Pharmacological Reports 66 (2014) 653–659

Original research article

Involvement of PKA, PKC, CAMK-II and MEK1/2 in the acuteantidepressant-like effect of creatine in mice

Mauricio P. Cunha, Josiane Budni, Francis L. Pazini, Agatha Oliveira, Julia M. Rosa,Mark W. Lopes, Rodrigo B. Leal, Ana Lucia S. Rodrigues *

Department of Biochemistry, Universidade Federal de Santa Catarina, Florianopolis, Brazil

A R T I C L E I N F O

Article history:

Received 18 April 2013

Received in revised form 13 March 2014

Accepted 17 March 2014

Available online 3 April 2014

Keywords:

Antidepressant

Creatine

Intracellular signaling

Kinases

Tail suspension test

A B S T R A C T

Background: The aim of this study was to investigate the involvement of signaling pathways on the

creatine antidepressant-like effect in the tail suspension test (TST) in mice.

Methods: The TST was used to assess the antidepressant-like properties of creatine.

Results: The anti-immobility effect of creatine (1 mg/kg, p.o.) in the TST was blocked by i.c.v.

pretreatment with H-89 (1 mg/site, PKA inhibitor), KN-62 (1 mg/site, CAMK-II inhibitor), chelerythrine

(1 mg/site, PKC inhibitor), U0126 (5 mg/site, MEK1/2 inhibitor) or PD09058 (5 mg/site, MEK1/2

inhibitor).

Conclusion: These results suggest that the antidepressant-like effect of creatine is dependent on PKA,

CaMK-II, PKC and MEK 1/2 activation.

� 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp.

z o.o. All rights reserved.

Contents lists available at ScienceDirect

Pharmacological Reports

jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate /p h arep

Introduction

There is evidence indicating that brain bioenergetics could be atherapeutic target for the treatment of depression [1]. Severalclinical trials and studies with animal models have reported thatcreatine exerts antidepressant properties [2–5]. For example, astudy demonstrated the adjunctive effect of creatine supplemen-tation for patients with antidepressant-resistant major depressivedisorder, as early as week 1 of treatment [5], indicating a rapidantidepressant effect. At preclinical level, our group recentlydemonstrated that a single dose of creatine decreases theimmobility time in the tail suspension test (TST), an indicativeof antidepressant property [2] by a mechanism dependent on thedopamine D1 and D2 receptors, a1-adrenoceptors and postsynaptic5-HT1A activation [2–4].

Abbreviations: CaMKII, Ca2+/calmodulin protein kinase II; CREB, cyclic adenosine

monophosphate response element binding protein; ERK1/2, extracellular signal-

regulated kinases; MEK1/2, mitogen-activated protein kinase kinase 1/2; PKA,

protein kinase A; PKC, protein kinase C.

* Corresponding author.

E-mail addresses: alsrodri@gmail.com,

analucia@mbox1.ufsc.br (A.L.S. Rodrigues).

http://dx.doi.org/10.1016/j.pharep.2014.03.004

1734-1140/� 2014 Institute of Pharmacology, Polish Academy of Sciences. Published b

Converging evidence also implicates signal transductioncascades, particularly protein kinases, as possible mediators ofthis vulnerability to depression. Protein kinases are key regulatorsof receptor–effector cascades, including the relationship betweenreceptor activation and gene expression. Four of these kinases,mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK), cyclic AMP-dependent protein kinase[also known as protein kinase A (PKA)], protein kinase C (PKC) andCa2+/calmodulin protein kinase II (CAMK-II), have been shown tobe abnormal in both central and peripheral tissue of depressivepatients and animals submitted to models of depression [6].Furthermore, the administration of inhibitors of these kinasesblocked the antidepressant of several compounds, such asmemantine [7].

The MEK-ERK cascade can be activated by serotonin andnorepinephrine via serotonin and/or a-adrenergic receptors,respectively, resulting from antidepressant treatment [6]. Addi-tionally, the activities of PKC and PKA are increased byantidepressant treatment [8,9]. Regarding the role of CAMK-II inmood regulation, there are some data showing that activation ofthis enzyme is implicated in the actions of antidepressants [7].

