Pharmacology, Biochemistry and Behavior 103 (2013) 589–596
`
`Contents lists available at SciVerse ScienceDirect
`
`Pharmacology, Biochemistry and Behavior
`
`j o u r n a l ho m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p h a r m bi oc h e m b e h
`
`Lithium and valproate modulate energy metabolism in an animal model of mania
`induced by methamphetamine
`Gustavo Feier a, Samira S. Valvassori a, Roger B. Varela a, Wilson R. Resende a, Daniela V. Bavaresco a,
`Meline O. Morais b, Giselli Scaini b, Monica L. Andersen c, Emilio L. Streck b, João Quevedo a,⁎
`a Laboratory of Neurosciences, National Institute for Translational Medicine (INCT-TM), and Center of Excellence in Applied Neurosciences of Santa Catarina (NENASC),
`Postgraduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, 88806‐000 Criciúma, SC, Brazil
`b Laboratory of Experimental Pathophysiology and National Institute for Translational Medicine (INCT-TM), Postgraduate Program in Health Sciences, Health Sciences Unit,
`University of Southern Santa Catarina, 88806‐000 Criciúma, SC, Brazil
`c Department of Psychobiology, Universidade Federal de São Paulo, 04024‐002 São Paulo, SP, Brazil
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 16 July 2012
`Received in revised form 7 September 2012
`Accepted 16 September 2012
`Available online 23 September 2012
`
`Keywords:
`m-AMPH
`Animal model of mania
`Krebs cycle enzymes
`Complexes of mitochondrial respiratory chain
`
`Studies have shown alterations in mitochondrial complexes of bipolar disorder (BD) patients. However,
`changes in the Krebs cycle enzymes have been little studied. The animal model of mania induced by amphet-
`amine has been widely used for the study of bipolar mania. The aim of this study is to assess behavioral and
`energy metabolism changes in an animal model of mania induced by methamphetamine (m-AMPH). Wistar
`rats were first given m-AMPH or saline for 14 days, and then, between days 8 and 14, rats were treated with
`lithium (Li), valproate (VPA), or saline (Sal). Locomotor behavior was assessed using the open-field task and
`activities of Krebs cycle enzymes (citrate synthase and succinate dehydrogenase), mitochondrial respiratory
`chain complexes (I, II, III, and IV), and creatine kinase measured in the brain structures (prefrontal, amygdala,
`hippocampus, and striatum). Li and VPA reversed m-AMPH-induced hyperactivity. The administration of
`m-AMPH inhibited the activities of Krebs cycle enzymes and complexes of the mitochondrial respiratory
`chain in all analyzed structures. Li and VPA reversed m-AMPH-induced energetic metabolism dysfunction;
`however, the effects of Li and VPA were dependent on the brain region analyzed. From the results obtained
`in this study, we suggested that the decreased Krebs cycle enzymes activity induced by m-AMPH may be
`inhibiting mitochondrial respiratory chain complexes. Therefore, changes in the Krebs cycle enzymes may
`also be involved in BD.
`
`© 2012 Published by Elsevier Inc.
`
`1. Introduction
`
`Although bipolar disorder (BD) is a common psychiatric disorder
`that leads to serious health problems, little is known about its patho-
`physiology. BD is a multifactorial illness and has diverse symptoms, in-
`cluding recurrences of mania, depression, and mixed states, which
`hampers the development of a suitable animal model (Machado-Vieira
`et al., 2004). Despite the difficulties inherent in modeling BD in animals,
`several behavioral animal models of mania or depression have been de-
`veloped in an attempt to mimic some aspect of behavioral changes
`
`Abbreviations: BD, Bipolar Disorder; AMPHs, Amphetamines; m-AMPH, Metamphetamine;
`d-AMPH, Dextroamphetamine; Li, Lithium; VPA, Valproate; Sal, Saline; SD, Succinate
`Dehydrogenase; DCIP, Succinate-2,6-dichloroindophenol; CK, Creatine Kinase; MM-CK,
`Dimeric MM-creatine kinase; ROS, Reactive Oxygen Species.
`⁎ Corresponding author at: Laboratório de Neurociências, PPGCS, UNASAU, Universidade
`do Extremo Sul Catarinense, 88806‐000 Criciúma, SC, Brazil. Fax: +55 48 3443 4817.
`E-mail address: quevedo@unesc.net (J. Quevedo).
`
`0091-3057/$ – see front matter © 2012 Published by Elsevier Inc.
