European Journal of Pharmacology 643 (2010) 232–238

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European Journal of Pharmacology

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Behavioural Pharmacology

Antidepressant-like effect of scopoletin, a coumarin isolated from Polygala sabulosa(Polygalaceae) in mice: Evidence for the involvement of monoaminergic systems

Juliano C. Capra a, Mauricio P. Cunha a, Daniele G. Machado a, Andrea D.E. Zomkowski a, Beatriz G. Mendes c,Adair Roberto S. Santos b, Moacir G. Pizzolatti c, Ana Lúcia S. Rodrigues a,⁎a Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, 88040-900, Florianópolis, SC, Brazilb Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, 88040-900, Florianópolis, SC, Brazilc Departamento de Química, Centro de Ciências Físicas e Matemáticas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, 88040-900, Florianópolis, SC, Brazil

⁎ Corresponding author. Tel.: +55 48 3721 5043; faxE-mail addresses: analucia@mbox1.ufsc.br, alsrodri@

0014-2999/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.ejphar.2010.06.043

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 April 2010Accepted 22 June 2010Available online 30 June 2010

Keywords:AntidepressantDopamineNoradrenalinePolygala sabulosaScopoletinSerotonin

The relationship between depression and monoaminergic systems has been hypothesized for many years. Inthis study, we have investigated the possible antidepressant-like effect of scopoletin, a coumarin fromPolygala sabulosa in the tail suspension test and forced swimming test. Moreover, the ability of scopoletin toreverse the depression-like behavior in the forced swimming test induced by immobility stress in mice wasevaluated. Scopoletin reduced the immobility time in the tail suspension test (10–100 mg/kg, p.o.), but not inthe forced swimming test. Fluoxetine (positive control) decreased the immobility time in the forcedswimming and tail suspension tests (20 mg/kg, p.o. and 10 mg/kg. p.o., respectively). Immobility stresscaused an increase in the immobility time in the forced swimming test (depression-like behavior), whichwas reversed by scopoletin (1–100 mg/kg, p.o.) and fluoxetine (10 mg/kg, p.o.). Scopoletin produced nopsychostimulant effect in the open-field test. The pretreatment of mice with ketanserin (5 mg/kg, i.p., apreferential 5-HT2A receptor antagonist), prazosin (1 mg/kg, i.p., an α1-adrenoceptor antagonist), yohimbine(1 mg/kg, i.p., an α2-adrenoceptor antagonist), haloperidol (0.2 mg/kg, i.p., a dopaminergic receptorantagonist), SCH23390 (0.05 mg/kg, s.c., a dopamine D1 receptor antagonist) or sulpiride (50 mg/kg, i.p., adopamine D2 receptor antagonist), but not WAY100635 (0.1 mg/kg, s.c., a selective 5-HT1A receptorantagonist) prevented the antidepressant-like effect of scopoletin (10 mg/kg, p.o.) in the tail suspension test.The results indicate that its antidepressant-like effect is dependent on the serotonergic (5-HT2A receptors),noradrenergic (α1- and α2-adrenoceptors) and dopaminergic (dopamine D1 and D2 receptors) systems.

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1. Introduction

Depression is a commonmood disorder associated with high ratesof chronicity, relapse, and recurrence; psychosocial and physicalimpairment; and suicide (Wong and Licinio, 2001). The World HealthOrganization ranks unipolar depression as the fourth most importantcause of mortality and disability (Murray and Lopez, 1997). There iswide consensus that stress exposure conceivably plays a causal role inthe etiology of depression (Caspi et al., 2003; Nestler et al., 2002).Stress induced pattern of changes in the neurotransmitter andneuroendocrine systems. Growing evidence indicates several classicalcandidates, including neurotransmitters and neuropeptides, as likelyintermediate links between stress exposure and depressive symptoms(Duman and Monteggia, 2006; Leonard, 2005; Nestler et al., 2002).Furthermore, many studies reveal important roles for monoaminergic

systems in the pathophysiology and treatment of depression (Coppen,1967; Elhwuegi, 2004; Schildkraut, 1965).