The cyclic adenosine monophosphate (cAMP) response elementbinding protein (CREB) is a widely expressed, activity-regulatedtranscription factor that functions in a variety of physiological

y Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Fig. 1. Involvement of the intracellular signaling pathway mediated by PKA on the

antidepressant-like effect of creatine in the TST in mice. Panel A represents the

experimental protocol. Effect of the pretreatment of mice with H-89 (a PKA

inhibitor, 1 mg/site, i.c.v.) on the anti-immobility action of creatine (1 mg/kg, p.o.) in

the TST (Panel B) and in the number of crossings in the open-field test (Panel C).

Values are expressed as the mean + S.E.M. (n = 7–12). **p � 0.01 compared with the

vehicle-treated control group; #p < 0.05 compared with the same group pre-treated

with vehicle (two-way ANOVA followed by Tukey’s HSD test).

M.P. Cunha et al. / Pharmacological Reports 66 (2014) 653–659654

processes [6]. Acute control over CREB activity is generally thoughtto result from regulatory phosphorylation [6]. Multiple kinasesphosphorylate CREB (Serine133) in response to extracellular stimuliincluding growth factors, depolarization, synaptic activity, hypox-ia, stress, drugs of abuse, light, learning tasks, chemical anti-depressants, and electroconvulsive seizure (ECS), suggesting thatCREB phosphorylation is a convergence intracellular point [6].

Interestingly, creatine alters the phosphorylation of severalkinases, as MEK/ERK 1/2, in C2C12 cells [10]. Furthermore, creatineincreases the phosphorylation of CAMK-2 and CREB in thehippocampus of rats [11]. Although creatine modulates severalkinases, there is no evidence demonstrating the role of theseintracellular targets for its antidepressant effect.

Taking into account that (i) antidepressant treatment results inthe increase of activity of several intracellular kinases (particularlyMEK/ERK1/2, CAMK-II, PKC and PKA [6]), (ii) depressive patientspresent kinase activity alterations in several brain tissues [6] and(iii) creatine exerts antidepressant activity in animal models [2–4]and increases the phosphorylation of several kinases in in vitro andin vivo studies [10,11], this study was aimed at investigating theinvolvement of the intracellular signaling pathways modulated byPKA, CAMK-II and MEK 1/2 and PKC in the antidepressant-likeeffect of creatine in the TST.

Materials and methods

Animals

Male Swiss mice (30–40 g) obtained from the Central Biotheryof Universidade Federal de Santa Catarina (UFSC) were used. Theywere housed in groups of fourteen animals per plastic cage undercontrolled conditions of light (from 07:00 to 19:00 h) andtemperature (21 � 1 8C). Mice were allowed free access to standardlaboratory food and tap water and adapted to the laboratoryenvironment for at least 1 week prior to the behavioral studies.Each experimental group consisted of 7–12 animals. Animals wererandomly distributed into specified experimental groups. All of themanipulations were conducted between 14:00 and 17:00 h, witheach animal used only once. All of the procedures in this study wereperformed in accordance with the National Institute of Health Guidefor the Care and Use of Laboratory Animals and approved by the EthicsCommittee of the Institution. All efforts were made to minimizeanimal suffering and reduce the number of animals used in theexperiments.

Drugs

The following drugs were used: creatine monohydrate,chelerythrine, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquino-linesulfonamide (H-89), PD098059, U0126 and 4-[2-[(5-isoquino-linyl-sulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl ester (KN-62) (Sigma Chemical Co., St. Louis, MO,USA). Creatine was diluted in water and administered orally (p.o.)via gavage. Chelerythrine was dissolved in saline and H-89,PD098059, U0126 and KN-62 were dissolved in saline containing1% DMSO. Creatine was diluted in water and administered orally(p.o.) via gavage in a constant volume of 10 ml/kg body weight.Chelerythrine, H-89, PD098059, U0126 and KN-62 were adminis-tered by intracerebroventricular (i.c.v.) injection. The controlgroups received the appropriate vehicle.