`http://dx.doi.org/10.1016/j.pbb.2012.09.010
`
`found in this psychiatric condition (Frey et al., 2006a; Jornada et al.,
`2010; Herman et al., 2007; Machado-Vieira et al., 2004).
`Several studies have suggested that dysfunctional cellular energy
`metabolism has a central role in BD, mainly in the mitochondria
`(Beech et al., 2010; Maurer et al., 2009; Wang, 2007; Anglin et al.,
`2012). Abnormalities in energy metabolism were found in functional
`assays and in magnetic resonance spectroscopy studies (Dager et al.,
`2004; Deicken et al., 1995; Frey et al., 2007; Regenold et al., 2009).
`In an animal model of mania, Li and VPA were able to reverse and pre-
`vent amphetamine-induced mitochondrial dysfunction, suggesting
`that one of the mechanisms of action in mood stabilizers may be de-
`creasing the amount of dopamine available, and stabilizing mitochon-
`drial function in the pathophysiology of BD (Valvassori et al., 2010).
`However, there are few studies evaluating changes in the enzymes
`of the Krebs cycle in bipolar disorder (Freitas et al., 2010; Fonseca et
`al., 2005).
`Dysfunctions in the Krebs cycle can be capable of altering the rate
`of brain metabolism and the production of free radicals. After glycol-
`ysis, pyruvate is decarboxylated to acetyl CoA by the pyruvate dehy-
`drogenase. The conversion of acetyl CoA to CO2 in the Krebs cycle
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`results in the production of NADH for the electron transport chain and
`subsequent production of ATP. Krebs cycle is a chemical system made
`up of several enzymes and steps. In the first step, citrate synthase (CS)
`catalyzes the condensation of oxaloacetate and the acetyl group of
`acetyl coenzyme-A (Shepherd and Garland, 1996). In the final step
`of Krebs cycle, malate dehydrogenase (MD) catalyzes the dehydroge-
`nation of l-malate to oxaloacetate (Kelly et al., 1989). Succinate dehy-
`drogenase (SD) is part of both the Krebs cycle and the respiratory
`chain (complex II); therefore, this enzyme is one of the most impor-
`tant markers of the mitochondrial ability to supply an adequate
`amount of ATP (Tyler, 1992).
`Oxidative phosphorylation is the next step following the Krebs
`cycle. In the oxidative phosphorylation, electrons are passed along a
`series of respiratory enzyme complexes (complexes I, II, III, and IV)
`located in the inner mitochondrial membrane, and the energy re-
`leased by this electron transfer is used to pump protons across the
`membrane. The resultant electrochemical gradient enables another
`complex, adenosine 5′-triphosphate (ATP) synthase, to synthesize
`ATP from ADP plus Pi (Horn and Barrientos, 2008).
`The symptoms of BD involve neurovegetative abnormalities, im-
`pulsivity and psychosis, suggesting that anterior limbic brain net-
`works controlling these behaviors are dysfunctional. One of the
`most important functions of the amygdala is modulate the limbic sys-
`tem, controlling an iterative circuit, prefrontal–striatal–thalamic,
`which control complex socioemotional behaviors (Strakowski et al.,
`2000, 2005).
`Thus, we examined the activities of mitochondrial enzymes in the
`Krebs cycle (CS, MD and SD) and respiratory enzyme complexes
`(complexes I, II, III and IV) in the amygdala, prefrontal, striatum,
`and hippocampus of rats submitted to an animal model of mania in-
`duced by methamphetamine.
`
`2. Experimental methods
`
`2.1. Animals
`
`The subjects were adult male Wistar rats (weighting 250–350 g)
`obtained from our breeding colony. The animals were housed five to
`a cage, with food and water available ad libitum and were maintained
`on a 12-h light/dark cycle (lights on at 7:00 a.m.) at a temperature of
`22±1 °C. All experimental procedures were performed in accordance
`with, and with the approval of the local Ethics Committee in the use
`of animals at the Universidade do Extremo Sul Catarinense. All exper-
`iments were performed at the same time during the day to avoid cir-
`cadian variations.
`
`2.2. Drugs and pharmacological procedures
`
`The animals received one daily intraperitoneal injection (i.p.) of
`m-AMPH 0.25 mg/kg or saline (Sal) for 14 days (45 animals per
`group). On the 8th day of treatment, the animals in the saline and
`d-AMPH group were divided in 3 groups (15 animals per group): 1)
`treatment with Li
`(47.5 mg/kg i.p.); 2)
`treatment with VPA
`(200 mg/kg i.p.) and 3) treatment with Sal for 7 days twice a day
`for all drugs. On the 15th day of treatment, the animals received a sin-
`gle injection of m-AMPH or Sal and locomotor activity was assessed
`2 h after the last injection. The rats were killed by decapitation imme-
`diately after the open-field task and amygdala, prefrontal, striatum
`and hippocampus were dissected, rapidly frozen and stored −70 ºC
`until assayed.