Herbal therapies may be effective alternatives in the treatment ofdepression, as in the case of St John's wort (Linde and Knuppel, 2005),and the search for novel pharmacotherapy approaches frommedicinalplants for psychiatric illnesses, including depression, has progressedsignificantly in the past decade (Zhang, 2004).

The plants of genus Polygala are widely distributed in SantaCatarina state, Brazil (Marques and Peixoto, 2007). Pharmacologicalstudies carried out with extracts from Polygala sabulosa show that thisplant exerts several biological effects, such as: antinociceptive (Ribaset al., 2008), anticonvulsant and anxiolytic-like effects (Duarte et al.,2008). Scopoletin (7-hydroxy-6-methoxycoumarin) is a coumarinpresent in Polygala sabulosa (Meotti et al., 2006) that has been shownto exert several biological activities, such as: anticholinesterasic(Orhan et al., 2008), antinociceptive (Ribas et al., 2008), anti-inflammatory (Kim et al., 2004), antithyroid, antioxidant, antihyper-glycemic (Panda and Kar, 2006), hypouricemic (Ding et al., 2005), andanti-tumoral (Liu et al., 2001).

233J.C. Capra et al. / European Journal of Pharmacology 643 (2010) 232–238

Considering that: a) depressive disorders have high incidence inthe world population, with impact in the social function and in the lifequality of patients (Wong and Licinio, 2001); b) treatment ofdepression with conventional antidepressants provides a completeremission just for 50–60% of the individuals (Nestler et al., 2002); andc) antidepressant therapy produces side effects that may reduce theadhesion of patients to treatment (MacGillivray et al., 2003; Sleathet al., 2003); it is necessary to develop novel strategies for thetreatment of depression.

Thus, this study aims to evaluate the antidepressant-like action ofscopoletin in mouse forced swimming test and tail suspension test,predictive models of antidepressant activity (Cryan et al., 2005; Steruet al., 1985) and to investigate the possible participation of themonoaminergic systems in its antidepressant-like action. Moreover,we examined the ability of scopoletin to reverse a depression-likebehavior induced by immobility stress, a model previously shown tobe responsible to antidepressants (Poleszak et al., 2006).

2. Materials and methods

2.1. Plant material and preparation of the extracts

Polygala sabulosa A.W. Bennett (Polygalaceae) was collected inRancho Queimado (Santa Catarina state, Brazil) and identified by Prof.Dr. Olavo de Araújo Guimarães. A voucher specimen was deposited atthe Herbarium of the Botany Department at Universidade Federal doParaná, Brazil, under the number 19640.

2.2. Extract and isolation

Scopoletin was isolated from crude ethanolic extract of de P.sabulosa as described previously (Meotti et al., 2006). Air-dried wholeplant was extracted with 96% ethanol and the crude hydroalcoholicextract was partitioned into hexane, CH2Cl2, EtOAc and n-BuOH togive five distinct hexane, CH2Cl2, AcOEt, n-BuOH and aqueous-solublefractions. The fractions 38–42, obtained from Si-gel chromatographyfractionation of the CH2Cl2 soluble fraction, was further processed byflash chromatography eluted with hexane–EtOAc 3:2 and thempurified by recrystallization on acetone to give scopoletin 98%.Detailed NMR spectroscopic analysis (1H and 13C) and comparisonof the physical and spectroscopic data with those reported in theliterature (Razdan et al., 1987) confirmed the structure of 7-hydroxy-6-methoxycoumarin (scopoletin), as depicted in Fig. 1.

2.3. Animals

Female Swiss mice (60 to 80 days old, weighing 30–45 g) weremaintained at constant room temperature (22–24 °C) with free accessto water and food, under a 12:12 h light/dark cycle (lights on at07:00 h). Mice were allowed to acclimatize to the holding room for24 h before the behavioral procedures. Animals were randomlydistributed into specified experimental groups. All experimentswere carried out between 11:00 and 16:00 h, with each animal usedonly once (N=5–8 animals per group). The procedures in this studywere performed in accordance with the National Institute of HealthGuide for the Care and Use of Laboratory Animals and approved by theEthics Committee of our Institution. All efforts weremade tominimizeanimals suffering and to reduce the number of animals used in theexperiments.