I.c.v. injections

I.c.v. administration was performed using a microsyringe(25 ml, Hamilton) connected to a 26-gauge stainless-steel needlethat was inserted perpendicularly 2 mm deep through the skull

according to the procedure as previously described [3]. Briefly, theanimals were lightly anesthetized with ether (i.e., enough toobserve loss of the postural reflex) and then gently restrained byhand for the i.c.v. injections. The injection site was sterilized usinggauze embedded in 70% ethanol. The needle was insertedunilaterally 1 mm into the midline point equidistant from eacheye at an equal distance between the eyes and the ears andperpendicular to the plane of the skull. A volume of 5 ml of eithersterile saline (vehicle) or drugs (dissolved in sterile saline) wasinjected directly into the left lateral ventricle. Mice exhibitednormal behavior within 1 min after injection. After completion ofthe experiments, all of the animals were decapitated and theirbrains were immediately examined. It was possible to confirm thesite of the i.c.v. injections by visual inspection of the dissectedbrain. This is a standard procedure commonly performed to

Fig. 2. Involvement of the intracellular signaling pathway mediated by CAMK-II on

the antidepressant-like effect of creatine in the TST in mice. Panel A represents the

experimental protocol. Effect of the pretreatment of mice with KN-62 (a CAMK-II

inhibitor, 1 mg/site, i.c.v.) on the anti-immobility action of creatine (1 mg/kg, p.o.) in

the TST (Panel B) and the number of crossings in the open-field test (Panel C). Values

are expressed as the mean + S.E.M. (n = 9–10). **p � 0.01 compared with the

vehicle-treated control group; #p < 0.05 compared with the same group pre-

treated with vehicle (two-way ANOVA followed by Tukey’s HSD test).

Fig. 3. Involvement of the intracellular signaling pathway mediated by PKC on the

antidepressant-like effect of creatine in the TST in mice. Panel A represents the

experimental protocol. Effect of the treatment of mice with chelerythrine (a PKC

inhibitor, 1 mg/site, i.c.v.) on the anti-immobility action of creatine (1 mg/kg, p.o.) in

the TST (Panel B) and the number of crossings in the open-field test (Panel C). Values

are expressed as the mean + S.E.M. (n = 8–9). **p � 0.01 compared with the vehicle-

treated control group; ##p < 0.01 compared with the same group pre-treated with

vehicle (two-way ANOVA followed by Tukey’s HSD test).

M.P. Cunha et al. / Pharmacological Reports 66 (2014) 653–659 655

determine the accuracy of the injection technique. Results frommice presenting either a misplaced injection site or any sign ofcerebral hemorrhage were excluded from the statistical analysis(less than 5% of the total animals used).

Experimental design

To test the hypothesis that the acute antidepressant-like effectof creatine in the TST is mediated through the activation of severalkinases, mice were treated by i.c.v. injection with either vehicle orwith H-89 (1 mg/site), KN-62 (1 mg/site), chelerythrine (1 mg/site),PD098059 (5 mg/site) or U0126 (5 mg/site), to test their effects onPKA, MEK/ERK, CAMK-II or PKC, respectively. Forty-five minutesafter creatine or vehicle administration, H-89, KN-62, chelerythr-ine, U0126, PD098059 or vehicle were injected, and 15 min later,the TST was performed (Figs. 1A, 2A, 3A and 4A, respectively). The

same protocol of the administration of the kinase inhibitors aloneor in combination with creatine was used to evaluate theirinfluence on the locomotor activity in the open-field test. The dosesof the drugs and protocols were selected on the basis of literaturedata for in vivo treatments [7].

Behavioral tests

Tail suspension test (TST)

The tail suspension test (TST) has become one of the most widelyused models for assessing antidepressant-like activity in mice. Thetest is based on the notion that animals subjected to the short-terminescapable stress of being suspended by their tail will develop animmobile posture. The total duration of immobility induced by tailsuspension was measured according to the method described bySteru et al. [12]. Briefly, acoustically and visually isolated mice were

Fig. 4. Involvement of the intracellular signaling pathway mediated by MEK/ERK 1/2 on the antidepressant-like effect of creatine in the TST in mice. Panel A represents the

experimental protocol. Effect of the pretreatment of mice with either U0126 (a MEK inhibitor, 5 mg/site, i.c.v.) or PD098059 (a MEK inhibitor, 5 mg/site, i.c.v.) on the anti-

immobility action of creatine (1 mg/kg, p.o.) in the TST (Panels B and D, respectively) and the number of crossings in the open-field test (Panels C and E, respectively). Values

are expressed as the mean + S.E.M. (n = 8–9). **p � 0.01 compared with the vehicle-treated control group; ##p < 0.01 compared with the same group pre-treated with vehicle

(two-way ANOVA followed by Tukey’s HSD test).