`The dose of Li and VPA was based on previous studies from our
`laboratory, since Li at 47.5 mg/kg and VPA at 200 mg/kg prevented
`and reversed the hyperactivity induced by amphetamine. In addition,
`the animals treated with Li had plasmatic levels of this drug between
`0.6 and 1.2 mEq/L, as recommended in the treatment of BD patients
`(Frey et al., 2006a).
`
`2.3. Locomotor activity
`
`Locomotor activity was assessed using the open-field task as pre-
`viously described (Barros et al., 2002; Frey et al., 2006a, 2006b,
`2006c). This task was performed in a 40×60 cm open field
`surrounded by 50 cm high walls, made of brown plywood, with the
`floor divided into 12 equal rectangles by black lines. The animals
`were gently placed on the left rear rectangle, and left free to explore
`the arena for 5 min. Crossings of the black lines (locomotor activity/
`horizontal activity) and rearings (exploratory activity/vertical activity)
`were counted.
`
`2.4. Tissue and homogenate preparation
`
`The prefrontal cortex, amygdala, hippocampus and striatum were
`removed and homogenized (1:10, w/v) in SETH buffer, pH 7.4
`(250 mM sucrose, 2 mM EDTA, 10 mM Trizma base, 50 IU/mL heparin).
`The homogenates were centrifuged at 800×g for 10 min at 4 °C and the
`supernatants kept at −70 °C until being used for enzyme activity deter-
`mination. The maximal period between homogenate preparation and
`enzyme analysis was always less than 5 days. The protein content was
`determined by the method described by Lowry et al. (1951) using bovine
`serum albumin as standard.
`
`2.5. Activities of enzymes of Krebs cycle
`
`2.5.1. Citrate synthase activity
`Citrate synthase activity was assayed according to the method de-
`scribed by Shepherd and Garland (1996). The reaction mixture
`contained 100 mM Tris, pH 8.0, 100 mM acetyl CoA, 100 mM
`5,5′-di-thiobis-(2-nitrobenzoic acid), 0.1% triton X-100, and 2–4 μg
`supernatant protein and was initiated with 100 μM oxaloacetate
`and monitored at 412 nm for 3 min at 25 °C.
`
`2.5.2. Malate dehydrogenase activity
`Malate dehydrogenase was measured as described by Kitto
`(1969). Aliquots (20 mg protein) were transferred into a medium
`containing 10 mM rotenone, 0.2% Triton X-100, 0.15 mM NADH,
`and 100 mM potassium phosphate buffer, pH 7.4, at 37 °C. The reac-
`tion was started by the addition of 0.33 mM oxaloacetate. Absorbance
`was monitored as described above.
`
`2.5.3. Succinate dehydrogenase activity
`Succinate dehydrogenase activity was determined according to the
`method of Fischer et al. (1985), and measured by following the decrease
`in absorbance due to the reduction of 2,6-di-chloro-indophenol
`(2,6-DCIP) at 600 nm with 700 nm as a reference wavelength (ε=
`19.1 mM−1 cm−1) in the presence of phenazine methosulfate (PMS).
`The reaction mixture consisting of 40 mM potassium phosphate,
`pH 7.4, 16 mM succinate and 8 μM 2,6-DCIP was pre-incubated with
`40–80 μg homogenate protein at 30 °C for 20 min. Subsequently,
`4 mM sodium azide, 7 μM rotenone and 40 μM 2,6-DCIP were added
`and the reaction was initiated by the addition of 1 mM PMS and was
`monitored for 5 min.
`
`2.6. Activities of mitochondrial respiratory chain enzymes
`
`2.6.1. Complex I activity
`NADH dehydrogenase (complex I) was evaluated according to
`Cassina and Radi (1996) by the determination of the rate of NADH-
`dependent ferricyanide reduction at λ=420 nm.
`
`2.6.2. Complex II activity
`The activities of succinate-2,6-dichloroindophenol (DCIP)-oxidoreductase
`(complex II) were determined by the method described by Fischer et al.
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`(1985). Complex II activity was measured by following the decrease in
`absorbance due to the reduction of 2,6-DCIP at λ=600 nm.
