Effects of Acute and Chronic Treatment Elicited by Lamotrigine on Behavior, Energy Metabolism, Neurotrophins and Signaling Cascades in Rats ⇑ Helena M

Neurochemistry International 59 (2011) 1163–1174

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Neurochemistry International

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Effects of acute and chronic treatment elicited by lamotrigine on behavior, energy metabolism, neurotrophins and signaling cascades in rats ⇑ Helena M. Abelaira a, Gislaine Z. Réus a, , Karine F. Ribeiro a, Giovanni Zappellini a, Gabriela K. Ferreira b, Lara M. Gomes b, Milena Carvalho-Silva b, Thais F. Luciano c, Scherolin O. Marques c, Emilio L. Streck b, Cláudio T. Souza c, João Quevedo a a Laboratório de Neurociências and Instituto Nacional de Ciência e Tecnologia Translacional em Medicina (INCT-TM), Programa de Pós-Graduação em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil b Laboratório de Bioenergética and Instituto Nacional de Ciência e Tecnologia Translacional em Medicina (INCT-TM), Programa de Pós-Graduação em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil c Laboratório de Fisiologia do Exercício, Programa de Pós-Graduação em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil article i nfo abstract

Article history: The present study was aimed to investigate the behavioral and molecular effects of lamotrigine. To this Received 20 September 2011 aim, Wistar rats were treated with lamotrigine (10 and 20 mg/kg) or imipramine (30 mg/kg) acutely and Received in revised form 13 October 2011 chronically. The behavior was assessed using forced swimming test. Brain-derived neurotrophic factor Accepted 15 October 2011 (BDNF), nerve growth factor (NGF), Proteina Kinase B (PKB, AKT), glycogen synthase kinase 3 (GSK-3) Available online 25 October 2011 and B-cell lymphoma 2 (Bcl-2) levels, citrate synthase, creatine kinase and mitochondrial chain (I, II, II–III and IV) activities were assessed in the brain. The results showed that both treatments reduced Keywords: the immobility time. The BDNF were increased in the prefrontal after acute treatment with lamotrigine Lamotrigine (20 mg/kg), and the BDNF and NGF were increased in the prefrontal after chronic treatment with lamo- Neurotrophins Signaling cascades trigine in all doses. The AKT increased and Bcl-2 and GSK-3 decreased after both treatments in all brain Energy metabolism areas. The citrate synthase and creatine kinase increased in the amygdala after acute treatment with Forced swimming test imipramine. Chronic treatment with imipramine and lamotrigine (10 mg/kg) increased the creatine Depression kinase in the hippocampus. The complex I was reduced and the complex II, II–III and IV were increased, but related with treatment and brain area. In conclusion, lamotrigine exerted antidepressant-like, which can be attributed to its effects on pathways related to depression, such as neurotrophins, metabolism energy and signaling cascade. Ó 2011 Published by Elsevier B.V. Open access under the Elsevier OA license.

1. Introduction Lamotrigine is an anticonvulsant drug that has shown efﬁcacy in the treatment of bipolar depression and resistant major Depression is a severe disorder that has enormous conse- depressive episodes (Bowden et al., 1999; Calabrese et al., 1999; quences for the individual’s quality of life, and it is among the most Frye et al., 2000; Barbosa et al., 2003). However, the mechanism prevalent forms of mental illness. Clinical symptoms like de- of antidepressant action of lamotrigine is still unclear. Although pressed mood, anhedonia, fatigue or loss of energy, feelings of the blockade of neuronal voltage-dependent sodium channels worthlessness or guilt, and the diminished ability to concentrate elicited by lamotrigine has an important role in its anticonvulsant or think are characteristics of depression. Despite the devastating effect, and it shares a common action with other mood stabilizing impact of depression, relatively little is known about the etiology anticonvulsants, the antiglutamatergic effect of lamotrigine has and pathogenesis of depression (Larsen et al., 2010). been implicated in its mood effect (Ketter et al., 2003). In addition to these effects, lamotrigine also blocks neuronal voltage-dependent calcium channels (Ketter et al., 2003) Moreover, the reduction of glutamate release induced by lamotrigine may be related Abbreviations: BDNF, brain-derived neurotrophic factor; NGF, nerve growth to the blockade of neuronal voltage-dependent sodium and factor; Bcl-2, B-cell lymphoma 2; AKT, Protein Kinase B; GSK-3, glycogen synthase calcium channels (Ketter et al., 2003). Reduced glutamatergic kinase 3. ⇑ Corresponding author. Fax: +55 48 3431 2736. neurotransmission has been related to an antidepressant effect. E-mail address: [email protected] (G.Z. Réus). For example, antagonists of the N-methyl-D-aspartate (NMDA)