M.P. Cunha et al. / Pharmacological Reports 66 (2014) 653–659656

suspended 50 cm above the floor by adhesive tape placedapproximately 1 cm from the tip of the tail. Immobility time wasrecorded during a 6 min period. Mice were considered immobileonly when they hung passively and completely motionless [2–4].

Open-field test

To assess the possible effects of creatine on locomotor activity,mice were evaluated in the open-field paradigm as previouslydescribed [2–4]. Mice were individually placed in a wooden box(40 cm � 60 cm � 50 cm) with the floor divided into 12 equalrectangles. The number of rectangles crossed by the animal with itsfour paws (crossing) was registered during a period of 6 min. The

number of crossings was considered to be indicative of locomotoractivity.

Immunobloting analysis

Mice were treated with creatine (1 mg/kg, p.o.) or vehicle. After1 h, animals were killed by decapitation and hippocampi wererapidly removed. During dissection hippocampi were maintainedin an ice-cold Krebs-Ringer bicarbonate buffer (KRB) with thefollowing compositions (in mM): 122 NaCl, 3 KCl, 1.2 MgSO4, 1.3CaCl2, 0.4 KH2PO4, 25 NaHCO3, and 10 D-glucose. The buffer wasbubbled with 95% O2 and 5% CO2 up to pH 7.4.

Fig. 5. Effect of creatine (1 mg/kg, p.o.) on CREB phosphorylation and CREB

immunocontent in the hippocampus of mice. A representative blot of hippocampal

immunoreactivity of phospho-CREB (P-CREB), total-CREB (T-CREB) and b-actin

(used as a load control) is shown in Panel A. The level of CREB phosphorylation

determined as a ratio of O.D. of the phosphorylated band over the O.D. of the total

band (Panel B) and CREB immunocontent (Panel C) are expressed as percentage of

the control (mean + S.E.M. of 6 mice; Student’s t-test).

M.P. Cunha et al. / Pharmacological Reports 66 (2014) 653–659 657

Hippocampi were solubilized with SDS-stopping solution(4% SDS, 2 mM EDTA, 8% b-mercaptoethanol, and 50 mM Tris,pH 6.8, 2 mM PMSF). Samples (60 mg of total protein/track) wereseparated by 10% SDS-PAGE and transferred to nitrocellulosemembranes. Membranes were blocked with 2% albumin (1 h) inTBS (10 mM Tris, 150 mM NaCl, pH 7.5), followed by three timeswashing with TBS-T (10 mM Tris, 150 mM NaCl, 0.05% Tween-20,pH 7.5). Membranes were incubated with primary antibodies anti-CREB, anti-pCREB (serine 133), anti-actin (Cell Signaling, dilution1:1000), overnight, at 4 8C, and then, they were exposed toappropriate peroxidase-conjugated secondary antibodies (1:5000)for 1 h at room temperature. Immunocomplexes were visualizedusing the enhancing chemiluminescense (ECL) detection system(Santa Cruz). Densitometric analysis was performed for thequantification of the immunoblottings using the Scion ImageSoftware (Scion Corporation).

Statistical analysis

The Kolmogorov–Smirnov test was used to evaluate thenormality assumption of behavioral and biochemical data. Allvariables in the present study showed a normal distribution.Comparisons between the experimental and control groups wereperformed by two-way ANOVA followed by Tukey’s HSD test(behavioral analysis) or Student’s-t test (immunoblotting analysis)when appropriate. A value of p < 0.05 was considered to bestatistically significant.