`
`3. Results
`
`2.6.3. Complex II–III activity
`The activity of succinate:cytochrome c oxidoreductase (complex
`III) was determined by the method described by Fischer et al.
`(1985). Complex II–III activity was measured by cytochrome c reduc-
`tion using succinate as substrate at λ=550 nm.
`
`2.6.4. Complex IV activity
`The activity of cytochrome c oxidase (complex IV) was assayed
`according to the method described by Rustin et al. (1994), measured
`by following the decrease in absorbance due to the oxidation of previ-
`ously reduced cytochrome c (prepared by reduction of cytochrome
`with NaBH4 and HCl) at λ=550 nm with 580 nm as the reference
`wavelength (ɛ=19.1 mM−1 cm−1). The activities of the mitochondri-
`al respiratory chain complexes were calculated as nmol·min−1·mg
`protein−1.
`
`2.7. Activity of creatine kinase enzyme
`
`Creatine kinase activity was measured in brain homogenates
`pretreated with 0.625 mM lauryl maltoside. The reaction mixture
`consisted of 60 mM Tris–HCl, pH 7.5, containing 7 mM phosphocrea-
`tine, 9 mM MgSO4 and approximately 0.4–1.2 μg protein in a final
`volume of 100 μL. After 15 min of pre-incubation at 37 °C, the reac-
`tion was started by the addition of 3.2 mmol of ADP plus 0.8 mmol
`of reduced glutathione. The reaction was stopped after 10 min by
`the addition of 1 μmol of p-hydroxymercuribenzoic acid. The creatine
`formed was estimated according to the colorimetric method of
`Hughes (1962). The color was developed by the addition of 100 μL
`2% α-naphthol and 100 μL 0.05% diacetyl in a final volume of 1 mL
`and read spectrophotometrically after 20 min at 540 nm. Results
`were expressed as units/min×mg protein.
`
`2.8. Statistical analysis
`
`Data were analyzed by two-way analysis of variance followed by
`the Tukey test when F was significant and are expressed as mean±
`standard deviation. All analyses were performed using the Statistical
`Package for the Social Science (SPSS; version 16.0) software.
`
`3.1. Li and VPA reversed hyperlocomotion induced by m-AMPH in rats
`
`Results for locomotor activity are shown in Fig. 1. For the analysis of
`locomotion (crossings), the two-way ANOVA revealed significant dif-
`ferences for m-AMPH administration [F(1.41)=28.5, p b0.001], treat-
`ment [F(2.41)=p b0.001], and m-AMPH administration×treatment
`interaction [F(2.41)=24.71, p b0.001].
`For the analysis of exploration (rearings), the two-way ANOVA also
`revealed significant differences for m-AMPH administration [F(1.41)=
`5.96, p=0.01], treatment [F(2.41)=11.14, p b0.001], and m-AMPH
`administration×treatment interaction [F(2.41)=4.3, p=0.02].
`Further analysis with Tukey post-hoc tests showed that the ad-
`ministration of m-AMPH increased locomotion and rearing behavior
`in rats treated with Sal. Treatment with Li and VPA reversed
`m-AMPH-related hyperlocomotion. Animals given Li or VPA, followed
`by m-AMPH, did not differ from the control group given Sal, followed
`by Sal.
`
`3.2. Li and VPA treatments attenuate m-AMPH-induced decreases in
`activities of enzymes of Krebs cycle in rat brains
`
`Results for citrate synthase activity are shown in Fig. 2A. The
`m-AMPH administration caused a marked decrease in the citrate
`synthase activity of rats' prefrontal, amygdala, hippocampus, and stri-
`atum. Seven days of treatment with Li reversed m-AMPH's effects on
`citrate synthase activity in the prefrontal and amygdala, and partially
`reversed in the hippocampus and striatum. The VPA treatment
`reversed m-AMPH's effects on citrate synthase activity in the
`amygdala and hippocampus and partially reversed in the prefrontal
`and striatum.
`Data from the two-way ANOVA for d-AMPH administration
`[prefrontal cortex: F(1.27) = 18.86, p > 0.001; amygdala: F(1.27) =
`7.19, p = 0.01; hippocampus: F(1.27) = 5.73, p = 0.02; striatum:
`F(1.27) = 6.2, p = 0.019], treatment [prefrontal cortex: F(4.14) =
`0.02, p = 0.02; amygdala: F(2.27) = 1.3, p = 0.28; hippocampus:
`F(2.27) = 2.36, p = 0.11; striatum: F(1.27) = 1.44, p = 0.25], and
`d-AMPH administration × treatment interaction [prefrontal cortex:
`F(2.27) = 12.01, amygdala: F(2.27) = 7.33, p = 0.002; p = 0.002;
`hippocampus: F(2.27) = 7.31, p = 0.02; striatum: F(2.27) = 2.83,
`p = 0.076].