Fig. 1. Chemical structure of scopoletin.

2.4. Drugs and treatment

The drugs used in the present study were: N-{2-[4-(2-methox-yphenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl)cyclohexanecarboxa-mide (WAY100635), ketanserin tartarate, prazosin hydrochloride,yohimbine hydrochloride, haloperidol, (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochlo-ride (SCH23390), sulpiride and fluoxetine (all from Sigma-AldrichChemical Company, St. Louis, MO, U.S.A.). All drugswere administeredby intraperitoneal (i.p.) route in a constant volume of 10 ml/kg bodyweight, except SCH23390 andWAY100635 that were administered bysubcutaneous (s.c.) route and fluoxetine that was administered byoral route (p.o.). Drugs were dissolved in saline except sulpiride thatwas diluted in saline with 5% dimethyl sulfoxide (DMSO) andfluoxetine that was dissolved in distillated water. Control animalsreceived appropriate vehicle. Scopoletin (1–100 mg/kg, p.o.) wasdissolved in distilled water and administered acutely by oral routethrough gavage (p.o.), 60 min before the forced swimming test, tailsuspension test or open-field test. Fluoxetine (10–20 mg/kg, p.o., aconventional antidepressant) was used as a positive control.

To address some of the mechanisms by which scopoletin causesantidepressant-like action in the tail suspension test, animals werepretreated with different pharmacological agents.

To investigate a possible contribution of the serotonergic system(5-HT receptor subtypes) in the antidepressant-like effect of thescopoletin in the tail suspension test, animals were pretreated withWAY100635 (0.1 mg/kg, s.c., a selective 5-HT1A receptor antagonist),ketanserin (5 mg/kg, i.p., a preferential 5-HT2A receptors antagonist),or vehicle and after 30 min, received scopoletin (10 mg/kg, p.o.) orvehicle injection before being tested in the tail suspension test 60 minlater.

To investigate the possible involvement of the noradrenergicsystem in the antidepressant-like effect of the scopoletin in the tailsuspension test, animals were pretreated with prazosin (1 mg/kg, i.p.,an α1-adrenoceptor antagonist), or yohimbine (1 mg/kg, i.p., an α2-adrenoceptor antagonist), or vehicle and after 30 min they receivedscopoletin (10 mg/kg, p.o.) or vehicle injection before being tested inthe tail suspension test 60 min later.

We also investigate the influence of the dopaminergic system inthe antidepressant-like effect of the scopoletin in the tail suspensiontest. Animals were pretreated with haloperidol (0.2 mg/kg, i.p., a non-selective dopaminergic receptor antagonist), SCH23390 (0.05 mg/kg,s.c., a dopamine D1 receptor antagonist) or sulpiride (50 mg/kg, i.p., adopamine D2 receptor antagonist), and after 30 min they receivedscopoletin (10 mg/kg, p.o.) or vehicle and were tested in the tailsuspension test 60 min later.

The administration schedule and the doses of the drugs used werechosen based on experiments previously performed in our laboratoryand literaturedata confirm theefficacyof the abovementionedprotocols(Binfaré et al., 2009; Dhingra and Valecha, 2007; Freitas et al., 2010;Machado et al., 2007; O'Neill et al., 2001; Redrobe and Bourin, 1997).

2.5. Behavioral analysis

2.5.1. Forced swimming testThe test was conducted using the method of Porsolt et al. (1977)

with minor modifications. This experimental model is based on theobservation that mice, when forced to swim in an inescapablecondition, after an initial period of agitation/flurry, adopt animmobility behavior. Thus, mice were individually forced to swim inan open cylindrical container (diameter 10 cm, height 25 cm),containing approximately 20 cm of water at 25±1 °C. The immobilitytime, defined as the absence of escape-oriented behaviors, such asswimming, was manually scored during 6 min, as described previ-ously (Kaster et al., 2007). Each mouse was judged immobile when itceased struggling and remained floating motionless in the water,

Fig. 2. Effect of treatment of mice with scopoletin (S) and fluoxetine (F) given orally(mg/kg) on the immobility time in the forced swimming test in non-stressed (panel A)and stressed (panel B) mice. Mice were immobilized for 2 h (acute stress) immediatelybefore the administration of scopoletin or fluoxetine (panel B). Each column representsthe mean+S.E.M. of 5–8 animals. ** Pb0.01 when compared with the non-stressedvehicle-treated control (V). # Pb0.01 when compared with the stressed vehicle-treatedcontrol (V). A) F4,27=0.44, P=0.78. B) F4,25=8.91, Pb0.01.