Results

Effect of treatment of mice with H-89 on the antidepressant-like effect

of creatine in the TST and open-field test

The result depicted in Fig. 1A shows that the anti-immobilityeffect of creatine (1 mg/kg, p.o.) was prevented by treating themice with the PKA inhibitor H-89 (1 mg/site, i.c.v.) in the TST. Atwo-way ANOVA showed significant differences for creatinetreatment alone (F(1,32) = 10.96; p < 0.01) and creatine � H-89treatment interaction (F(1,32) = 7.50; p < 0.05), but not H-89treatment alone (F(1,32) = 2.93; p = 0.09). The number of crossingsin the open-field test was not altered by the treatment withcreatine and/or H-89 treatment (p > 0.05; Fig. 1B).

Effect of treatment of mice with KN-62 (a CAMK-II inhibitor) on the

antidepressant-like effect of creatine in the TST and in the open-field

test

The result depicted in Fig. 2A shows that the anti-immobilityeffect of creatine (1 mg/kg, p.o.) was prevented by treatment ofmice with the CAMK-II inhibitor KN-62 (1 mg/site, i.c.v.) in the TST.A two-way ANOVA showed significant differences for creatinetreatment alone (F(1,30) = 17.12; p < 0.01) and creatine � KN-62treatment interaction (F(1,30) = 4.84; p < 0.05), but not KN-62treatment alone (F(1,30) = 3.42; p = 0.07). The number of crossingsin the open-field test was not altered by the treatment withcreatine and/or KN-62 treatment (p > 0.05; Fig. 2B).

Involvement of PKC in the antidepressant-like effect of the creatine

The result depicted in Fig. 3A shows that the anti-immobilityeffect of creatine (1 mg/kg, p.o.) was prevented by treating themice with the PKC inhibitor chelerythrine (1 mg/site, i.c.v.) in theTST. A two-way ANOVA showed significant differences forcreatine treatment alone (F(1,32) = 8.38; p < 0.01), chelerythrinetreatment alone (F(1,32) = 18.40; p < 0.01), and creati-ne � chelerythrine treatments (F(1,32) = 10.81; p < 0.01). The

number of crossings in the open-field test was not altered by thetreatment with creatine and/or chelerythrine treatment(p > 0.05; Fig. 3B).

Involvement of MEK/ERK 1/2 in the antidepressant-like effect of the

creatine

The result depicted in Fig. 4A shows that the anti-immobilityeffect of creatine (1 mg/kg, p.o.) was prevented by treating themice with the MEK 1/2 inhibitor U0126 (5 mg/site, i.c.v.) in the TST.A two-way ANOVA showed significant differences for creatinetreatment alone (F(1,30) = 10.19; p < 0.01), U0126 treatment alone(F(1,30) = 17.96; p < 0.01), and creatine � U0126 treatment inter-action (F(1,30) = 7.31; p < 0.05). Furthermore, the antidepressant-like effect of creatine (1 mg/kg, p.o.) was prevented by treatment ofmice with the MEK1 inhibitor PD098059 (5 mg/site, i.c.v.) in theTST (Fig. 4C). A two-way ANOVA showed significant differences forcreatine treatment alone (F(1, 29) = 10.02; p < 0.01), PD098059treatment alone (F(1,29) = 19.21; p < 0.01), and creati-ne � PD098059 treatment interaction (F(1,29) = 7.65; p < 0.01).The number of crossings in the open-field test was not altered bythe treatment with creatine and/or U0126 or PD098059 treatment(p > 0.05; Fig. 4B and D, respectively).

M.P. Cunha et al. / Pharmacological Reports 66 (2014) 653–659658

Creatine did not alter CREB phosphorylation in the hippocampus of

mice

The Student’s-t test revealed that creatine did not alter CREBphosphorylation and total CREB immunocontent in the hippocam-pus of mice, as compared to control group (Fig. 5B and C,respectively; p > 0.05).