`
`Fig. 1. Open field test. Free movement in the open field.
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`Fig. 2. Activities of enzymes of Krebs cycle. A) Citrate synthase activity in the prefrontal, amygdala, hippocampus, and striatum. B) Succinate dehydrogenase activity in the prefrontal,
`amygdala, hippocampus, and striatum. C) Malate dehydrogenase activity in the prefrontal, amygdala, hippocampus, and striatum. *Different from Sal+Sal group. #Different from
`m-AMPH+Sal group.
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`Results for succinate dehydrogenase activity are shown in Fig. 2B.
`The m-AMPH administration decreased succinate dehydrogenase
`activity in all brain structures evaluated. Treatment with Li reversed
`the reduction of succinate dehydrogenase activity induced by
`m-AMPH in the amygdala and hippocampus, but not in the striatum.
`In the prefrontal, the Li administration of the m-AMPH pretreated
`rats increased succinate dehydrogenase activity when compared to
`the control group. In the hippocampus, succinate dehydrogenase ac-
`tivity was significantly decreased in the Sal + Li group. Seven days of
`treatment with VPA reversed m-AMPH's effects on succinate
`dehydrogenase in the striatum, but not in the prefrontal and
`amygdala. In the hippocampus, succinate dehydrogenase activity
`was significantly increased in the VPA + m-AMPH group. We also
`found that in treatment using VPA alone, succinate dehydrogenase
`activity was significantly enhanced in the prefrontal when compared
`to the control group.
`Data from the two-way ANOVA for d-AMPH administration [pre-
`frontal cortex: F(1.23)=21.95, p>0.001; amygdala: F(1.23)=
`23.78, p b0.001; hippocampus: F(1.23)=1.15, p=0.29; striatum:
`F(1.23)=32.87, pb0.001], treatment [prefrontal cortex: F(2.23)=
`36.76, pb0.001; amygdala: F(2.23)=33.57, pb0.001; hippocampus:
`F(2.23)=17.51, pb0.001; striatum: F(2.23)=4.65, p=0.02], and
`d-AMPH administration×treatment
`interaction [prefrontal cortex:
`F(2.23)=4.23, p=0.02, amygdala: F(2.23)=8.71, p=0.001; hippo-
`campus: F(2.23)=47.94, pb0.001; striatum: F(2.23)=13.53, pb0.001].
`Results for malate dehydrogenase activity are shown in Fig. 2C.
`After m-AMPH administration, malate dehydrogenase activity also
`decreased in the amygdala, hippocampus, and striatum. The Li treat-
`ment reversed m-AMPH's effects on the malate dehydrogenase
`activity in the hippocampus and striatum. The VPA treatment re-
`versed m-AMPH's effects on malate dehydrogenase activity in the
`hippocampus and partially in the amygdala and striatum.
`Data from the two-way ANOVA for d-AMPH administration [pre-
`frontal cortex: F(1.23)=0.11, p=0.71; amygdala: F(1.23)=162.21,
`p b0.001;
`hippocampus:
`F(1.23)=16.14,
`p b0.001;
`striatum:
`F(1.23)=40.91, p b0.001], treatment [prefrontal cortex: F(2.23)=
`5.18, p=0.01; amygdala: F(2.23)=39.73, p=0.18; hippocampus:
`F(2.23)=12.5, p b0.001; striatum: F(2.23)=9.31, p=0.001], and
`d-AMPH administration×treatment interaction [prefrontal cortex:
`F(2.23)=5.06, p=0.01, amygdala: F(2.23)=14.09, p b0.001; hippo-
`campus: F(2.23)=5.41, p=0.01; striatum: F(2.23)=11.54, p b0.001].
`
`Data from the two-way ANOVA for d-AMPH administration [pre-
`frontal cortex: F(1.21)=13.18, p=0.001; amygdala: F(1.21)=
`24.88, p b0.001; hippocampus: F(1.21)=13.38, p=0.001; striatum:
`F(1.21)=2.1, p=0.16],
`treatment
`[prefrontal cortex: F(2.21)=
`32.22, p b0.001; amygdala: F(2.21)=20.64, p b0.001; hippocampus:
`F(2.21)=4.42, p=0.02; striatum: F(2.21)=10.63, p b0.001], and
`d-AMPH administration×treatment interaction [prefrontal cortex:
`F(2.21)=21.11, p b0.001, amygdala: F(2.21)=20.64, p b0.001; hip-
`pocampus: F(2.21)=5.92, p=0.009;
`striatum: F(2.21)=16.14,
`p b0.001].
