (Kuczenski and Segal, 1997; Gerasimov et al., 2000), and also enhancing locomotion after repeated administration (Yang et al., 2006). It is believed that both the drug-induced pleasurable effects and withdrawal-induced aversive ones are mainly due to cellular and molecular alterations induced by the drugs in these reward systems, of which the ventral tegmental area, the nucleus accumbens and the prefrontal cortex are the main representatives (Koob et al., 1998). To go to one step further in this matter, in the second experiment of our study, we evaluated the effects of withdrawal from MP on Fos expression, induced by EPM aversive cues, in brainstem areas chiefly associated with the modulation/expression of unconditioned anxiety and fear. Data obtained showed that rats under MP withdrawal have increased Fos-immunolabeling in all regions studied, including all columns of the periaqueductal gray, the DpSC, the CIC, and the DR, a group of mesencephalic structures belonging to the well-known brain aversion system (Graeff, 1990). The activation of these brainstem regions was also observed in several studies using Fos-protein expression as a neuronal marker of anxiety-like states (Senba et al., 1993; Sandner et al., 1993; Silveira et al., 1995; Beck and Fibiger, 1995; Beckett et al., 1997) and after intraperitoneal injection of drugs that elicit panic-like symptoms (Singewald and Sharp, 2000). In addition, we have previously demonstrated that withdrawal of other classes of drugs, such as benzodiazepines and opiates (Fontanesi et al., 2007; Avila et al., 2008) promotes comparable Fos-immunolabeling. Since this group of structures is also activated in rats during experimentally induced anxiety, and given the fact that anxiety induced by drug withdrawal shares relatively similar properties to that elicited by anxiety- and fear-related stimuli, it is conceivable that both classes of unconditioned aversive stimuli produce a similar pattern of neural activation. Considering the statements above one question that deserves attention is whether MP could affect anxiety and neural reactivity after a single acute administration. With regard to this point studies on this subject are lacking. However, psychostimulants and dopamine receptor agonists have been shown to alter the expression of immediately-early genes as c-fos, Fos-B and Zif 268 in the neurons of the cortical and subcortical regions. For example, the study of Lin et al. (1996) showed increased Fos expression in cats after oral administration of 2.5 mg/kg of MP. Significant

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Fos immunoreactivity was observed on several brainstem areas related to the modulation/expression of anxiety- and fear-related behaviors, including the periaqueductal gray, superior and inferior colliculus. In another study, Trinh et al. (2003) showed that a single high dose of MP (30 mg/kg) could induce significant elevation in c-Fos immunoreactivity in several cortical and subcortical regions of wild type mice, among them the basolateral amygdala, periaqueductal gray and dorsal raphe. Whether these results reflect an increase in anxiety levels induced by acute treatment is still a matter of debate, since in these studies the animals were tested in the absence of a stressor stimulus or a behavioral test in order to assessing anxiety-like behaviors. A brain aversion system made up of the DPAG, dorsomedial hypothalamus and amygdala, has been associated with unconditioned fear (Graeff et al., 1986; Graeff, 1990, 2004). The electrical or chemical stimulation of these regions causes a characteristic pattern of active defense, with alertness, freezing and escape responses, along with autonomic changes (Graeff et al., 1986; Brandão et al., 2003; Borelli et al., 2004; Graeff, 2004). With regard to the DPAG, whether or not the results obtained on Fos-immunolabeling represent an aversive component of MP withdrawal, a decreased threshold of electrical stimulation of this structure should be observed in MP-withdrawn rats submitted to this procedure. In fact, the results from experiment three corroborated our hypothesis, showing that rats under this condition experience a negative affective state, possibly raised by the sensitization of the midbrain tectum promoted by the MP treatment-break, since rats under MP withdrawal had significantly decreased electrical stimulation thresholds for this structure. This result suggests that a chronic MP treatment-break in adult rats alters the neuronal substrates of the brain aversion system, which is known to be involved in the expression of the emotional, autonomic and motor components of anxiety and fear-like behaviors. In other words, analogous neuronal activation underlying the expression of these emotional states, induced by aversive stimuli, seems to be occurring in MP-withdrawn rats. Curiously, rats tested under withdrawal have increased thresholds for escape behavior. However, an increase in the escape threshold induced by electrical stimulation of the DPAG cannot be interpreted as a general depressor effect on motor activity. Instead, it is believed that higher freezing responses in relation to DPAG-evoked stimulation make the animals less able to exert further physical activity in response to DPAG electrical stimulation at the escape threshold (Oliveira et al., 2007). As suggested by the results obtained on Fos immunohistochemistry and DPAG electrical stimulation, withdrawal from MP enhances the neural sensitivity of mesencephalic regions mainly involved in the modulation and expression of anxiety- and fearrelated behaviors. To date, studies on the effects of withdrawal from MP on brainstem activity are lacking, staying the mesocorticolimbic dopaminergic pathway as the main focus for the MP pharmacological activity. Since in our study no pharmacological intervention was made in order to achieve the real nature of MP effects on anxiety and brainstem neural sensitization during the “washout” phase, any statement on the subject should be conducted carefully. MP functions in a manner similar to other stimulants as amphetamine and cocaine by raising extracellular dopamine and noradrenaline levels (Wise and Bozarth, 1987; Gatley et al., 1996; Koob et al., 1998; Askenasy et al., 2007), but differs in that it has little or no effect on serotonin levels (Kuczenski and Segal, 1997). In our study, significant neural sensitization of dorsal midbrain was observed following MP treatment-break. However, the study of Unis et al. (1985) showed no specific binding for MP in the brainstem. On the other hand, this encephalic region has the highest specific binding for [3H] amphetamine (Hauger et al., 1984). This result indicates that, in our study, the increased levels of anxiety