Discussion

The potential role of metabolic impairments in the pathophysi-ology of depression is motivating researchers to evaluate thetreatment efficacy of creatine, a naturally occurring energetic andneuroprotective compound found in brain and muscle tissues.Specifically, the antidepressant-like effect of oral creatine is shownin the TST and forced swimming test in rodents [2–4]. In thepresent study, we also demonstrated the antidepressant-like effectof creatine administered by oral route 60 min before of TST. It is inline with the fact that acute systemic administration of creatine inrodents elicits several central effects such as anticonvulsant [13]and spatial learning enhancement [11]. Furthermore, in a previousstudy from our group we demonstrated the creatine administeredi.c.v. exerts antidepressant-like effect in the TST, suggesting thecentral antidepressant-like effect of creatine [3]. Moreover, a singlesystemic administration of creatine was reported to increasestriatal creatine and phosphocreatine content and creatine kinaseactivity in the brain of rats [13].

In the present study, we demonstrated that creatine producesan antidepressant-like effect in the TST by acting through the PKA,PKC, CAMK-II and MEK/ERK 1/2 signaling pathways. The PKAcascade is known to be associated with the pathogenesis andtreatment of depression [9]. This study shows the reduction ofimmobility time in TST elicited by creatine was fully abolished bythe pretreatment of mice with H-89, an isoquinolinesulfonamidethat acts as a competitive inhibitor against ATP for binding to thecatalytic subunit of PKA. The PKA inhibitor H-89 was also reportedto abolish the antidepressant effect of fluoxetine and electro-acupuncture (two strategies widely used in the treatment ofdepression), in chronic mild-stressed rats [14]. However, aprevious study showed that H-89 did not alter the facilitatoryeffect of creatine in the spatial memory in the Barnes maze test[11]. This may be due to the difference in the behavioral paradigmsemployed in the present study that investigated the antidepres-sant profile of creatine compared to the one used in previous studythat investigated the facilitatory effect of creatine in the spatiallearning retention.

CAMK-II, the most abundant protein kinase in the brain, hasbeen shown to be activated by long-term treatment with someantidepressant drugs, suggesting that modulation of CAMK-II mayalso have important beneficial effects in the treatment ofdepressive disorders [6]. The present study provides evidencethat CAMK-II plays a role in mediating the antidepressant-likeeffect elicited by the acute treatment of creatine in the TST becauseKN-62, an inhibitor that interacts with the regulatory domain ofCAMK-II and blocks the active form of this enzyme, was able tocompletely abolish the antidepressant-like effect of creatine in theTST. Our results are in agreement with a neurochemical study thatrevealed that treatment with creatine in rats increased the levels ofpCAMK-II [11]. Moreover, KN-62 suppresses the enhanced spatiallearning retention induced by creatine, suggesting that CAMK-IImay also have a crucial role in mediating the effects of creatine incognitive function [11].

Another protein kinase implicated in the pathophysiology ofdepression is PKC, whose activity was reported to be decreased inthe prefrontal cortex and hippocampus of teenage suicide victims[8] and in animal models of depression [8]. Interestingly, acute

administration of the PKC activator bryostatin-1 in naive ratsresulted in an antidepressant-like activity, which was abolished byco-administration of a PKC inhibitor [15]. Our data demonstratedthat the antidepressant-like effect of creatine was also preventedby the pretreatment with chelerythrine, a benzophenanthridinealkaloid that is a selective and potent competitive inhibitor to theATP binding site on the catalytic domain of PKC, suggesting thatthis signaling pathway is involved in antidepressant-like action ofcreatine in the TST.

There are reports showing that fluoxetine, a selective serotoninreuptake inhibitor, promotes rapid activation of the MAPK cascadein cultured astrocytes [16]. In our study, we demonstrate theinvolvement of the MEK/ERK pathway in the acute antidepres-sant-like effect of creatine in the TST. Pretreatment of mice withthe MEK 1/2 inhibitors PD098059 and U0126 at doses that had noeffect on the TST significantly inhibited the anti-immobility effectof creatine. The MEK 1/2 inhibitors employed in this study displaydistinct mechanisms of action. PD098059 inhibits MEK1 andMEK2 activation by binding to the inactive enzyme, whichprevents its activation by Raf kinase and subsequently inhibitsactivation of ERK1/2 [17]. U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) is a selective and potentinhibitor of MEK1 activity in vitro as well as of the activation ofERK1/2 by MEK1/2 in vivo by directly inhibiting the catalyticactivity of the active enzyme [18]. Thus, U0126 acts one stepfurther downstream of PD098059 and blocks MEK1/2 activity. Ourresults indicate that the acute antidepressant-like effect ofcreatine could be due to the activation of the MEK/ERK pathway.Reinforcing this notion, a study from Deldicque et al. [10] alsodemonstrated that creatine treatment increases the ERK 1/2activity in the C2C12 cells.