0197-0186/Ó 2011 Published by Elsevier B.V. Open access under the Elsevier OA license. doi:10.1016/j.neuint.2011.10.007 1164 H.M. Abelaira et al. / Neurochemistry International 59 (2011) 1163–1174 complex exhibit an antidepressant-like effect in animal models 2. Material and methods of depression (Paul and Skolnick, 2003; Réus et al., 2010, 2011). Moreover the lamotrigine presents effects in dopami- 2.1. Animals nergic, adrenergic, muscarinic, opioid, adenosine, serotonin (5HT3) and 5HT1A receptors (for a review see: Goldsmith et al., Male Adult Wistar rats (60 days old) were obtained from the 2003). breeding colony at UNESC (Universidade do Extremo Sul Catarin- Evidence indicates that neurotrophins such as brain-derived ense, Criciúma, SC, Brazil). They were housed five per cage with neurotrophic factor (BDNF) and nerve growth factor (NGF) may food and water available ad libitum and were maintained on a play a role in the pathophysiology of depression and that antide- 12-h light/dark cycle (lights on at 7:00 a.m.). All experimental pro- pressants may in part exert their effects through the regulation cedures involving animals were performed in accordance with the of BDNF and NGF. Several clinical studies have reported that serum NIH Guide for the Care and Usage of Laboratory Animals and under BDNF levels are decreased in depressed patients, and that they can the Brazilian Society for Neuroscience and Behavior (SBNeC) rec- be normalized by antidepressant treatment (Brunoni et al., 2008; ommendations for animal care, and with approval by the local Gervasoni et al., 2005). Ethics Committee under protocol number 01/2011. The understanding of the signaling pathways in neurons or the investigation of new components with already discovered ones can 2.2. Drugs and treatments be considered as the basis to finding molecular–biological causes of neuropsychiatric diseases (D’Sa and Duman, 2002). Protein Lamotrigine was purchased from Sigma (Brazil) and imipra- Kinase B (PKB, AKT), glycogen synthase kinase 3 (GSK-3) and com- mine, a classic antidepressant was purchased from Novartis Phar- ponents regulating programmed cell death, especially the family of maceutical Industry (São Paulo, Brazil). Different groups of rats B-cell lymphoma 2 (Bcl-2) proteins and various mitochondrial (n = 15 each) were administered intraperitoneally (i.p.) with saline factors, are intensively studied because of the known biochemical (control group), different doses of lamotrigine (10 mg/kg and effects of the long-term administration of antidepressants and 20 mg/kg) or imipramine (30 mg/kg) (positive control) in one sin- mood stabilizers, with regard to the supposed role of the survival, gle dose (acute treatment) or over the course of 14 days, once a day plasticity and metabolism of neurons during mental and neurode- (chronic treatment), the protocols being in accordance with previ- generative diseases (Jope and Roh, 2006). Recently, it has been pro- ous study executed by Kaster et al. (2007). All treatments were posed that antidepressants may exert their long-term therapeutic administered in a volume of 1 ml/kg. The behavior tests (open-field effects by triggering cellular mechanisms that promote neuronal and forced swimming tests) were evaluated one hour after the plasticity (Manji et al., 2003) and neuroprotective pathways by administration of the last injection. increasing the neurogenesis in the hippocampus (Malberg et al., 2000). Most cellular energy is obtained through oxidative phosphory- 2.3. Open-field test lation, a process requiring the action of various respiratory enzyme complexes located in a special structure of the inner mitochondrial This apparatus consists of a 45 Â 60 cm brown plywood arena membrane, the mitochondrial respiratory chain. It is well de- surrounded by 50 cm high wooden walls and containing a frontal scribed that mitochondrial dysfunction has been implicated in glass wall. The floor of the open field was divided into nine rectan- the pathogenesis of a number of diseases affecting the brain, such gles (15 Â 20 cm each) by black lines. Animals were gently placed as dementia, cerebral ischemia, Alzheimer’s disease and Parkin- on the left rear quadrant and left to explore the arena. In a separate son’s disease (Blass, 2001; Brennan et al., 1985; Heales et al., series of experiments, rats were acutely treated with lamotrigine 1999; Schurr, 2002; Monsalve et al., 2007). Several recent works (10 and 20 mg/kg), imipramine (30 mg/kg) and saline 60 min be- also support the hypothesis that metabolism impairment is in- fore exposure to the open-field apparatus and after 12 days of volved in the pathophysiology of depression (Tretter et al., 2007; chronic treatment, rats were exposed to the open-field apparatus. Petrosillo et al., 2008; Kanarik et al., 2008; Stanyer et al., The numbers of horizontal (crossings) and vertical (rearings) activ- 2008).The enzyme creatine kinase (CK), catalyses the reversible ities performed by each rat during the 5 min observation period transphosphorylation of creatine by adenosine triphosphate and were counted by an expert observer, in order to assess the possible plays a key role in energy buffering and energy transport, particu- effects of drug treatment on spontaneous locomotor activity. larly in cells with high and fluctuating energy requirements, including neurons (Andres et al., 2008). It is also known that a dim- 2.4. Forced swimming test inution of CK activity may potentially impair energy homeostasis, contributing to cell death (Aksenov et al., 2000; David et al., The forced swimming test was conducted according to previous 1998) In addition, citrate synthase has been used as a quantitative reports (Garcia et al., 2008a,b; Porsolt et al., 1977; Detke et al., enzyme marker for the presence of intact mitochondria (Marco 1995). The test involves two individual exposures to a cylindrical et al., 1974), which may be related with mood disorders (Agostinho tank filled with water, in which the rats cannot touch the bottom et al., 2009). of the tank or escape. The tank is made of transparent Plexiglas, Therefore, considering that neutrophins, energy metabolism 80 cm tall, 30 cm in diameter, and filled with water (22–23 °C) to and cell signaling cascades are all involved in the pathophysiology a depth of 40 cm. For the first exposure, rats without drug treat- of mood disorders and that there are still no studies showing the ment were placed in the water for 15 min (pre-test session). consistent effects of lamotrigine on these targets, the present study Twenty-four hours later, rats were once again placed in the water was aimed to investigate the behavioral and physiological effects for a 5 min session (test session), and the immobility time of rats of acute and chronic administration of lamotrigine in rats. The were recorded in seconds. Rats were treated with lamotrigine, behavioral effects were evaluated in the open field and forced imipramine or saline only 60 min before the second exposure to swimming tests. Additionally, creatine kinase citrate, synthase the cylindrical tank of water (test session). On day 13 of chronic activities and mitochondrial respiratory chain (I, II, II–III and IV) treatment, 1 h after drug administration, the rats were individually activities; Bcl-2, AKT and Gsk-3 expression; and BDNF and NGF placed in the cylinder containing water for 15 min (pre-test ses- protein levels were assessed in the prefrontal cortex, hippocampus sion). On the 14th