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making only those movements necessary to keep its head abovewater. Conventional antidepressants decrease the immobility time inthis test (Porsolt et al., 1977).

2.5.2. Tail suspension testThe tail suspension test has become one of the most widely used

models for assessing antidepressant-like activity in mice. The test isbased on the fact that animals subjected to the short-term inescapablestress of being suspended by their tail will develop an immobileposture. The total duration of immobility induced by tail suspensionwas measured according to the method described by Steru et al.(1985). Briefly, mice both acoustically and visually isolated weresuspended 50 cm above the floor by adhesive tape placed approxi-mately 1 cm from the tip of the tail. Immobility time was manuallyrecorded during a 6 min period (Machado et al., 2007, 2008). Micewere considered immobile only when they hung passively or staycompletely motionless. Conventional antidepressants decrease theimmobility time in this test (Cunha et al., 2008; Steru et al., 1985).

2.5.3. Open-field testTo assess the possible effect of the scopoletin on exploratory

behavior, the animals were submitted to the open-field paradigm aspreviously described (Cunha et al., 2008). Mice were individuallyplaced in a wooden box (40×60×50 cm) with the floor of the arenadivided into 12 equal squares. The number of squares crossed with allpaws (crossing) and the number of rising with the front paws(rearing) were registered during a period of 6 min (Cunha et al., 2008;Machado et al., 2007). The number of rectangles crossed by the animalwith its four paws was considered as indicative of locomotor activityand the number of rearings was indicative of the exploratorybehavior. The floor of the open-field apparatus was cleaned with10% ethanol between tests.

2.6. Immobility stress (restraint)

Mice were immobilized in a well ventilated Plexiglas tube (10 cmlong, 2.8 cm in diameter, and 0.5 cm wall) for 2 h at a time (acutestress). Animals were not physically compressed and did notexperience pain. Immediately after restraint, mice were administeredwith scopoletin (1–100 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.) orvehicle and moved back to the home cages. Non-stressed mice wereplaced separately in different cage for 2 h and then moved back totheir home cages. The evaluation of the depressant-like behavior ofpreviously stressed mice was performed in the forced swimming testwhich was carried out 1 h after administration of scopoletin,fluoxetine or vehicle, as described by Poleszak et al. (2006).

2.7. Statistical analysis

Comparisons between experimental and control groups wereperformed by one-way (dose–response curve of scopoletin in theforced swimming test, tail suspension test and open-field behavior) ortwo-way (experiments dealing with the involvement of the mono-aminergic system in the effect of scopoletin in the tail suspension test)Analysis of Variance (ANOVA) followed by Tukey's HSD test whenappropriate. A value of Pb0.05 was considered to be significant.

3. Results

3.1. Effect of acute treatment with scopoletin on the immobility time inthe forced swimming test of mice submitted to immobility stress ornon-stressed mice

Fig. 2A depicts the effects of the acute administration of scopoletinat the doses of 0.1, 1, 10 and 100 mg/kg p.o. on the immobility time inthe forced swimming test in non-stressed mice. The administration of

scopoletin by p.o. route at the dose range of 0.1–100 mg/kg did notalter the immobility time of the non-stressed mice in the forcedswimming test.

Mice previously submitted to immobility stress for 2 h increasedthe immobility time in the forced swimming test (14.5%), which isconsistent with a depressant-like behavior. Scopoletin reversed thedepressant-like effect of the immobility stress at the doses of 1, 10 and100 mg/kg p.o. (Fig. 2B).

Fluoxetine, used as a positive control, decreased the immobilitytime in non-stressed mice at the dose of 20 mg/kg p.o. (39.0% ofreduction), but not at the dose of 10 mg/kg p.o. (Fig. 2A). However,fluoxetine at the dose of 10 mg/kg p.o. was able to prevent thedepressant-like behavior in the forced swimming test induced byimmobility stress (Fig. 2B).