In a previous study, we demonstrated that the antidepressant-like effect of creatine is dependent on a1-adrenoceptor activation[3]. Interestingly, selective a1-adrenoceptors agonist phenyleph-rine increased levels of phosphorylated ERK 1/2, and U0126blocked this effect [19]. This suggests that a1-adrenoceptoractivation induced by creatine may play a role in ERK 1/2phosphorylation.

We also demonstrated that the antidepressant-like effect ofcreatine is dependent on activation of the dopamine D1 and D2

receptors [2]. D1 receptor stimulation activates signaling pathwaysmediated by PKA and PKC [20]. Moreover, D2 receptors activate theintracellular targets PKC, MEK/ERK 1/2 and CAMK-II [21]. Wesuggest that the activation of multiple intracellular signalingpathways mediated by PKA, PKC, CAMK-II and ERK 1/2 and theirinfluence on the antidepressant-like effect of the creatine could bedependent, at least in part, on the dopamine D1 and D2 receptoractivation.

The reversal of the antidepressant-like effect of creatine in theTST observed here in the presence of signaling pathway inhibitorsis not due to any alteration in the locomotor activity because theadministration of H-89, KN-62, chelerythrine, U0126 andPD098059 either alone or in combination with creatine did notsignificantly alter the ambulation of mice in the open-field test.

Interestingly, in spite of the behavioral studies indicating therole of the signaling pathways in the behavioral response tocreatine in the TST, no alteration in the hippocampal CREBphosphorylation was observed as a consequence of creatineadministration. However, we should take into account that thebiochemical measurements were performed in the whole hippo-campus 60 min after creatine administration. We could not ruleout that biochemical alterations in this parameter may occur inhippocampal sub-regions or even in different cell types of thecentral nervous system [6]. Besides, considering that phosphor-ylation is a transient event, it is possible that a shorter time pointmay reveal changes on CREB phosphorylation.

M.P. Cunha et al. / Pharmacological Reports 66 (2014) 653–659 659

The results of this study provide clear and novel data on theputative participation of PKA, CAMK-II, PKC and MEK/ERK in theantidepressant effect of creatine, reinforcing the notion that theseare important targets for antidepressant activity. Furthermore, ourresults also reinforce literature data that have shown TST as auseful tool for the evaluation of intracellular targets involved in thebehavioral effects of antidepressant compounds.

Conflicts of interest

The authors declare no conflicts of interest.

Funding body

This study was supported by grants from the Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) no. 307687/2009-0, Coordenacao de Aperfeicoamento de Pessoal de EnsinoSuperior (CAPES), Fundacao de Apoio a Pesquisa Cientıfica eTecnologica do Estado de Santa Catarina (FAPESC) no. 1347/2010-1, Rede Instituto Brasileiro de Neurociencia (IBN-Net/CNPq), andthe Nucleo de Excelencia em Neurociencias Aplicadas de SantaCatarina (NENASC) Project/PRONEX Program (CNPq/FAPESC).

References

[1] Strakowski SM. Bioenergetics for depression: something different for depres-sion. Am J Psychiatry 2012;169:891–3.

[2] Cunha MP, Machado DG, Capra JC, Jacinto J, Bettio LE, Rodrigues ALS. Antide-pressant-like effect of creatine in mice involves dopaminergic activation. JPsychopharmacol 2012;26:1489–501.

[3] Cunha MP, Pazini FL, Oliveira A, Bettio LE, Rosa JM, Machado DG, et al. Theactivation of a1-adrenoceptors is implicated in the antidepressant-like effectof creatine in the tail suspension test. Prog Neuropsychopharmacol BiolPsychiatry 2013;44C:39–50.

