Journal of Psychiatric Research 44 (2010) 506–510

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Journal of Psychiatric Research

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Effects of mood stabilizers on hippocampus and amygdala BDNF levels in ananimal model of mania induced by ouabain

Luciano K. Jornada a, Morgana Moretti a, Samira S. Valvassori a, Camila L. Ferreira a, Peterson T. Padilha a,Camila O. Arent a, Gabriel R. Fries b, Flavio Kapczinski b, João Quevedo a,*

a Laboratory of Neurosciences and National Science and Technology Institute for Translational Medicine, Postgraduate Program in Health Sciences, Health Sciences Unit,University of Southern Santa Catarina, 88808-000 Criciúma, SC, Brazilb Bipolar Disorders Program, Laboratory of Molecular Psychiatry and National Science and Technology Institute for Translational Medicine,Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS, Brazil

a r t i c l e i n f o

Article history:Received 17 September 2009Received in revised form 29 October 2009Accepted 3 November 2009

Keywords:OuabainManiaBDNFLocomotor activityMood stabilizersAnimal model

0022-3956/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.jpsychires.2009.11.002

* Corresponding author. Laboratório de Neurociêncsidade do Extremo Sul Catarinense, 88806-000 Crici3431 2578; fax: +55 48 3443 4817.

E-mail address: quevedo@unesc.net (J. Quevedo).

a b s t r a c t

There is a body of evidence suggesting that BDNF is involved in bipolar disorder (BD) pathogenesis. Intra-cerebroventricular (ICV) injection of ouabain (OUA), a specific Na+/K+ ATPase inhibitor, induces hyperlo-comotion in rats, and has been used as an animal model of mania. The present study aims to investigatethe effects of the lithium (Li) and valproate (VPT) in an animal model of mania induced by ouabain. In thereversal model, animals received a single ICV injection of OUA or cerebrospinal fluid (aCSF). From the dayfollowing the ICV injection, the rats were treated for 6 days with intraperitoneal (IP) injections of saline(SAL), Li or VPT twice a day. In the maintenance treatment (prevention model), the rats received IP injec-tions of Li, VPT, or SAL twice a day for 12 days. In the 7th day of treatment the animals received a singleICV injection of either OUA or aCSF. After the ICV injection, the treatment with the mood stabilizers con-tinued for more 6 days. Locomotor activity was measured using the open-field test and BDNF levels weremeasured in rat hippocampus and amygdala by sandwich-ELISA. Li and VPT reversed OUA-related hyper-active behavior in the open-field test in both experiments. OUA decreased BDNF levels in first and secondexperiments in hippocampus and amygdala and Li treatment, but not VPT reversed and prevented theimpairment in BDNF expression after OUA administration in these cerebral areas. Our results suggest thatthe present model fulfills adequate face, construct and predictive validity as an animal model of mania.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Bipolar disorder (BD) is a common, chronic and highly debilitat-ing disease, that affects millions of individuals worldwide and isassociated with higher rates of suicide and general medical ill-nesses (Belmaker, 2004; Schloesser et al., 2008). BD is character-ized by a complex alternating clinical course, with recurrentmood switches including manic, depressive, and mixed episodes(Goodwin and Jamison, 1990), nevertheless, the unique hallmarkof this disease is acute mania (Belmaker, 2004), showing symp-toms such as irritable mood, psychomotor activation, reducedneeded for sleep, and excessive involvement in potentially prob-lematic behavior (El-Mallakh et al., 2003). Mood-stabilizing drugs,mainly lithium and valproate, are considered first line agents foracute mania treatment (Yatham et al., 2005), controlling symptomsand preventing new episodes.

ll rights reserved.

ias, PPGCS, UNASAU, Univer-úma, SC, Brazil. Tel.: +55 48

One of the main difficulties in developing treatments for BD isthe absence of an animal model, which makes hard the full under-standing of the illness pathophysiology and pathogenesis (Katoet al., 2007). Animal model of human disease should meet threesets of criteria: have similar behavioral manifestations as the hu-man disease (face validity), share pathophysiological characteris-tics of the human condition (construct validity), and improvewith medications that improve the symptoms seen in afflicted hu-mans (predictive validity) (Machado-Vieira et al., 2004). The intra-cerebroventricular (ICV) administration of ouabain (a potent Na+/K+-ATPase inhibitor) in rats mimics a widely reproduced biologicabnormality in mania and has been used by many authors as a toolfor the study of BD (Herman et al., 2007; El-Mallakh et al., 2003;Riegel et al., 2009).