day the rats received the last intraperitoneal and amygdala. drug treatment, and after 1 h they were again subjected to the H.M. Abelaira et al. / Neurochemistry International 59 (2011) 1163–1174 1165 forced swimming test for a 5-min session (test session). During the standard. Creatine kinase activity was measured in brain homoge- test session immobility time was recorded. After the behavioral nates pre-treated with 0.625 mM lauryl maltoside. The reaction tests, in both acute and chronic treatments, all rats were killed mixture consisted of 60 mM Tris–HCl, pH 7.5, containing 7 mM by decapitation and the skulls were immediately removed. The phosphocreatine, 9 mM MgSO4 and approximately 0.4–1.2 lg pro- prefrontal cortex, hippocampus and amygdala were quickly iso- tein in a final volume of 100 lL. After 15 min of preincubation at lated by hand dissection using a magnifying glass and a thin brush, 37 °C, the reaction was started by the addition of 0.3 lmol of the dissection being based on histological distinctions described by ADP plus 0.08 lmol of reduced glutathione. The reaction was Paxinos and Watson (1986). stopped after 10 min by the addition of 1 lmol of hydroxymercu- ribenzoic acid. The creatine formed was estimated according to 2.5. Neurotrophins measurement the colorimetric method of Hughes (1962). The color was developed by the addition of 100 lL2%a-naphthol and 100 lL 0.05% The BDNF and NGF levels in the prefrontal cortex, hippocampus diacetyl in a final volume of 1 mL and read spectrophotometrically and amygdala (n = 6–8 each) were measured by sandwich-ELISA, after 20 min at 540 nm. Results were described as according to the manufacturer’s instructions (Chemicon, USA for nmolminÀ1 mgproteinÀ1. BDNF and Millipore, USA & Canada for NGF). Briefly, the rat prefrontal cortex, hippocampus and amygdala were homogenized in 2.8. Citrate synthase activity phosphate buffer solution (PBS) with protease inhibitor cocktail (Sigma). Microtiter plates (96-well flat-bottom) were coated for The prefrontal cortex, hippocampus and amygdala (n = 5 each) 24 h with the samples diluted 1:2 in sample diluent and the stan- were homogenized (1:10, w/v) in SETH buffer (0.25 M sucrose, dard curve ranged from 7.8 to 500 pg/ml of BNDF and NGF. The 1 mM EDTA, 10 mM Tris–HCl, pH 7.4). The homogenates were cen- plates were then washed four times with sample diluent and a trifuged at 800Âg for 10 min and the supernatants were kept at monoclonal anti-BNDF, and an anti-NGF rabbit antibody (diluted À70 °C until it will be used for enzyme activity determination. Pro- 1:1000 in sample diluent) was added to each well and incubated tein content was determined by the method described by Lowry for 3 h at room temperature. After washing, a peroxidase conju- et al. (1951) using bovine serum albumin as standard. Citrate syn- gated anti-rabbit antibody (diluted 1:1000) was added to each well thase activity was assayed according to the method described by and incubated at room temperature for 1 h. After the addition of Shepherd and Garland (1969). The reaction mixture contained the streptavidin-enzyme, substrate and stop solutions, the amount 100 mM Tris, pH 8.0, 100 mM acetyl CoA, 100 mM 5,5-di-thiobis of each neurotrophin was determined by absorbance in 450 nm. (2-nitrobenzoic acid), 0.1% triton X-100, and 2–4 g supernatant The standard curve demonstrates a direct relationship between protein and was initiated with 100 Moxaloacetate and monitored Optical Density (OD) and the concentration. Total protein was at 412 nm for 3 min at 25 °C (the final volume of reaction mixture measured by Lowry’s method using bovine serum albumin as a was 0.3 mL). standard, as previously described by Lowry et al. (1951). 2.9. Protein analysis by immunoblotting 2.6. Respiratory chain enzyme activities The Prefrontal cortex, hippocampus and amygdala tissues (n =5 The homogenates (n = 5 each) were centrifuged at 800g for each) were excised. The tissues were homogenized immediately in 10 min and the supernatants kept at À70 °C until used for enzyme extraction buffer (mM) (1% Triton-X 100, 100 Tris, pH 7.4, contain- activity determination. The maximal period between homogenate ing 100 sodium pyrophosphate, 100 sodium fluoride, 10 EDTA, 10 preparation and enzyme analysis was always less than 5 days. Pro- sodium vanadate, 2 PMSF and 0.1 mg of aprotinin/ml) at 4 °C with tein content was determined by the method described by Lowry a Polytron PTA 20S generator (Brinkmann Instruments model PT et al. (1951) using bovine serum albumin as standard. NADH dehy- 10/35) operated at maximum speed for 30 s. The extracts were drogenase (complex I) was evaluated by the method described by centrifuged at 11,000 rpm and 4 °C in a Beckman 70.1 Ti rotor (Palo Cassina and Radi (1996) by the rate of NADH-dependent ferricya- Alto, CA) for 40 min to remove insoluble material, and the superna- nide reduction at 420 nm. The activity of succinate: Cytochrome c tants of these tissues were used for protein quantification, using oxidoreductase (complexes II and II–III) were determined according the Bradford method. Proteins were denatured by boiling in to the method of Fischer et al, measured by Cytochrome c reduction (Laemmli, 1970) sample buffer containing 100 mM DTT (De Souza from succinate. The activity of Cytochrome c oxidase (complex IV) et al., 2003). After this, 0.2 mg of protein extracts obtained from was assayed according to the method described by Rustin et al, mea- each tissue were separated by SDS–PAGE, transferred to nitrocellu- sured by following the decrease in absorbance due to the oxidation lose membranes and blotted with anti-AKT, anti-Bcl-2 and anti- of previously reduced Cytochrome c at 550 nm with 580 nm as ref- GSK-3b. Antibodies were from Santa Cruz Biotechnology (Santa erence wavelength (e = 19.1 mMÀ1 cmÀ1). The reaction buffer con- Cruz, CA, USA). Chemiluminescent detection was performed with tained 10 mM potassium phosphate, pH 7.0, 0.6 mM n-dodecyl-D- horseradish peroxidase-conjugate secondary antibodies. Visualiza- maltoside, 2–4 lgÀ1 homogenate protein and the reaction was initi- tion of the protein bands was performed by exposure of the mem- ated with addition of 0.7 lgÀ1 reduced cytochrome c. The activity of branes to RX-films. The original membrane was stripped and complex IV was measured at 25 °C for 10 min. The activities of the reblotted with actin loading protein (bands not showing). After mitochondrial respiratory chain complexes were described as transfer, the membrane was stained with Ponceau and bands were nmolminÀ1 mgproteinÀ1. visualized, photographed and quantified before the primary antibody, to control the transfer. Band intensities were quantitated 2.7. Creatine kinase activity by optical densitometry (Scion Image software, ScionCorp, Freder- ick, MD) of the developed autoradiographs. The homogenates (n = 5 each) were centrifuged at 800g for 10 min. and the supernatants kept at À70 °C until used for creatine 2.10. Statistical analysis kinase activity determination. The maximal period between homogenate preparation and enzyme analysis was always less All data are presented as mean ± SEM. Differences among exper- than 5 days. Protein content was determined by the method imental groups in the forced swimming and open field tests and in described by Lowry et al. (1951) using bovine serum albumin as the assessment of the biochemical analysis were determined by 1166 H.M. Abelaira et al. / Neurochemistry International 59 (2011) 1163–1174