[4] Cunha MP, Pazini FL, Oliveira A, Machado DG, Rodrigues ALS. Evidence for theinvolvement of 5-HT1A receptor in the acute antidepressant-like effect ofcreatine in mice. Brain Res Bull 2013;95:61–9.

[5] Lyoo IK, Yoon S, Kim TS, Hwang J, Kim JE, Won W, et al. A randomized, double-blind placebo-controlled trial of oral creatine monohydrate augmentation for

enhanced response to a selective serotonin reuptake inhibitor in women withmajor depressive disorder. Am J Psychiatry 2012;169:937–45.

[6] D’Sa C, Duman RS. Antidepressants and neuroplasticity. Bipolar Disord2002;4:183–94.

[7] Almeida RC, Souza DG, Soletti RC, Lopez MG, Rodrigues ALS, Gabilan NH.Involvement of PKA, MAPK/ERK and CaMKII, but not PKC in the acute antide-pressant-like effect of memantine in mice. Neurosci Lett 2006;395:93–7.

[8] Abrial E, Lucas G, Scarna H, Haddjeri N, Lambas-Senas L. A role for the PKCsignaling system in the pathophysiology and treatment of mood disorders:involvement of a functional imbalance? Mol Neurobiol 2011;44:407–19.

[9] Ozawa H, Rasenick MM. Chronic electroconvulsive treatment augments cou-pling of the GTP-binding protein Gs to the catalytic moiety of adenylyl cyclasein a manner similar to that seen with chronic antidepressant drugs. J Neu-rochem 1991;56:330–8.

[10] Deldicque L, Theisen D, Bertrand L, Hespel P, Hue L, Francaux M. Creatineenhances differentiation of myogenic C2C12 cells by activating both p38 andAkt/PKB pathways. Am J Physiol Cell Physiol 2007;293:1263–71.

[11] Souza MA, Magni DV, Guerra GP, Oliveira MS, Furian AF, Pereira L, et al.Involvement of hippocampal CAMKII/CREB signaling in the spatial memoryretention induced by creatine. Amino Acids 2012;43:2491–503.

[12] Steru L, Chermat R, Thierry B, Simon P. The tail suspension test: a new method forscreening antidepressants in mice. Psychopharmacology (Berl) 1985;85:367–70.

[13] Royes LF, Fighera MR, Furian AF, Oliveira MS, de Carvalho Myskiw C, Fiorenza NG,et al. Effectiveness of creatine monohydrate on seizures and oxidative damageinduced by methylmalonate. Pharmacol Biochem Behav 2006;83:136–44.

[14] Liu JH, Wu ZF, Sun J, Jiang L, Jiang S, Fu WB. Role of AC-cAMP-PKA cascade inantidepressant action of electroacupuncture treatment in rats. Evid BasedComplement Alternat Med 2012;2012:932414.

[15] Sun MK, Alkon DL. Protein kinase C isozymes: memory therapeutic potential.Curr Drug Targets CNS Neurol Disord 2005;4:541–52.

[16] Mercier G, Lennon AM, Renouf B, Dessouroux A, Ramauge M, Courtin F, et al.MAP kinase activation by fluoxetine and its relation to gene expression incultured rat astrocytes. J Mol Neurosci 2004;24:207–16.

[17] Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of themitogen-activated protein kinase cascade. Proc Natl Acad Sci USA1995;92:7686–9.

[18] Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, et al.Identification of a novel inhibitor of mitogen-activated protein kinase kinase.J Biol Chem 1998;273:18623–32.

[19] Bauer RM, Strittmatter F, Gratzke C, Gottinger J, Schlenker B, Reich O, et al.Coupling of a1-adrenoceptors to ERK1/2 in the human prostate. Urol Int2011;86:427–33.

[20] Neve KA, Seamans JK, Trantham-Davidson H. Dopamine receptor signaling.J Recept Signal Transduct Res 2004;24:165–205.

[21] Yan Z, Feng J, Fienberg AA, Greengard P. D2 dopamine receptors inducemitogen-activated protein kinase and cAMP response element-binding pro-tein phosphorylation in neurons. Proc Natl Acad Sci USA 1999;96:11607–12.