Studies have showed direct and indirect evidence of decreaseactivity of the electrogenic sodium, and potassium-activated aden-osine triphosphatase membrane pump (Na, K-ATPase or Na pump)in acute mania relative to recovered euthymic bipolar individuals(Reddy et al., 1992; Scott and Reading, 1978; Hokin-Neaversonand Jefferson, 1989). Ouabain ICV administration in rats greatlyincreases locomotion, which may persist for several days after a

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single injection (Decker et al., 2000; Ruktanochai et al., 1998; Rie-gel et al., 2009), suggesting that the inhibition of Na, K-ATPase inthe brain causes hyperactivity. These behavioral changes inducedby ouabain can be preventable by mood stabilizing agents suchas lithium, haloperidol and carbamazepine, (El-Mallakh et al.,2006; Li et al., 1997; Hennion et al., 2003). Together, this informa-tion supports the hypothesis that ICV administration of ouabain inrats can be considered a valid animal model of mania, since it mim-ics behavioral and biological aspects of a maniac state.

Brain-derived neurotrophic factor (BDNF) is important for neu-rogenesis, differentiation and neuronal survival and is highly ex-pressed in brain areas that are known to regulate cognitive andemotional behavior, such as hippocampus and amygdala (Strakow-ski et al., 2005). A body of evidence suggesting that BDNF is in-volved in BD. Cunha and colleagues showed that serum BDNF isdecreased during manic and depressive episodes in BD patients,and that serum BDNF was negatively correlated with the severityof manic and depressive symptoms (Cunha et al., 2006). Also, thechronic administration of lithium (Frey et al., 2006a) or valproateincreased BDNF expression in the hippocampus and temporal cor-tex in rat’s brain, suggesting that these mood stabilizers might pro-duce a neurotrophic effect (Einat et al., 2003; Fukumoto et al.,2001).

The present study aims to investigate if the administration oflithium and valproate reverses and prevents the behavioral effectsof ICV administration of ouabain in rats, and the effects of ouabainand mood stabilizers on BDNF expression in rat hippocampus andamygdala.

6 days

OUA

3 days

Surgery Mood Stabilizers

Scheme 1.

2. Methods

2.1. Animals

We conducted the study using adult male Wistar rats (250–300 g) obtained from our breeding colony. The animals werehoused 5 to a cage, on a 12-h light/dark cycle (lights on at 7:00am), with free access to food and water. All experimental proce-dures were carried out in accordance with the National Institutesof Health Guide for the Care and Use of Laboratory Animals andthe Brazilian Society for Neuroscience and Behaviour (SBNeC). Thisstudy was approved by the local ethics committe (Comitê de Éticano Uso de Animais da Universidade do Extremo Sul Catarinense),and all efforts were made to minimize animal suffering, to reducethe number of animals used, and to utilize alternatives to in vivotechniques.

2.2. Surgical procedure

Animals were intraperitoneally anesthetized with ketamine(80 mg/kg) and xylasine (10 mg/kg). In a stereotaxic apparatus,the skin of the rat skull was removed and a 27 gauge 9 mm guidecannula was placed at 0.9 mm posterior to bregma, 1.5 mm rightfrom de midline and 1.0 mm above the lateral brain ventricle.Through a 2 mm hole made at the cranial bone, a cannula was im-planted 2.6 mm ventral to the superior surface of the skull, andfixed with jeweler acrylic cement. Animals recovered from surgeryfor 3 days.

2.3. Treatment

2.3.1. Reversal modelWe designed our first model to reproduce the management of

an acute manic episode. Animals (n = 72) received a single ICVinjection of 5 ll of ouabain 10�3 M dissolved in artificial cerebro-spinal fluid (aCSF), or 5 ll of aCSF alone on the 4th day following

surgery (El-Mallakh et al., 2003; Riegel et al., 2009). (Note: El-Mal-lakh et al. (2003) published reports in which the ouabain dose wasreported as 10�5 M are in error; the dose used in those studies was10�3 M). A 30 gauge cannula was placed into the guide cannula andconnected by a polyethylene tube to a microsyringe. The tip of thecannula infusion protruded 1.0 mm beyond the cannula guide aim-ing the right lateral brain ventricle. From the day following theinjection of ouabain or aCSF, the rats were treated for 6 days withintraperitoneal (IP) injections of saline, lithium or valproate into 6experimental groups of 12 animals per group: aCSF ICV + saline IP(aCSF + SAL), aCSF ICV + lithium IP (aCSF + Li), aCSF ICV + valproateIP (aCSF + VPT), ouabain ICV + saline IP (OUA + SAL), ouabainICV + lithium IP (OUA + Li), ouabain ICV + valproate IP (OUA + VPT).Animals in the Li group received intraperitoneal injections of lith-ium (47.5 mg/kg) and those in the VPT group received valproate(200 mg/kg) twice a day. We measured locomotor activity twice:immediately after ICV injection of ouabain or saline (data notshown) and 7 days after ouabain administration – the day follow-ing the end of treatment with Li, VPT or SAL (see Scheme 1).