one-way ANOVA, followed by Tukey post-hoc test when ANOVA p = 0.62 Fig. 2A), compared with saline. In the amygdala (F(3–16) = was signiﬁcant; P values <0.05 were considered to be statistically 2.676; p = 0.82; Fig. 3B) and in the hippocampus (F(3–16) =1.693; signiﬁcant. p =0.20;Fig. 2A), there were no alterations in the BDNF levels after chronic treatment. 3. Results The acute treatment did not alter the NGF protein levels in the

prefrontal cortex (F(3–16) = 1.024; p = 0.40 Fig. 2B), in the amyg- The effects of the acute and chronic administration of lamotri- dala (F(3–16) = 3.076; p = 0.58 Fig. 2B) or in the hippocampus gine on the immobility times are illustrated in Fig. 1A. In the acute (F(3–16) = 0.095; p = 0.96 Fig. 2B). The chronic treatment increased

(F(3–21) = 6.148; p = 0.04 Fig. 1A) and chronic (F(3–66) = 6.222; the NGF levels in the prefrontal cortex with lamotrigine at the p = 0.01 Fig. 1A) treatments we observed a decrease in the immo- dose of 10 and 20 mg/kg (F(3–15) = 8.982; p = 0.01 Fig. 2B), com- bility time with imipramine at the dose of 30 mg/kg and lamotri- pared with saline, but the NGF protein levels did not alter in the gine at the doses of 10 and 20 mg/kg, compared with saline. prefrontal cortex with imipramine at the dose of 30 mg/kg

Interestingly, in the open-ﬁeld test both acute and chronic (F(3–15) = 8.982; p = 0.57 Fig. 2B). The amygdala (F(3–16) = 0,230; treatments with imipramine or lamotrigine did not modify the p = 0.87 Fig. 2B) and the hippocampus (F(3–16) = 3.2080; p = 0.51 number of crossings (acute; F(3–55) = 0.595; p = 0.62; Fig. 1B; Fig. 2B) did not have alterations in the BDNF levels after chronic chronic; F(3–53) = 3.411; p = 0.24 Fig. 1B) and rearings (acute; treatment. F(3–55) = 0.393; p = 0.75; chronic; F(3–53) = 0.844; p = 0.47 Fig. 1B), The acute treatment increased the citrate synthase activity in compared with saline. the amygdala with imipramine at the dose of 30 mg/kg

With regards to the acute treatment, there was an increase the (F(3–10) = 6.474; p = 0.02 Fig. 3A) compared with saline. In the pre- BDNF levels in the prefrontal cortex with lamotrigine at the dose frontal cortex and hippocampus there were no alterations in the of 20 mg/kg (F(3–16) = 5.501; p = 0,009 Fig. 2A), compared with sal- citrate synthase activity after acute treatment. The chronic treat- ine, but BDNF protein levels did not alter in the prefrontal cortex ment did not alter the citrate synthase activity in the prefrontal with imipramine at the dose of 30 mg/kg (F(3–16) = 5.501; p = 0.22 cortex (F(3–11) = 0.460; p = 0.71 Fig. 3A), amygdala (F(3–12) = 2.676; Fig. 2A) and with lamotrigine at the dose of 10 mg/kg p = 0.94 Fig. 3A) or hippocampus (F(3–12) = 3.079; p = 0.68

(F(3–16) = 5.501; p = 0.91 Fig. 2A), compared with saline. The Fig. 3A). amygdala (F(3–16) = 1.292; p = 0,31 Fig. 2A) and the hippocampus The acute treatment increased the creatine kinase activity in the (F(3–16) = 2.844; p = 0.71 Fig. 2A) did not have any alterations in their amygdala with imipramine at the dose of 30 mg/kg (F(3–15) = 5.415; BDNF levels after acute treatment. In the chronic treatment data, we p = 0.01 Fig. 3B), compared with saline. The chronic treatment in- found an increase occurred in the BDNF levels in the prefrontal cor- creased the creatine kinase activity in the hippocampus with imip- tex with lamotrigine at the dose of 10 and 20 mg/kg (F(3–16) = 8.478; ramine at the dose of 30 mg/kg and lamotrigine at the dose of p = 0.01 Fig. 2A), compared with saline, but BDNF protein levels did 10 mg/kg (F(3–15) = 7.967; p = 0.02 Fig. 3B), compared with control not alter in the prefrontal cortex with imipramine (F(3–16) = 8.478; group.

A 180 160 140 120 100 * 80 * * Sal 60 * Imipramine 30 40 Immobility time (s) * Lamotrigine 10 20 * Lamotrigine 20

Acute Chronic B 90 80 70 60 50 40 30 Sal 20 Imipramine 30 Lamotrigine 10 10 Lamotrigine 20 Number of crossings and rearings Crossing Rearing Crossing Rearing

Acute Chronic

Fig. 1. The effects of administration of acute and chronic doses of lamotrigine and imipramine on the immobility time (A) of rats subjected to the forced swimming test and on the number of crossings and rearings (B) of rats subjected to the open ﬁeld test. Bars represent means ± S.E.M. (n = 15). ⁄P < 0.05 compared to saline group according to ANOVA followed by Tukey post-hoc test. H.M. Abelaira et al. / Neurochemistry International 59 (2011) 1163–1174 1167

A 2 1.8 1.6 1.4 1.2 1 0.8 BDNF pg/µg 0.6 Saline * Imipramine 30 0.4 * * 0.2 Lamotrigine 10 Lamotrigine 20 0 Prefontal Amygdala Hippocampus Prefontal Amygdala Hippocampus cortex cortex

Acute Chronic

B 0.14

0.12

0.1

0.08

0.06 NGF activity 0.04 * Saline Imipramine 30 [nmol/min X mg protein] * 0.02 Lamotrigine 10 Lamotrigine 20 0 Prefontal Amygdala Hippocampus Prefontal Amygdala Hippocampus cortex cortex

Acute Chronic

Fig. 2. The effects of administration of acute and chronic does of lamotrigine and imipramine on the BDNF protein levels (A) and NGF protein levels (B) in the prefrontal cortex, hippocampus and amygdala. Bars represent means ± S.E.M. (n = 6–8). ⁄P < 0.05 compared to saline group according to ANOVA followed by Tukey post-hoc test.