3.2. Effect of acute treatment with scopoletin on the immobility time inthe tail suspension test

Fig. 3 shows the effect of the acute administration of scopoletin atthe doses of 0.1, 1, 10 and 100 mg/kg, p.o. on the immobility time inthe tail suspension test. Scopoletin significantly reduced the immo-bility time in tail suspension test at doses of 10 and 100 mg/kg ascompared to the control group (32.7% and 26.7% of reduction,respectively). Also, fluoxetine (10 mg/kg, p.o.) produced a significantreduction (41.7%) in the immobility time in the tail suspension test.

3.3. Effect of acute treatment with scopoletin on the locomotor activity inthe open-field test of stressed or non-stressed mice

Table 1 presents the effects of the acute administration ofscopoletin at the doses of 0.1, 1, 10 and 100 mg/kg p.o. on the open-

Table 2Effect of acute treatment with scopoletin on the locomotor activity in the open-field testof mice submitted to immobility stress.

Compound Dose (mg/kg, p.o.) Number of crossings

Naive – 72.3±11.0Vehicle – 76.2±3.9Scopoletin 1 80.2±6.8Scopoletin 10 74.5±2.9Scopoletin 100 71.4±11.1

Results are expressed as mean±S.E.M. of 6–7 animals. F4,27=0.44, P=0.78.

Fig. 3. Effect of treatment of mice with scopoletin (S) and fluoxetine (F) given orally(mg/kg) on the immobility time in the tail suspension test. Each column represents themean+S.E.M. of 5–8 animals. ** Pb0.01 when compared with the vehicle-treatedcontrol. F4,27=0.44, P=0.78.

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field test in non-stressed animals. The administration of scopoletin byp.o. route at the dose range of 0.1–100 mg/kg did not alter the numberof crossings of the non-stressed mice in the open-field test.

Table 2 presents the effects of the acute administration ofscopoletin at the doses of 1, 10 and 100 mg/kg p.o. on the open-field test in stressed mice. The administration of scopoletin by p.o.route at the dose range of 1–100 mg/kg did not alter the number ofcrossings of the immobility stress mice in the open-field test.Furthermore, immobility stress did not alter the number of crossingsof mice, as compared to naive animals.

3.4. Investigation of some possible mechanisms underlying theantidepressant-like effect of scopoletin in the tail suspension test

Considering the results previously shown, the dose of 10 mg/kgscopoletin was chosen for the investigation of participation of themonoaminergic system in its antidepressant-like action in the tailsuspension test.

3.4.1. Involvement of serotonergic system in the antidepressant-likeeffect of scopoletin in the tail suspension test

Fig. 4A shows that the pretreatment of mice with the selective5HT1A receptor antagonist WAY100635 (0.1 mg/kg, s.c.) did notprevent the antidepressant-like action of scopoletin (10 mg/kg, p.o.)in the tail suspension test. Fig. 4B, otherwise, shows that thepretreatment ofmicewith the preferential 5-HT2A receptor antagonistketanserin (5 mg/kg, i.p.) prevented the antidepressant-like action ofscopoletin in the tail suspension test.

3.4.2. Involvement of noradrenergic system in the antidepressant-likeeffect of scopoletin in the tail suspension test

The results depicted in Fig. 5A and B, show that pretreatment ofmicewith either the α1-adrenoceptor antagonist prazosin (1 mg/kg, i.p.) orα2-adrenoceptor antagonist yohimbine (1 mg/kg, i.p.) was able toreverse the antidepressant-like effect of the scopoletin (10 mg/kg, p.o.)in the tail suspension test, respectively.

Table 1Effect of acute treatment with scopoletin on the locomotor activity in the open-fieldtest.

Compound Dose (mg/kg, p.o.) Number of crossings

Vehicle – 81.4±8.5Scopoletin 0.1 81.5±11.0Scopoletin 1 98.7±8.7Scopoletin 10 89.7±7.7Scopoletin 100 81.7±11.4

Results are expressed as mean±S.E.M. of 6–8 animals. F4,27=0.71, P=0.59.