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induced during MP withdrawal, and attributed to midbrain regions sensitization, could be mainly due to changes in dopaminergic and noradrenergic pathways within cortical and subcortical limbic structures that act as modulators of unconditioned anxiety and fear behaviors organized in the brainstem. For example, projections arising predominantly from the medial prefrontal cortex target, through the basolateral amygdala, specific columns of the dorsal aspects of the PAG. This neuronal pathway, particularly important in the integration of cognitive-affective information linked to aversive states (Reep and Winans, 1982; Floyd et al., 2000), is glutamatergic in its nature (Del Arco and Mora, 2009), is modulated by dopaminergic neurons in PFC (Pierce and Kumaresan, 2006), and seems to be down-regulated during psychostimulants withdrawal (Goldstein and Volkow, 2002). After acute administration, MP induces locomotor activation and stereotyped behavior that resemble that seen in cocaine- and amphetamine-pretreated rats. After chronic administration, sensitization of the MP locomotor-stimulating effects and rewarding properties is observed. In our study, in order to achieve an additional measure of anxiety and to analyze the influence of MP treatment or withdrawal on motor response, rats were submitted to the startle response procedure. In connection with this, it is well known that both humans and animals, when facing fearand anxiety-inducing stimuli or after the administration of anxiogenic drugs, exhibit an increased startle reaction (Davis et al., 1979, 1993; Liebsch et al., 1998; Winslow et al., 2002). In the same way, the startle response is overdone in clinical patients with anxiety disorders (Grillon, 2002). In addition, withdrawal from some drugs of abuse also has the ability to increase the startle response (Vivian et al., 1994; Avila et al., 2008). Our results showed that, although there were no differences in locomotion in the plus-maze test, as revealed by the results obtained on the difference in frequency of closed arm entries between saline and MP groups, MP increased the startle amplitude of rats tested under its chronic effects, in a manner analogous to that described by Conti et al. (2006), who found that increased startle was obtained after acute administration of a higher MP dose, similar to that used here. This effect was likely to be mediated by the action of MP on nigro-striatal dopamine receptors, since the acoustic startle reflex is under the influence of dopamine systems (Davis, 1980). Future studies should try to elucidate why MP has different effects on locomotor behavior (as seen in the plus-maze) and motor response (as noted in the startle response test). There is a high-co-occurrence of juvenile mania and ADHD (Biederman et al., 1996; Giedd, 2000). Because of this high-rate of co-occurrence, and the difficult in differentiating their clinical presentations, children with bipolar disorders are commonly treated with stimulants prior to the onset of the disease or early in its course (Biederman et al., 1996), and several case reports indicate that psychostimulants may worsen its symptoms (Koehler-Troy et al., 1986; Rosse et al., 1997). In addition, anxiety comorbidities have a high prevalence among bipolar patients (Pini et al., 1997; Simon et al., 2004). In our study, except for the startle response test, withdrawal from MP increased all measures related to the expression of anxiety-like behaviors. The results obtained on Fosimmunolabeling showed that this increased levels of aversion was correlated with the increased neural reactivity of brainstem regions directly linked to the expression of unconditioned anxiety and fear-related behaviors. This is important to note because children with the predisposition for developing bipolar disorder will exhibit increased frequency, severity and duration of their affective symptoms (DelBello et al., 2001; Kauer-Sant’Anna et al., 2009) when treated with stimulants, being in risk for develop bipolar disorder in younger age and more likely to exhibit substance abuse and even suicide behavior (Young et al., 1993; Simon et al., 2007; Otto et al., 2006).

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