The acute treatment decreased the mitochondrial complex I imipramine at the dose of 30 mg/kg and in the amygdala activity in the prefrontal cortex with imipramine at the dose of (F(3–14) = 10.512; p = 0.001 Fig. 4C) with all treatments, but did not 30 mg/kg and lamotrigine at the dose of 10 mg/kg (F(3–14) = alter in the prefrontal cortex (F(3–15) = 4.175; p > 0.05 Fig. 4C) 10.859; p < 0.001 Fig. 4A) compared with control group. The chronic and in the hippocampus (F(3–13) = 10.168; p > 0.05 Fig. 4C). The treatment did not alter the mitochondrial complex I activity in the acute administration increased the mitochondrial complex IV prefrontal cortex (F(3–14) = 0.570; p = 0.64 Fig. 4A), amygdala activity in the hippocampus (F(3–13) = 18.471; p < 0,001 Fig. 4D) (F(3–14) = 2.599; p = 0.09 Fig. 4A) or hippocampus (F(3–12) = 0.875; with all treatments, compared with saline, but did not alter in the p = 0.48 Fig. 4A). The acute administration increased the mitochon- prefrontal cortex (F(3–12) = 0.828; p = 0.50 Fig. 4D) and amygdala drial complex II activity in the amygdala with imipramine at the (F(3–11) = 4,514; p = 0,27 Fig. 4D). The chronic treatment did dose of 30 mg/kg and lamotrigine at the dose of 20 mg/kg not alter the mitochondrial complex IV activity in the prefrontal

(F(3–13) = 21.798; p < 0.001 Fig. 4B), and in the hippocampus with cortex (F(3–13) = 0.689; p = 0.57 Fig. 4D), amygdala (F(3–16) = lamotrigine at the dose of 10 mg/kg (F(3–11) = 5.643; p = 0,02 3.666; p = 0.35 Fig. 4D) or hippocampus (F(3–11) = 2.317; p = 0.13 Fig. 4B) compared with saline. The chronic treatment increased Fig. 4D). the mitochondrial complex II activity in the prefrontal cortex The acute treatment decreased the Bcl-2 protein levels in the

(F(3–15) = 19.218; p < 0,001 Fig. 4B) and hippocampus (F(3–12) = prefrontal cortex (F(3–12) = 106.818; p < 0,001 Fig. 5A) and in the hip- 4.471; p = 0,03 Fig. 4B) with imipramine at the dose of 30 mg/kg, pocampus (F(3–12) = 265,226; p < 0,001 Fig. 5A) with imipramine at compared with saline. The acute administration did not alter the the dose of 30 mg/kg and lamotrigine at the dose of 20 mg/kg, and mitochondrial complex II–III activity in the prefrontal cortex also in the amygdala (F(3–12) = 87.304; p < 0.001 Fig. 5A) with all (F(3–16) = 0,759; p = 0,53 Fig. 4C), amygdala (F(3–16) = 2.451; treatments, compared with saline. The chronic treatment decreased p = 0.10 Fig. 4C) and hippocampus (F(3–16) = 1.519; p = 0,24 the Bcl-2 protein levels in the prefrontal cortex (F(3–12) = 310.093; Fig. 4C). The chronic treatment increased the mitochondrial com- p < 0.001 Fig. 5A), amygdala (F(3–12) = 238.818; p < 0.001 Fig. 5A) plex II-III activity in the prefrontal cortex (F(3–15) = 4.175; p = 0,03 and hippocampus (F(3–12) = 557.669; p < 0.001 Fig. 5A) with all Fig. 4C) and hippocampus (F(3–13) = 10.168; p = 0.001 Fig. 4C) with treatments. 1168 H.M. Abelaira et al. / Neurochemistry International 59 (2011) 1163–1174

6 5 * 4 3 Saline

Citrate synthase activity 2 Imipramine 30 [nmol TBN/nm X mg protein] 1 Lamotrigine 10 Lamotrigine 20 0 Prefontal Amygdala Hippocampus Prefontal Amygdala Hippocampus cortex cortex

Acute Chronic

2 1.8 * 1.6 1.4 1.2 1 0.8 0.6 * * Saline Imipramine 30 Creatine kinase activity

[nmol/min x mg protein] 0.4 Lamotrigine 10 0.2 Lamotrigine 20 0 Prefontal cortex Amygdala Hippocampus Prefontal cortex Amygdala Hippocampus

Acute Chronic

Fig. 3. The effects of administration of acute and chronic doses of lamotrigine and imipramine on the citrate synthase (A) and creatine kinase activities (B) in the prefrontal cortex, hippocampus and amygdala. Bars represent means ± S.E.M. (n = 5). ⁄P < 0.05 compared to saline group according to ANOVA followed by Tukey post-hoc test.