3.4.3. Involvement of dopaminergic system in the antidepressant-likeeffect of scopoletin in the tail suspension test

Fig. 6A shows that the anti-immobility effect of scopoletin (10 mg/kg, p.o.) was prevented by pretreatment of mice with the non-selective dopamine receptor antagonist haloperidol (0.2 mg/kg, i.p.).In addition, the pretreatment of mice with the dopamine D1 receptorantagonist SCH23390 (0.05 mg/kg, s.c.) and with the D2 receptorantagonist sulpiride (50 mg/kg, i.p.) prevented the antidepressant-like action of scopoletin in the tail suspension test (Fig. 6B and C,respectively).

Fig. 4. Effect of pretreatment of mice withWAY100635 (0.1 mg/kg, s.c., panel A) or withketanserin (5 mg/kg, i.p., panel B) on the anti-immobility action of scopoletin (10 mg/kg, p.o.) in the tail suspension test. Each column represents the mean+S.E.M. of 6–8animals. * Pb0.05 or ** Pb0.01 when compared with the vehicle-treated control.# Pb0.05 as compared with scopoletin alone. A) WAY100635 pretreatment: F1,31=0.58,P=0.45; scopoletin treatment: F1,31=21.13, Pb0.01; scopoletin×WAY100635 interac-tion: F1,31=1.00, P=0.33. B) Ketanserin pretreatment: F1,21=2.26, P=0.15; scopoletintreatment: F1,21=5.89, Pb0.05; scopoletin×ketanserin interaction: F1,21=8.14, Pb0.01.

Fig. 5. Effect of pretreatment of mice with prazosin (1 mg/kg, i.p., panel A) or withyohimbine (5 mg/kg, i.p., panel B) on the anti-immobility effect of scopoletin (10 mg/kg, p.o.) in the tail suspension test. Each column represents the mean+S.E.M. of 6–8animals. ** Pb0.01 when compared with the vehicle-treated control. ## Pb0.01 ascompared with scopoletin alone. A) Prazosin pretreatment: F1,21=23.77, Pb0.01;scopoletin treatment: F1,21=13.65, Pb0.01; scopoletin×prazosin interaction: F1,21=6.13, Pb0.05. B)Yohimbine treatment: F1,24=12.36,Pb0.01; scopoletin treatment: F1,24=12.45, Pb0.01; scopoletin×yohimbine interaction: F1,24=4.54, Pb0.05.

Fig. 6. Effect of pretreatment of mice with haloperidol (0.2 mg/kg, i.p., panel A),SCH23390 (0.05 mg/kg, s.c., panel B) or sulpiride (50 mg/kg, i.p., panel C) on the anti-immobility effect of scopoletin (10 mg/kg, p.o.) in the tail suspension test. Each columnrepresents the mean+S.E.M. of 6–8 animals. ** Pb0.01 when compared with the vehicle-treated control. ## Pb0.01 as compared with scopoletin alone. A) Haloperidol pre-treatment: F1,27=29.71, Pb0.01; scopoletin treatment: F1,27=3.69, P=0.07; scopoletin×haloperidol interaction: F1,27=19.55, Pb0.01. B) SCH23390 pretreatment: F1,29=8.36,Pb0.01; scopoletin treatment: F1,29=17.00, Pb0.01; scopoletin×SCH23390 interaction:F1,29=4.46, Pb0.05. C) Sulpiride pretreatment: F1,25=23.29, Pb0.01; scopoletin treat-ment: F1,25=8.52, Pb0.01; scopoletin×sulpiride interaction: F1,25=6.12, Pb0.05.