`Results for complex II–III activity are shown in Fig. 3C. The Li and
`VPA treatments attenuate the m-AMPH-induced decrease of complex
`II–III activity in the hippocampus and striatum. Seven days of treat-
`ment with VPA attenuates m-AMPH's effects on complex II–III activity
`in the prefrontal and amygdala; adversely, Li administration potenti-
`ates the m-AMPH's effects in these cerebral structures.
`Data from the two-way ANOVA for d-AMPH administration [pre-
`frontal cortex: F(1.25)=240.9, p b0.001; amygdala: F(1.25)=
`414.15, p b0.001; hippocampus: F(1.25)=98.6, p b0.001; striatum:
`F(1.25)=152.73, p b0.001], treatment [prefrontal cortex: F(2.25)=
`16, p b0.001; amygdala: F(2.25)=30.25, p b0.001; hippocampus:
`F(2.25)=8.55, p=0.001; striatum: F(2.25)=3.57, p=0.04], and
`d-AMPH administration×treatment interaction [prefrontal cortex:
`F(2.25)=12.11, p b0.001; amygdala: F(2.25)=48.2, p b0.001; hippo-
`campus: F(2.25)=9.59, p b0.001; striatum: F(2.25)=4.6, p=0.01].
`Results for complex IV activity are shown in Fig. 3D. Treatment
`with Li and VPA reversed m-AMPH-related complex IV dysfunction
`in the amygdala and striatum. In the prefrontal, VPA, but not Li, re-
`versed m-AMPH's effects on complex IV activity. In the amygdala, Li
`and VPA partially reversed the m-AMPH's effects on complex IV
`activity.
`Data from the two-way ANOVA for d-AMPH administration [pre-
`frontal cortex: F(1.25)=137.29, p b0.001; amygdala: F(1.25)=
`15.97, p b0.001; hippocampus: F(1.25)=48.15, p b0.001; striatum:
`F(1.26)=3.74, p=0.06], treatment [prefrontal cortex: F(2.25)=
`31.31, p b0.001; amygdala: F(2.25)=25.29, p b0.001; hippocampus:
`F(2.25)=6.78, p=0.004; striatum: F(2.26)=30.40, p b0.001], and
`d-AMPH administration×treatment interaction [prefrontal cortex:
`F(2.25)=460.32, p b0.001, amygdala: F(2.25)=15.29, p b0.001; hip-
`pocampus: F(2.25)=0.015, p=0.98;
`striatum: F(2.26)=17.28,
`p b0.001].
`
`3.3. Li and VPA treatments attenuate m-AMPH-induced decreases in the
`activities of respiratory chain complexes in rat brain
`
`3.4. Effects of Li and VPA on creatine kinase activity in the rats submitted
`to animal model of mania induced by m-AMPH
`
`Administration of m-AMPH inhibited the respiratory chain
`complexes (I, II, II–III, and IV) in all brain structure evaluated.
`Results for complex I activity are shown in Fig. 3A. The Li and VPA
`administration reversed m-AMPH's effects on complex I activity in
`the prefrontal and amygdala of rats. The VPA treatment, but not the
`Li, reversed the m-AMPH-induce decrease of complex I activity in
`the hippocampus and striatum.
`Data from the two-way ANOVA for d-AMPH administration
`[prefrontal cortex: F(1.27) = 25.54, p b0.001; amygdala: F(1.27) =
`19.56, p b0.001; hippocampus: F(1.27) = 107.71, p b0.001; striatum:
`F(1.27) = 64.99, p b0.001], treatment [prefrontal cortex: F(2.27) =
`546.93, p b0.001; amygdala: F(2.27) = 14.95, p b0.001; hippocam-
`pus: F(2.27) = 23.92, p b0.001; striatum: F(2.27) = 23.25, p b0.001],
`and d-AMPH administration×treatment interaction [prefrontal cortex:
`F(2.27)=595.32, pb0.001, amygdala: F(2.27)=18.15, pb0.001; hippo-
`campus: F(2.27)=42.37, pb0.001; striatum: F(2.27)=31.55, pb0.001].