2.3.2. Maintenance modelWe designed the second model to mimic the maintenance

phase of the treatment of BD (prevention model). After recoveryfrom surgery, animals (n = 72) received IP injections of either Li(47.5 mg/kg), VPT (200 mg/kg) or SAL twice a day for 12 days. Inthe 7 days of treatment with SAL, Li or VPT the animals receiveda single ICV injection of either OUA (10�3 M) or aCSF, totaling sixexperimental groups of 12 animals per group: SAL + aCSF, Li + aCSF,VPT + aCSF, SAL + OUA, Li + OUA, VPT + OUA. After the OUA or aCSFinjection, the treatment with mood stabilizer continued for 6 days.

After administration of aCSF or OUA, animals were evaluated inthe open-field test on two occasions: immediately after (data notshown) and 7 days after ICV injection (see Scheme 2).

2.4. Behavioral assessment

We used the open field task to assess locomotor activity. Thetask was performed in a 40 � 60 cm open field surrounded by50 cm-high walls made of brown plywood with a frontal glass wall.The floor of the open field was divided into 9 equal rectangles withblack lines. The animals were gently placed on the left rear rectan-gle and were allowed to explore the arena. Crossings of the blacklines were counted for 5 min.

The rats were sacrificed by decapitation right after the lastopen-field evaluation and the hippocampus and amygdale weredissected, rapidly frozen, and stored at �80 �C until assayed.

2.5. BDNF level measurement

BDNF levels in hippocampus and amygdala, were measured byanti-BDNF sandwich-ELISA, according to the manufacturer instruc-tions (Chemicon, USA). Briefly, brain slices were homogenized inphosphate buffer solution (PBS) with 1 mM phenylmethylsulfonylfluoride (PMSF) and 1 mM (EGTA). Microtiter plates (96-well flat-bottom) were coated for 24 h with the samples diluted 1:2 in sam-ple diluent and standard curve ranged from 7.8 to 500 pg/ml ofBNDF. The plates were then washed four times with sample diluentand a monoclonal anti-BNDF rabbit antibody diluted 1:1000 insample diluent was added to each well and incubated for 3 h at

3 days

Mood Stabilizers OUA Surgery Mood Stabilizers

7 th day

12 days

Scheme 2.

Fig. 1. Reversal Model – Number of crossings in rats subjected to the open-field testduring 5 min (A) and BDNF levels in rat hippocampus (B) and amygdala (C). Barsrepresent means ± standard error of means (15 animals each group for crossingsand 5 animals each group for BDNF). *P < 0.05 vs. aCSF + SAL group, according toANOVA followed by the Tukey test. #P < 0.05 compared with OUA + SAL groupaccording to ANOVA followed by the Tukey test (crossings: dF = 5, F = 153.586,P < 0.0001; BDNF hippocampus: dF = 5, F = 368.367, P < 0.0001; BDNF amygdala:dF = 5, F = 235.519, P < 0.0001). &P < 0.05 vs. aCSF + SAL group according Indepen-dent-Sample T Test (BDNF hippocampus: t = 2.905, dF = 15, P = 0.011; BDNFamygdala: t = 3.697, dF = 16, P = 0.002).

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room temperature. After washing, a peroxidase conjugated anti-rabbit antibody (diluted 1: 1000) was added to each well and incu-bated at room temperature for 1 h. After addition of streptavidin-enzyme, substrate and stop solution, the amount of BDNF wasdetermined by absorbance in 450 nm. The standard curve demon-strates a direct relationship between Optical Density (OD) andBDNF concentration. Total protein was measured by Lowry’s meth-od using bovine serum albumin as a standard (Frey et al., 2006b).

2.6. Statistical analysis

Behavioral and biochemical data are presented as mean andstandard error of the mean. Differences among the experimentalgroups were determined by one-way analysis of variance (ANOVA)followed by the Tukey post hoc test. When necessary, we used theIndependent-Sample T Test to compare the groups versus aCSF + -saline. In all comparisons, statistical significance was set atP < 0.05.