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4. Discussion

Animal models predictive of antidepressant action have beenextensively used in the development of novel therapeutic compoundsand for understanding the neural substrates underlying depressivebehavior. The predictive validity of animal models of depression isdetermined solely by their response to antidepressant drugs (Cryanet al., 2002, 2005). The forced swimming test and tail suspension testare themost common predictive tests for screening of antidepressant-like activity of drugs. In both tests, animals are placed in aninescapable situation and the antidepressant-like activity is expressedby the decrease of immobility when compared with control groups(Porsolt et al., 1977; Steru et al., 1985). Furthermore, both tests aresensitive to all major classes of antidepressant drugs, includingtricyclics, serotonin reuptake inhibitors, monoamine oxidase inhibi-tors, and atypicals (Porsolt et al., 1977; Steru et al., 1985). In thepresent study we provide convincing evidence that the coumarinscopoletin administered by oral route produces an antidepressant-like effect in the tail suspension test. However, scopoletin was devoidof effect in the forced swimming test in non-stressed animals.Similarly, studies have shown that some compounds produceantidepressant-like effects in the tail suspension test, but not in theforced swimming test (Binfaré et al., 2009; Machado et al., 2008). Theunderlying principle measuring the lack of active coping behavior isidentical in the tail suspension test and forced swimming test, but

their variability in response to certain antidepressant agents indicatespotentially different substrates and neurochemical pathways medi-ating performance in these tests (Bai et al., 2001).

Depression is a disorder arising from strong negative environ-mental stressors (Caspi et al., 2003; van Praag, 2005). In an attempt tomimic excessive human day-to-day stress, several animal modelshave been developed. Methods using acute stressors, as restraintstress, rely on the ability of a sequence of acute stressor to produce

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behavioral changes (depression-like effect) that are reversible byantidepressant treatment (Poleszak et al., 2006). In the present study,scopoletin reversed the depression-like behavior assessed in theforced swimming test of mice previously exposed to restraint stressfor 2 h, an effect comparable to fluoxetine (selective serotoninreuptake inhibitor antidepressant). It is noteworthy that scopoletinproduced this effect at a dose that did not produce antidepressant-likeeffect in forced swimming test in non-stressed mice. This finding is inline with the fact that imipramine and magnesium were reported toreverse the depression-like behavior induced by the same protocol ofimmobility stress, at sub-effective doses, that did not produceantidepressant-like effect in the forced swimming test in non-stressedmice (Poleszak et al., 2006). Indeed, the sensitization to drugs byprevious stress exposure has been reported (Breese et al., 2004;Espejo and Gil, 1997). Our finding supports the assumption that singlerestraint stress is able to induce behavioral alteration in mice that leadto a greater sensitivity to the antidepressant action of scopoletin andfluoxetine in the forced swimming test. Moreover, this findingsuggests that scopoletin may be further investigated as a potentialsubstance for the treatment of stress-induced depression.

The predictive tests for antidepressant-like activity and models ofdepression have some drawbacks represented by the possibility ofobtaining some false results. The reduction in the immobility time inthe tail suspension test elicited by scopoletin cannot be attributable toa psychostimulant action of this compound. This conclusion derivesfrom the fact that in our study scopoletin administered by p.o. route ata dose that produced a significant decrease in the immobility time inthe tail suspension test did not significantly alter the locomotor andexploratory activity in the open-field paradigm. Moreover, theimmobility stress increased the immobility time of mice in the forcedswimming test without changing ambulation and rearing responses inthe open-field test (specific depression-like effect).

The dysregulation of the central nervous system involving theneurotransmitters noradrenaline, serotonin and dopamine has beensuggested to play a role in the pathogenesis of depression (mono-amine hypothesis) (Coppen, 1967; Schildkraut, 1965). Consideringthat the monoaminergic systems are directly implicated in patho-physiology and pharmacotherapeutic approach of major depression,the present study also was aimed at investigating the influence ofpharmacological agents that modulate the monoaminergic systems inthe antidepressant-like effect of scopoletin in the tail suspension test.

The serotonergic system has been recognized as playing animportant role in the etiology of depression and drugs acting on theserotonergic system have been largely implicated in the treatment ofdepressive disorders (Blier and Ward, 2003; Elhwuegi, 2004).Therefore, the involvement of 5-HT receptors subtypes in the anti-immobility effect of scopoletin was investigated.

Although the 5-HT1A partial agonists, such as buspirone, induceantidepressant effects (Blier and Ward, 2003; Borsini, 1995), in ourstudy, the pretreatment of mice with the 5-HT1A antagonistWAY100635 did not prevent the anti-immobility effect elicited byscopoletin in the tail suspension test, suggesting that these receptorsare not involved in the antidepressant-like effect of this coumarin.