`Results for complex II activity are shown in Fig. 3B. The Li and VPA
`treatments reversed m-AMPH's effects on complex II activity in the
`hippocampus and striatum. In the prefrontal and amygdala, VPA,
`but not Li, significantly increased the complex II activity when com-
`pared to the control group.
`
`No significant difference was found in the analysis of creatine
`kinase (Data not shown).
`
`4. Discussion
`
`Initially, we can reproduce data from previous studies in our labo-
`ratory, in which Li and VPA reversed the hyperactivity induced by
`m-AMPH at 0.25 mg/kg (da-Rosa et al., in press). It is known that
`mood stabilizers reverse and prevent hyperactivity induced by
`d-AMPH at 2 mg/kg (Frey et al., 2006a, 2006b, 2006c, 2006d).
`Accumulating evidence suggests that energetic metabolism dys-
`function contributes to the pathogenesis of BD. Impairment of com-
`plex I was found in the prefrontal cortex of bipolar patients
`(Andreazza et al., 2010). In a large-scale DNA microarray analysis of
`postmortem brains, it was shown that a global down-regulation of mi-
`tochondrial genes, such as those encoding respiratory chain compo-
`nents is in BD (Iwamoto et al., 2005). In an elegant study, Cataldo et
`al. (2010) have demonstrated that mitochondria from patients with
`BD have size and distributional abnormalities compared with the
`control group. In the brain, individual mitochondria profiles had sig-
`nificantly smaller areas, on average, in BD samples. In peripheral
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`activityintheprefrontal,amygdala,hippocampus,andstriatum.D)ComplexIVactivityintheprefrontal,amygdala,hippocampus,andstriatum.*DifferentfromSal+Salgroup.#Differentfromm-AMPH+Salgroup.
`Fig.3.Activitiesofmitochondrialrespiratorychainenzymes.A)ComplexIactivityintheprefrontal,amygdala,hippocampus,andstriatum.B)ComplexIIactivityintheprefrontal,amygdala,hippocampus,andstriatum.C)ComplexII–III
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`cells, mitochondria in BD samples were concentrated proportionately
`more within the perinuclear region than in the distal processes. These
`changes in mitochondrial morphology and distribution appear to be
`linked to diverse cellular events, such as differentiation, aging, and
`apoptosis. In addition, abnormalities in the energy metabolism of
`BD patients were also found in functional assays and magnetic reso-
`nance spectroscopy studies (Dager et al., 2004; Deicken et al., 1995;
`Frey et al., 2007; Regenold et al., 2009).
`In previous studies, we have demonstrated that d-AMPH at 2 mg/kg
`inhibited citrate synthase (Corrêa et al., 2007) and mitochondrial respi-
`ratory chain complex I, II, II–III, and IV (Valvassori et al., 2010) activities
`in the brain of rats, which was reversed by Li and VPA. As described
`above, changes in energy metabolism are strongly linked to BD; there-
`fore, the development of animal models with this feature is interesting
`to study the action mechanisms of classical drugs on this system and
`still to test new drugs.
`Here, we demonstrated that chronic administration of m-AMPH at
`0.25 mg/kg was able to inhibit the cycle Krebs enzymes activities, cit-
`rate synthase, succinate dehydrogenase, and malate dehydrogenase.
`Moreover, we demonstrated that m-AMPH at 0.25 mg/kg inhibits mi-
`tochondrial respiratory chain complexes (I, II, II-III, and IV) in all brain
`structures evaluated, as well as d-AMPH at 2 mg/kg, as demonstrated
`in previous studies (Valvassori et al., 2010). Dysfunctions in the Krebs
`cycle can be capable of altering the function of the mitochondrial re-
`spiratory chain complexes, and consequently, the rate of brain metab-
`olism (Shepherd and Garland, 1996). Recently, it was shown that
`m-AMPH administration decreased Krebs cycle intermediates in the
`urine and increased glucose in the plasma of rats (Shima et al.,
`2011). Kim et al. (2009) has demonstrated dose-dependent frontal
`hypometabolism on fluorodeoxy-D-glucose-positron emission to-
`mography (FDG-PET) in methamphetamine abusers. Impaired energy
`metabolism results in depletion of ATP in cells, and ATP depletion as a
`consequence of mitochondrial toxicity may be related to a number of
`m-AMPH-induced toxicities. From the results obtained in this study,
`we suggest that the decreased Krebs cycle enzyme activity induced
`by m-AMPH can inhibit the mitochondrial respiratory chain complex
`activity, which is involved in several psychiatric disorders (Manji et
`al., 2012).