3. Results

In the first experiment (reversal model), OUA increased locomo-tor behavior in saline-treated rats immediately after the ICVadministration (data not shown), effect that persists for 7 daysafter a single injection and both Li and VPT reversed OUA-relatedhyperactive behavior (Fig. 1A). The administration of Li or VPT inaCSF-treated animals did not change behavioral measures, indicat-ing that the effects of mood stabilizers in OUA-treated rats werenot associated with sedation. OUA administration decreased BDNFlevels in rat hippocampus (Fig. 1B) and amygdala (Fig. 1C), how-ever, Li treatment, but not VPT reversed the impairment in BDNFexpression after OUA administration in these cerebral areas. Fur-ther analysis with Independent-Sample T Test showed that InaCSF-treated animals, the chronic use of Li increased BDNF levelsin both structures evaluated when compared with aCSF + salinegroup, while VPT had no influence in BDNF levels nor in hippocam-pus neither amygdale. Fig. 2 summarizes the results of mainte-nance model. OUA increased locomotor behavior immediatelyafter the ICV administration (data not shown), effect that persistsfor 7 days after a single injection and both Li and VPT pretreatmentwere able to prevent OUA-related hyperactivity (Fig. 2A). aCSFadministration in mood stabilizer-pretreated animals demon-strated no behavioral effect. In this experiment, OUA decreasedBDNF levels in hippocampus (Fig. 2B) and amygdala (Fig. 2C) of sal-ine pretreated rats, while Li, but not VPT pretreatment preventedthe BDNF impairment in these cerebral structures after OUAadministration. In aCSF-treated animals, the chronic use of Li in-creased BDNF expression in hippocampus and amygdala, whileVPT had no influence in BDNF levels in both structures.

4. Discussion

The main findings of the present study can be summarized asfollow: (a) ouabain increased locomotor behavior that persists for7 days following a single ICV injection in saline-treated rats in bothtreatment, maintenance and reversion, (b) substantial reduction inthe locomotion was evident in OUA group treated with Li and VPT,

preventing and reversing OUA-related hyperactivity, (c) OUA de-creased significantly BDNF levels in saline-treated rats in hippo-campus and amygdala in both treatment, prevention and

Fig. 2. Maintenance model – Number of crossings in rats subjected to the open-field test during 5 min (A) and BDNF levels in rat hippocampus (B) and amygdala (C)7 days after a single injection of ouabain or aCSF. Bars represent means ± standarderror of means (15 animals each group for crossings and 5 animals each group forBDNF). *P < 0.05 vs. aCSF + SAL group, according to ANOVA followed by the Tukeytest. #P < 0.05 compared with OUA + SAL group. (Crossings: dF = 5, F = 5.851,P < 0.001; BDNF hippocampus: dF = 5, F = 75.156, P < 0.0001; BDNF amygdala:dF = 5, F = 135.897, P < 0.0001).

L.K. Jornada et al. / Journal of Psychiatric Research 44 (2010) 506–510 509

reversion, (d) Li, but not VPT treatment, increased BDNF levels inaCSF-treated; moreover revert the OUA-induced decrease of BDNFexpression in hippocampus and amygdala of rats.

The present data are in line with previous findings that showedconsistent hyperlocomotor effects of OUA in rats (Decker et al.,2000; Ruktanochai et al., 1998; El-Mallakh et al., 2006; Li et al.,1997; Hennion et al., 2003); however, our work includes acute(reversion) and maintenance treatment (prevention). Besides, El-Mallakh and colleagues (2006) evaluated the efficacy of olanzapineand haloperidol in animal model of mania induced by ouabain, but

it is noteworthy that haldol and olanzapine are not first line drugsto the treatment of this disease. Here we showed that OUA-in-duced hyperlocomotion can be prevented and reversed with classi-cal mood stabilizers drugs such as Li and VPT. In addition, at the fisttime, our results demonstrated changes in BDNF expression in thisanimal model of mania.