The acute treatment increased the AKT protein levels in the pre- 4. Discussion frontal cortex (F(3–12) = 49.088; p = 0.000 Fig. 5B) with imipramine at the dose of 30 mg/kg, in the amygdala (F(3–12) = 70.335; p < 0.001 Depression is a clinically and biologically heterogeneous dis- Fig. 5B) with lamotrigine at the dose of 20 mg/kg and in the hippo- ease, with 10–30% of women and 7–15% of men likely to suffer campus (F(3–12) = 21.011; p = 0.009 Fig. 5B), with imipramine at the from depression in their life-time (Briley and Moret, 2000). How- dose of 30 mg/kg and with lamotrigine at the dose of 20 mg/kg, ever, combinations of multiple genetic factors may be involved in compared with saline. The acute treatment also decreased the AKT the development of depression, because a defect in a single gene protein levels in the amygdala with imipramine at the dose of usually fails to induce the expression of multifaceted symptoms 30 mg/kg (F(3–12) = 70.335; p = 0.04 Fig. 5B) and in the hippocampus of depression (Larsen et al., 2010). Also, various non-genetic factors with lamotrigine at the dose of 10 mg/kg (F(3–12) = 21.011; p = 0.04 such as stress, affective trauma, viral infection, and neurodevelop- Fig. 5B). The chronic treatment increased the AKT protein levels in mental abnormalities increase the complexity of the pathogenesis the prefrontal cortex (F(3–12) = 121.938; p < 0,001 Fig. 5B), of the disease. Thus, extensive studies have led to a variety of amygdala (F(3–12) = 83.853; p < 0.001 Fig. 5A) and hippocampus hypotheses for the molecular mechanism of depression, but a def- (F(3–12) = 58.262; p < 0,001 Fig. 5B) after all treatments. inite pathogenic mechanism has yet to be defined. The acute treatment decreased the GSK-3 protein levels in the The behavioral effects induced by imipramine in rats reported prefrontal cortex with imipramine at the dose of 30 mg/kg and in the present study are in agreement with literature data, which lamotrigine at the dose of 20 mg/kg (F(3–12) = 126.185; p < 0.001 support an antidepressant action for imipramine in basic and clin- Fig. 5C), compared with saline. In the amygdala (F(3–12) = ical studies. In fact, findings from our group have demonstrated 328.079; p < 0.001 Fig. 5C) the GSK-3 protein levels decreased with that a single injection of imipramine (10 and 20 mg/kg) and all doses, and in the hippocampus with imipramine at the dose of chronic administration of imipramine (10, 20 and 30 mg/kg) de- 30 mg/kg (F(3–12) = 80.214; p < 0.001 Fig. 5C) after acute treatment, creased the immobility time of rats in the forced swimming test, compared with saline. The chronic treatment decreased the GSK-3 without modifying the locomotor activity (Garcia et al., 2008a,b). protein levels in the prefrontal cortex (F(3–12) = 168.217; p = 0.001 Our results showed that acute and chronic treatment with lamotri- Fig. 5C) and in the amygdala (F(3–12) = 535.095; p < 0.001 Fig. 5C) gine decreased the immobility time of rats in the forced swimming with all doses, and in the hippocampus (F(3–12) = 596.903; test, without changing locomotor activity in open field test p < 0.001 Fig. 5C) with imipramine at the dose of 30 mg/kg and compared to saline. Consistent with our study, Consoni et al. lamotrigine at the dose of 20 mg/kg. (2006) showed that lamotrigine (10 mg/kg) decreased immobility H.M. Abelaira et al. / Neurochemistry International 59 (2011) 1163–1174 1169

A 2000 1800 1600 1400 1200 1000 Saline 800 * * 600 Imipramine 30

Complex I activity 400 Lamotrigine 10

[nmol/min X mg protein] 200 Lamotrigine 20 0 cortex cortex Prefontal Prefontal Amygdala Amygdala Hippocampus Hippocampus Acute Chronic

B 12 * 10

8 * * 6 * * Saline 4 Imipramine 30

Complex II activity 2 Lamotrigine 10 [nmol/min X mg protein] Lamotrigine 20 0 cortex cortex Prefontal Prefontal Amygdala Amygdala Hippocampus Hippocampus Acute Chronic

C 8 7 * 6 5 * * 4 * * 3 Saline 2

Complex II-IIII activity II-IIII Complex Imipramine 30 [nmol/min X mg protein] 1 Lamotrigine 10 Lamotrigine 20 0 Prefontal Amygdala Hippocampus Prefontal Amygdala Hippocampus cortex cortex

Acute Chronic

D 400 350

300

250 200 * * 150 * Saline

Complex IV activity activity IV Complex 100 Imipramine 30 [nmol/min X mg protein] X mg [nmol/min 50 Lamotrigine 10 Lamotrigine 20 0 Prefontal Amygdala Hippocampus Prefontal Amygdala Hippocampus cortex cortex

Acute Chronic

Fig. 4. The effects of administration of acute and chronic doses of lamotrigine and imipramine on the mitochondrial respiratory chain complexes I (A), II (B), II–III (C) IV (D) in the prefrontal cortex, hippocampus and amygdala. Bars represent means ± S.E.M. (n = 5). ⁄P < 0.05 compared to saline group according to ANOVA followed by Tukey post-hoc test. 1170 H.M. Abelaira et al. / Neurochemistry International 59 (2011) 1163–1174

A 6000

5000 * 4000 * * * 3000 * * * * *

(Arbitrary units) (Arbitrary 2000 Bcl-2 expression * * * * * * Saline * Imipramine 30 1000 Lamotrigine 10 Lamotrigine 20 0 Prefontal Amygdala Hipocamppus Prefontal Amygdala Hipocamppus cortex cortex

Acute Chronic

B 6000 *

5000 * * * * * * * * * * ** 4000 *

3000 * AKT expression (Arbritary units) 2000 Saline 1000 Imipramine 30 Lamotrigine 10 Lamotrigine 20 0 Prefontal cortex Amygdala Hippocampus Prefontal cortex Amygdala Hippocampus

Acute Chronic

C 8000

7000 * 6000 * 5000

4000 * * * * * 3000 * (Arbitrary units) Gsk-3 expression * * Saline 2000 * * * * Imipramine 30 1000 Lamotrigine 10 Lamotrigine 20 0 Prefontal Amygdala Hippocampus Prefontal Amygdala Hippocampus cortex cortex