`Another significant finding in this study is that mood stabilizers, Li
`and VPA, attenuated the AMPH's effects on Krebs cycle enzyme activ-
`ity, and consequently, reduced the impairment on respiratory chain
`complex activity. Kazuno et al. (2008) have showed that VPA may
`stabilize intracellular calcium in cells with high mitochondrial calci-
`um levels. The excessive release of glutamate by m-AMPH may in-
`duce neuronal damage through receptor-mediated intracellular
`Ca2+ overload and increased reactive oxygen species (ROS) produc-
`tion (Arundine and Tymianski, 2004; Choi, 1995; Lu et al., 2008).
`The Li and VPA treatments exert neuroprotective effects against cyto-
`toxicity by inhibiting the glutamate-induced increase of intracellular
`free Ca2+ concentration (Shao et al., 2005). It is well known that
`m-AMPH-induced increased DA oxidized metabolites inhibit the mi-
`tochondrial
`respiratory system, both in vivo and in vitro
`(Przedborski et al., 1993). The Li and VPA treatments inhibit the do-
`paminergic system by a mechanism of second messenger activation,
`which is activated by D2 receptors (Yatham et al., 2002). Together
`with our results, these studies suggest that mood stabilizers, Li and
`VPA, attenuate the glutamate and DA systems, protecting the mito-
`chondria against m-AMPH-induced damage.
`Taking into account that mitochondrial dysfunction plays a critical
`role in BD, improving mitochondrial function could ameliorate the
`neural plasticity and cellular resilience associated with this disorder.
`Previous studies have demonstrated that chronic treatment with Li
`or VPA enhanced mitochondrial function, as determined by mito-
`chondrial membrane potential and mitochondrial oxidation in
`SH-SY5Y cells (Bachmann et al., 2009a, 2009b).
`In addition,
`Bachmann et al. (2009a, 2009b) have found that treatment with Li
`
`or VPA prevented the m-AMPH-induced reduction of mitochondrial
`cytochrome c, the mitochondrial anti-apoptotic Bcl-2/Bax ratio, and
`mitochondrial cytochrome oxidase (COX) activity. Bcl-2 attenuates
`apoptosis by sequestering proforms of caspases (death-driving cyste-
`ine proteases), preventing the release of mitochondrial apoptogenic
`(programmed cell death) factors, such as calcium, cytochrome c,
`and apoptosis-inducing factor, into the cytoplasm, and enhancing mi-
`tochondrial calcium uptake. Several preclinical and clinical studies
`have shown that Li and VPA regulate numerous factors involved in
`cell survival pathways, including brain-derived neurotrophic factor
`(BDNF), bcl-2, and mitogen-activated protein kinases (MAPK) (Frey
`et al., 2006a; Bachmann et al., 2009a, 2009b; Suwalska et al., 2010;
`Chiu and Chuang, 2010). We suggested that these drugs increase
`neurotrophic factors that ameliorate impairments of mitochondrial
`functions, and consequently, improve cellular plasticity and resilience
`underlying the pathophysiology of BD.
`It should be noted that, in the present study, the effects of Li and
`VPA on some enzymes and in some brain areas are similar. However,
`for other enzymes and in some brain areas, VPA, but not Li, amelio-
`rates the effects of m-AMPH. The present data is in line with previous
`studies that showed heterogeneity of oxidative stress parameters
`across brain regions and treatment regimens (Musavi and Kakkar,
`1998, 2000 and Musavi and Kakkar, 2003). As an example, Li and
`VPA showed a treatment and region-specific effect on oxidative stress
`in an animal model of mania induced (Frey et al., 2006c). There is no
`specific explanation for this phenomenon; regions of the central ner-
`vous system can respond distinctly (Sullivan et al., 2005), and the ac-
`tivity of energetic metabolism enzymes was analyzed in different
`brain regions, which in part represent different cell types, indicating
`heterogeneity in terms of physiological and metabolic characteristics
`(Lai et al., 1977, Sims, 1991; Sonnewald et al., 1998).
`From the results obtained in this study, we suggest that the de-
`creased Krebs cycle enzyme activity induced by m-AMPH can inhibit
`mitochondrial respiratory chain complexes. Li and VPA reversed
`m-AMPH-induced energetic metabolism dysfunction. However, the
`effects of Li and VPA were dependent on the brain region analyzed.
`Therefore, we suggest that changes in the Krebs cycle enzymes may
`be involved in BD, and in the therapeutic effects of mood stabilizers.
`More studies are needed to better characterize the role of Krebs en-
`zyme cycle in this disorder.
`
`Acknow