The literature suggests that a small decrease in Na pump activ-ity may result in neuronal hyperexcitability, giving rise to manicsymptoms, such as hyperactivity (El-Mallakh, 1983). The ICVinjection of ouabain induces hyperlocomotion in rodents, a com-mon symptom of manic patients. Numerous laboratories havedemonstrated that the most commonly used mood-stabilizingdrugs, lithium and valproate have an important neuroprotectiverole in acute and chronic treatment, attenuating neurodegenera-tive actions caused by several diseases (for a review see: Roweand Chuang (2004), Vajda (2002)). Many studies show that theneuroprotective effect of Li and VPT may be responsible for theirtherapeutic effects (Li et al., 2002). One of the mechanisms ofthese drugs is the increase of BDNF levels, a polypeptide thathas an important role in the survival and differentiation neuronalin adults (Huang and Reichardt, 2001), acting on dependentmechanisms of plasticity, such as potential long-term learningand memory (Huang and Reichardt, 2001; Malcangio and Less-mann, 2003; Schinder and Poo, 2000). BDNF is decreased in man-iac patients (Cunha et al., 2006) and these changes in BDNF levelswere negatively correlated with the severity of manic and depres-sive symptoms. In addition, our group has showed that BDNF isdecreased in a dopaminergic model of mania induced by amphet-amine (Frey et al., 2006b), which reinforced that the BDNF mayplay a role in the BD.

Here, we demonstrated that ICV injection of OUA-inducedhyperlocomotion, that can be prevented and reversed by Li andVPT, as in our previous findings in the animal model of mania in-duced by amphetamine (Frey et al. 2006b). Our data showed thatOUA administration decreased BDNF levels in rat hippocampusand amygdala, which might be prevented and reversed by Li, butnot VPT. This discrepancy suggests that, when co-administeredwith OUA, Li may increase BDNF content in an earlier time coursethan VPT. Moreover, in maintenance and reversal model, our datesdemonstrated that in aCSF-treated animals, the use of Li increasedBDNF expression in hippocampus and amygdala. Regarding thesedata, is valid accentuate that when evaluated by one-way analysisof variance (ANOVA), no differences were found between aCSF + -SAL versus aCSF + Li groups, however, when we used a T Test, a sig-nificant result was achieved.

A very interesting finding that we can observe in this paper isthat treatment with Li blocks, or reverses, the decrease in BDNFproduced by OUA. However, what appears to be happening is thatLi treatment alone, both in the reversal and maintenance modelelevates BDNF significantly, and that this elevation essentiallycounteracts the effects of OUA. Conversely, valproate treatmentdoes not have this effect; it does not elevate BDNF when givenalone, and it does not block the OUA-induced decrease in BDNF.Together these data may suggest two hypotheses: (1) Li and VPThave different mechanism of action for the control of manic symp-toms and that (2) is not necessary to reverse OUA-induced de-creases in brain BDNF in order for the drugs in question toreverse OUA-induced mania.

In conclusion, we propose that the persistent increase in the ratspontaneous locomotion induced by OUA is associated with de-creased BDNF levels in the hippocampus and amygdala, effects thatcan be reversed and prevented by mood stabilizers Li and VPA,indicating a good face, predictive and construct validity. Thus, oua-bain-induced mania-like behavior may provide a useful model totest the hypothesis of BDNF levels dysfunction is associated topathophysiology of bipolar disorder. Further studies aiming to

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investigate the signaling cascades and other drugs in this psychiat-ric disorder are worth of doing.

Role of funding source

This research was supported by grants from CNPq (Flavio Kapc-zinski and João Quevedo), FAPESC (João Quevedo), and UNESC (JoãoQuevedo). Flavio Kapczinski and João Quevedo are CNPq ResearchFellow. Morgana Moretti and Samira S. Valvassori are holders ofCAPES studentships, Camila O. Arent and Camila L. Ferreira isholder of a CNPq Studentship.

The National Council for Scientific and Technological Develop-ment (CNPq) is an agency linked to the Ministry of Science andTechnology (MCT), dedicated to the promotion of scientific andtechnological research and to the formation of human resourcesfor research in the country. Its history is directly linked to the sci-entific and technological development of Brazil.

The FAPESC aims to support and promote the scientific andtechnological research for the advancement of all areas of knowl-edge for regional balance, sustainable development and improvingthe quality of life of people of the Santa Catarina state with respectfor ethical values and based on the principles established by arts.

Coordination of Improvement of Higher Education Personnel(CAPES) plays a role in the expansion and consolidation of post-graduate (masters and doctorate) in all states of Brazil.

Contributors

Luciano K. Jornada designed the study, wrote the protocol andthe first draft of the manuscript. Samira S. Valvassori undertookthe statistical analysis. Gabriel Fries made the biochemical analy-sis. Morgana Moretti and Camila L. Ferreira were responsible forthe surgery and pharmacological treatment. Camila O. Arent andPeterson Padilha made the behavioral tests. All authors contrib-uted to and have approved the final manuscript.

Conflict of interest statement

None declared.

Acknowledgements

We thank CNPq, FAPESC, CAPES, Instituto Cérebro e Mente andUNESC for financial support.

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