Acute Chronic

Fig. 5. The effects of administration of acute and chronic doses of lamotrigine and imipramine on the Bcl-2 (A), AKT (5B) and GSK-3 (C) levels in the prefrontal cortex, hippocampus and amygdala. Bars represent means ± S.E.M. (n = 5). ⁄P < 0.05 compared to saline group according to ANOVA followed by Tukey post-hoc test. H.M. Abelaira et al. / Neurochemistry International 59 (2011) 1163–1174 1171 and increased climbing scores, a similar pattern to nortriptyline, in 2000; Konradi et al., 2004). Our findings showed that imipramine addition, lamotrigine neither changed locomotion in the open-field increased the citrate synthase activity in the amygdala after acute test nor impaired habituation. Kaster et al. (2007) also showed that treatment, but not in the chronic treatment. In fact, a previous study lamotrigine (20–30 mg/kg) decreased the immobility time in the already showed that the acute administration (but not chronic), forced swimming test. Still, Mikulecká et al. (2004) showed that with antipsychotic olanzapine and antidepressant fluoxetine in- administration of lamotrigine (10 and/or 20 mg/kg for 6 consecu- creased citrate synthase activity in the brain areas tive days) did not change motor abilities and behavior in an open (Agostinho et al., 2009), suggesting that the results could be related field. However, recently Barbee et al. (2011) in a double-blind pla- to desensitization to the effects of the repeated administration of cebo-controlled evaluating patients with treatment-resistent drugs, or to an adaptation mechanism. depression showed that there was no difference between lamotri- Considering that metabolism impairment is probably involved gine and placebo groups. The authors suggesting that lamotrigine’s in the pathophysiology of depressive disorders, an increase in cre- efficacy might focus on specific subgroups with depression. atine kinase activity by antidepressants may be an important The precise molecular mechanisms responsible for the antide- mechanism of action of these drugs. In the present work we pressant action of lamotrigine are still not fully understood. Indeed showed that CK was increased in the amygdala after the adminis- studies have suggested that Antiepileptic drugs, such as lamotri- tration of imipramine in the acute treatment and in the hippocam- gine presents targets of action in the synapse, which could be rel- pus after the administration of imipramine and lamotrigine in the evant in epilepsy and other disorders. The mechanisms of action chronic treatment. Another study showed that CK activity was in- including, modulating ion channels and receptors and intracellular creased after the chronic administration of paroxetine (Santos signaling pathways (Johannessen, 2008; Mazza et al., 2007). Inter- et al., 2009). Assis et al. (2009) also reported that the acute admin- estingly, evidence suggests that a variety of intracellular pathways istration of ketamine and imipramine increased creatine kinase and signal transduction cascades are involved in both the patho- activity in the rat brain. On the other hand, the chronic administra- physiology and treatment of depression (Coyle and Duman, tion of nortriptiline and venlafaxine did not affect CK activity in the 2003; Duman, 1998; Duman et al., 1997; Vaidya et al., 2007). Many rat brain (Santos et al., 2009). Some other studies also point to the antidepressant drugs acutely increase monoamine levels, but the possibility that drugs used in the treatment of such disorders mod- requirement for chronic treatment has led to the hypothesis that ulate energy metabolism (Búrigo et al., 2006; Gamaro et al., 2003; long-term adaptations are necessary for the therapeutic actions Streck et al., 2006). Studies have reported brain energy metabolism of these treatments (Duman et al., 1994). Among the many long impairment in an animal model of mania induced by amphet- term targets of antidepressant treatments may be the regulation amine. It has been demonstrated that the administration of of neurotrophins, such as brain-derived neurotrophic factor (BDNF) amphetamine inhibited citrate synthase (Corrêa et al., 2007) and and nerve growth factor (NGF). creatine kinase (Streck et al., 2008) activity in the brain of rats. Our results showed that the acute and chronic treatments with Thus, it is possible that diminution of brain energy metabolism is lamotrigine increased the BDNF levels in the prefrontal cortex. involved in the pathophysiology of psychiatric disorders (Fattal Consistent with this result, Li et al. (2010) showed that the chronic et al., 2006; Madrigal et al., 2001). treatment with lamotrigine (30 mg/kg) increased BDNF protein In this context, Gardner et al. (2003) showed a significant de- expression in the prefrontal cortex, but contrarily to our result crease of mitochondrial ATP production rates and mitochondrial the BDNF protein expression was also increased in the hippocam- enzyme ratios in muscle in major depressive disorder patients, pus. We cannot explain why such discrepancies occur, but they when compared to controls. In the present study we demonstrated may be related to the dosage used. In addition, a study by our that both acute and chronic treatments with imipramine or lamo- group showed that acute administration of ketamine at the higher trigine altered respiratory chain complexes in rat brain, however dose 15 mg/kg, but not in lower doses, increased BDNF protein lev- these alterations were different with relation to protocols (acute els in the rat hippocampus. Our results also showed that chronic, or chronic), complex and brain area. but not acute; treatment with lamotrigine increased the NGF levels Furthermore, study from our group also showed that mitochon- in the prefrontal cortex. Another result showed that in rats, treat- drial respiratory chain complexes I, III and IV were inhibited after ment with lithium at various dosages increased NGF in the hippo- chronic mild stress in the cerebral cortex and cerebellum, and campus, amygdala, frontal cortex, and limbic forebrain, whereas acute administration of ketamine reversed this inhibition (Rezin NGF in the striatum, midbrain, and hypothalamus was unchanged et al., 2009). Madrigal et al. (2001) also reported that complexes (Hellweg et al., 2002). Our results showed that imipramine did not I–III and II–III of mitochondrial respiratory chain were inhibited alter de BDNF and NGF levels, suggesting that the antidepressant in rat brain after chronic stress (immobilization for six hours over effects of lamotrigine may be related, at least in part, by its action 21 days). Additionally, Ben-Shachar and Karry (2008) demon- on the neurotrophins, which was not observed with the classic strated reductions in mRNA and protein of complex I subunits antidepressant. NDUFV1, NDUFV2 and NADUFS1 in the postmortem cerebellum It is important that others studies have been shown effects of from patients with depression. Hroudova and Fisar (2010) using imipramine on the BDNF. In fact, chronic treatment with imipra- an in vitro study from pig brain, demonstrated that the complex mine increased BDNF mRNA levels in the dentate gyrus of the dor- I, II and IV activity decreased with antidepressants and mood sal hippocampus (Larsen et al., 2010). Réus et al. (2011) also stabilizers, suggesting in this study that antidepressants generally pointed to increase on the BDNF levels with imipramine in the pre- act as inhibitors of electron transport chain. Our findings also frontal cortex, hippocampus and amygdala by imunoblot, its showed an inhibitory effect on the activity of complex I, but effects were more pronounced when co-administrated with keta- contrarily to this, lamotrigine and imipramine increased the activ- mine, an antagonist of NMDA receptor. In contrast, others no have ities of complexes II, II-III and IV, suggesting that the increase in been shown effects of imipramine on the BDNF levels in the hippo- the complexes II, II-III and IV activity may be related, at least in campus (Garcia et al., 2008a,b; Fortunato et al., 2010), suggesting part to compensating the decrease of complex I activity. Such, that imipramine presents effects on the BDNF related with brain the effects of lamotrigine and imipramine on the mitochondrial area and technical used. respiratory chain could be positive, taking into account that there There is a strong body of evidence suggesting that dysfunction in is impairment in energy metabolism related to depression brain metabolism is related to neuropsychiatry disorders, such as, (Ben-Shachar and Karry, 2008; Rezin et al., 2009; Madrigal et al., depression and bipolar disorder (Albert et al., 2002; Kato and Kato, 2001). 1172 H.M. Abelaira et al. / Neurochemistry International 59 (2011) 1163–1174