The 5-HT2 receptors are also widely distributed throughout thebrain, in a pattern suggesting that their activation may be implicatedin the regulation of mood disorders (Celada et al., 2004). In our study,the pretreatment of mice with the preferential 5-HT2A receptorantagonist ketanserin prevented the antidepressant-like effect ofscopoletin (10 mg/kg, p.o.) in the tail suspension test, suggesting apossible participation of 5-HT2A receptors in this effect.

Depression is also associated with a hypofunction of thenoradrenergic system (Elhwuegi, 2004). Most of the antidepressantsuntil the 1980s were shown to be more potent at blocking reuptake ofnoradrenaline than at blocking reuptake of serotonin (Richelson andPfenning, 1984). Furthermore, many antidepressants, such as dox-epin, amitriptyline and nefazodone, have significant α1-adrenoceptor

affinity (Richelson, 2001). Moreover, α1- and α2-adrenoceptors havebeen shown to underlie some of the antidepressant-like responses ofdrugs in behavioral models of antidepressant activity (Kaster et al.,2007). In our study, prazosin, an α1-adrenoceptor antagonist, andyohimbine, an α2-adrenoceptor antagonist, were able to reverse theantidepressant-like effect of the scopoletin. This result is in agreementwith the fact that the antidepressant-like effects of several com-pounds and plant extracts in the tail suspension test were shown to bereversed by prazosin and yohimbine (Binfaré et al., 2009; Freitas et al.,2010; Machado et al., 2007; Nishizawa et al., 2007). Furthermore,phenylephrine, a α1-adrenoceptor agonist, and clonidine, an α2-adrenoceptor agonist, caused a reduction in the immobility time inthe forced swimming test (Kitada et al., 1983; O'Neill et al., 2001) andwere able to cause a synergistic antidepressant-like effect withlamotrigine in the mouse forced swimming test (Kaster et al., 2007).Hence, our result suggests that scopoletin may exert its effect inthe tail suspension test by activating postsynaptic α1- and α2-adrenoceptors.

The dopaminergic system is also implicated in regulation of mood.Since dopamine is the main neurotransmitter involved in themesolimbic reward pathway, it has been proposed that an increasein dopaminergic neurotransmission might counteract the anhedonia,which is a prominent symptom of depression (D'Aquila et al., 2000).Furthermore, some substances that modulate the dopaminergicsystem are being used as antidepressants, such as bupropion (Dhillonet al., 2008). In the present work we observed that in addition tohaloperidol, both the selective dopamine D1 receptor antagonistSCH23390 and the dopamine D2 receptor antagonist sulpiride signifi-cantly antagonized the anti-immobility effect of scopoletin in the tailsuspension test, suggesting that an activation of dopamine D1 and D2

receptors may mediate the antidepressant effects of scopoletin. Indeed,dopamine D1 and D2 receptors have been suggested to play a role in themechanismof action of several compounds and plant extracts in the tailsuspension test (Dhingra and Valecha, 2007; Freitas et al., 2010; Hiranoet al., 2007; Machado et al., 2007; Nishizawa et al., 2007).

In conclusion, the present study indicates that the coumarinscopoletin produces a specific antidepressant-like effect in the tailsuspension test, an animal model predictive of antidepressant activityand was also able to reverse a depressant-like behavior induced byacute immobility stress. In addition, this work provides evidence thatthe antidepressant-like effect of scopoletin in the tail suspension testis dependent on the interaction with the serotonergic (5-HT2A/2Creceptors), noradrenergic (α1- and α2-adrenoceptor) and dopami-nergic (D1 and D2 receptors) systems. Our results suggest thatscopoletin shares with established antidepressants some pharmaco-logical effects, at the preclinical level. Hence, we suggest thatscopoletin should be further evaluated as an agent for the treatmentof depression.

Acknowledgements

The present study was supported by grants from FAPESC, CNPq,CAPES, and FINEP-IBN-Net (Brazil) 01.06.0842-00.

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