A balance between cell death and cell proliferation must be wood and Agam 2003; De Sarno et al., 2002). Aubry et al. (2009) maintained to ensure the health of every human being. Recent showed that lithium, valproate, olanzapine and clozapine enhance findings indicate that approximately one-half of all major human proliferation and protect cells against serum withdrawal-induced diseases are a consequence of abnormal apoptosis (Reed, 2002). injury. These drugs also activated AKT-1 and GSK-3b phosphoryla- Neurodegeneration mediated by apoptosis can be initiated by tion (Aubry et al., 2009). Interestingly, gene expression of AKT-1 Bax translocation from the cytosol to the mitochondria, where it mRNA and protein, but not GSK-3b, was increased. Another study affects membrane permeability and permits cytochrome c release showed that sertraline potently inhibited the phosphorylation of and subsequent activation of caspases (Yang et al., 1995; Ghribi AKT and caused cell death. (Reed, 2002). Lamotrigine has a potent et al., 2001). Inappropriate apoptosis can cause autoimmune and activity dependent on ion channels (i.e., Na+ and Ca+) and could have neurodegenerative disorders, as well as heart disease, while resis- an indirect action on signal transduction (Xie and Hagan, 1998). Con- tance to apoptosis can promote cancer and impede the effective- sistent with our results, lamotrigine had an indirect action on AKT ness of cancer therapeutics. Our results demonstrate that protein levels. Whether lamotrigine has direct actions on these imipramine and lamotrigine decreased the Bcl-2 expression in intracellular signaling molecules has not been extensively studied the prefrontal cortex, amygdala and hippocampus in the acute to date. To our knowledge, no other previous assay has tested such and chronic treatments. Peng et al. (2008) showed that 3 lM imip- a complex mechanism. ramine treatment significantly up-regulated the mRNA and protein Reduced glutamatergic neurotransmission has been related to expression and Bcl-2 in day-7 imipramine-treated neural stem the antidepressant effect of lamotrigine. In fact, electrophysiologi- cells (NSCs). Huang et al. (2007) also showed that desipramine in- cal studies in the amygdala (Wang et al., 2002) and in the striatum creased the Bcl-2 expression in day 3 DP-treated NSCs. Another (Calabrese et al., 1999) showed that lamotrigine reduced excitatory study demonstrated that by the fourteenth day, (but not acute post-synaptic potential mediated by glutamate, an effect reversed treatment with citalopram), imipramine and amitriptyline in mice when exogenous glutamate was applied, findings consistent with had significantly elevated the hippocampal Bcl-2 protein expres- the proposal that lamotrigine had an inhibitory action on gluta- sion as compared to vehicle treated animals. (Murray and Hutson, mate release. Functional antagonists of the N-methyl-d-aspartate 2007), suggested that their elevation reflects a compensatory (NMDA) complex exhibit an antidepressant- like effect in animal mechanism. Consistent with our results, another study showed a models of depression. In adition, NMDA receptor antagonists have significant decrease of antiapoptotic Bcl-2 protein upon fluoxetine demonstrated alter neutrophins (Réus et al., 2010), and energy treatment, which is a known molecular event in the initiation of metabolism (Rezin et al., 2009; Assis et al., 2009) suggesting that apoptosis, suggesting that the effects of fluoxetine over antiapo- changes are mediated by glutamate action through NMDA recep- ptotic Bcl-2 may be interpreted as activation of apoptotic response. tor, thus, the effects exerted by lamotrigine in these pathways, GSK-3 (isoform b) is an important regulator of glycogen synthe- may be related, at least in part to its action on the glutamatergic sis, gene transcription, synaptic plasticity, apoptosis (cell death), system. cellular structure and resilience. It has been suggested that GSK- 3 regulates behavior by affecting b-catenin, glutamate receptors, circadian rhythms, and serotonergic neurotransmission (Beaulieu 5. Conclusions et al., 2008). All of these have been implicated in the pathophysiology of severe mood disorders. Our results showed a decreased in In conclusion, this is the first study that directly compares the the prefrontal cortex, amygdala and hippocampus with imipra- effects of acute and chronic lamotrigine treatment depressive-like mine and lamotrigine in the acute and chronic treatments. Another symptoms together with the effects on neurotrophins, metabolism study showed that lithium induces neurotrophic and neuroprotec- energy, signaling cascade. The behavioral effects of lamotrigine can tive effects in rodents, partly due to GSK-3b inhibition (Gould and be attributed to its action on neurochemistry pathways related to Manji, 2005). Li et al. (2004) also showed that treatment with depression. However, the results findings in the present research monoamine reuptake inhibitors fluoxetine and imipramine also in- were in preclinical study and we suggest clinical studies evaluating creased the level of Gsk-3. Valproate was initially reported to inhi- serum or postmortem brain from patients with major depression bit GSK-3b activity in SH-SY5Y cells (Chen et al., 1999; Chuang, and to evaluate whether lamotrigine could be a new option for this 2005), but these effects have not been confirmed in neuronal cells impairment disorder. (Gurvich and Klein, 2002). In general, increased activity of GSK-3 is proapoptotic, whereas inhibiting GSK-3 prevents apoptosis. Thus, Acknowledgements we suggest that in our study the effects of lamotrigine and imipramine were antiapototic, since both inhibited GSK-3. This study was supported in part by grants from ‘Conselho Nac- Accumulating evidence suggests that impaired AKT and/or ERK ional de Desenvolvimento Científico e Tecnológico’ (CNPq-Brazil – signaling contributes to the pathogenesis of schizophrenia, bipolar J.Q., C.T.S. and E.L.S.), from the Instituto Cérebro e Mente (J.Q.) and disorder and major depression and pharmacological studies indicate UNESC (J.Q., C.T.S. and E.L.S.). J.Q. and E.L.S. are recipients of CNPq that antipsychotics activate these signaling pathways in vivo or (Brazil) Productivity Fellowships. G.Z.R. is holder of a CAPES in vitro (Arguello and Gogos, 2008; Beaulieu et al., 2006; Lu et al., studentship. 2004; Zhang et al., 2005). Previous reports have demonstrated that AKT is not only involved in cell growth but is also involved in glucose metabolism/uptake (Hajduch et al., 2001; Lawlor and Alessi, 2001). 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