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Neuroscience 171 (2010) 1216–1227

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IGH-INTENSITY PHYSICAL EXERCISE DISRUPTS IMPLICIT MEMORYN MICE: INVOLVEMENT OF THE STRIATAL GLUTATHIONE

NTIOXIDANT SYSTEM AND INTRACELLULAR SIGNALING

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. M. CORDOVA,c G. MANCINI,c R. WALZ,b,d

. F. DE BEM,c A. LATINI,b,c R. B. LEAL,c R. A. PINHOe

ND R. D. S. PREDIGERa,b*

Departamento de Farmacologia, Universidade Federal de Santa Ca-arina, 88049-900 Florianópolis, SC, Brazil

Centro de Neurociências Aplicadas (CeNAp), Hospital Universitário,niversidade Federal de Santa Catarina, 88040-900 Florianópolis,C, Brazil

Departamento de Bioquímica, Universidade Federal de Santa Cata-ina, 88040-900 Florianópolis, SC, Brazil

Departamento de Clínica Médica, Hospital Universitário, Univer-idade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil

Laboratório de Fisiologia e Bioquímica do Exercício, Universidade doxtremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil

bstract—Physical exercise is a widely accepted behavioraltrategy to enhance overall health, including mental function.owever, there is controversial evidence showing brain mi-

ochondrial dysfunction, oxidative damage and decreasedeurotrophin levels after high-intensity exercise, which pre-umably worsens cognitive performance. Here we investi-ated learning and memory performance dependent on dif-erent brain regions, glutathione antioxidant system, and ex-racellular signal-regulated protein kinase 1/2 (ERK1/2),erine/threonine protein kinase (AKT), cAMP response ele-ent binding (CREB) and dopamine- and cyclic AMP-regu-

ated phosphoprotein (DARPP)-32 signaling in adult Swissice submitted to 9 weeks of high-intensity exercise. The

xercise did not alter the animals’ performance in the refer-nce and working memory versions of the water maze task.n the other hand, we observed a significant impairment in

he procedural memory (an implicit memory that depends onasal ganglia) accompanied by a reduced antioxidant capac-

ty and ERK1/2 and CREB signaling in this region. In addition,e found increased striatal DARPP-32-Thr-75 phosphoryla-

ion in trained mice. These findings indicate an increasedulnerability of the striatum to high-intensity exercise asso-iated with the disruption of implicit memory in mice andccompanied by alteration of signaling proteins involved inhe plasticity of this brain structure. © 2010 IBRO. Published bylsevier Ltd. All rights reserved.

Correspondence to: R. D. S. Prediger, Laboratório Experimental deoenças Neurodegenerativas, Departamento de Farmacologia, Uni-ersidade Federal de Santa Catarina, Campus Trindade, 88049-900,lorianópolis, SC, Brazil. Tel: �55-48-3721-9764; fax: �55-48-3337-479.-mail address: ruidsp@hotmail.com (R. D. S. Prediger).bbreviations: BDNF, brain-derived neurotrophic factor; CREB, cAMP

esponse element binding; DARPP, dopamine- and cyclic AMP-regu-ated phosphoprotein; ERK1/2, extracellular signal-regulated proteininase; GPX, GSH peroxidase; GR, GSH reductase; GSH, glutathi-

tne; MLSS, maximal lactate steady state; ROS, reactive oxygen spe-ies.

306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rightoi:10.1016/j.neuroscience.2010.09.053

1216

ey words: physical exercise, treadmill, memory, hippocam-us, striatum, oxidative stress.

hysical exercise improves mental function and contrib-tes to neuronal plasticity (van Praag et al., 1999; Trejo etl., 2008; Figueiredo et al., 2010), as documented on agednd diseased brain (Gomez-Pinilla et al., 1998; Schmidt-ieber et al., 2004; Aguiar et al., 2009). However, thesexercise-induced outcomes are dependent on the animal’sealth, brain region, parameters of physical training, suchs ergometer utilized (running wheel and treadmill thatermit running movement), and the intensity and durationf physical training (low- to moderate-intensity programs)Radak et al., 2001a).

These exercise parameters modulate the formation ofitochondrial reactive oxygen species (ROS) as a conse-uence of the increased oxygen intake by the tissuesDroge, 2002; Aguiar et al., 2008b) leading to oxidativeignaling optimization in several tissues (Radak et al.,001b; Aguiar Jr and Pinho, 2010), regulating cell signal-

ng pathways and gene expression (Droge, 2002; Aguiar Jrnd Pinho, 2010). However, depending on concentration,

ocation and context, ROS can be either “friends” or “foes”egarding overall brain functions (Droge, 2002). Therefore,he complex neurobiology of exercise generally demon-trates U-shaped dose–response curves, where low dosesre stimulatory and high doses inhibitory (Hu et al., 2009).

Concerning high-intensity exercise, direct evidence ofncreased exercise-induced ROS production is still scarceut it is supported by oxidative stress imbalance in severalissues after exercise (Sen, 1995; Droge, 2002; Aguiar etl., 2008c). In the pro-oxidant brain (Aguiar Jr and Pinho,010), even the increase in downstream ROS scavengingnzymes [e.g. GSH peroxidase (GPX) and reductaseGR)] and small antioxidant molecules [e.g. glutathioneGSH)] are not sufficient to prevent mitochondrial dysfunc-ion and oxidative damage after high-intensity exerciseRosa et al., 2007; Aguiar et al., 2008b). Moreover, therere few studies reporting the possible intracellular signal-

ng mechanisms involved in the neurotrophins and mito-hondrial dysfunction (Aguiar et al., 2008a,b; Siamilis etl., 2009), interruption of hippocampal neurogenesis (Lout al., 2008), and cognitive impairments (Grebot et al.,003; Rhodes et al., 2003; Rosa et al., 2007; Taverniers etl., 2010) observed in response to high-intensity exercise.

In this study, we used a high-intensity exercise para-igm in mice to investigate the associative mnemonic func-

ion of the basal ganglia and the cognitive memory systems reserved.

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A. S. Aguiar Jr et al. / Neuroscience 171 (2010) 1216–1227 1217

f the medial temporal lobe in which the hippocampus is aajor component (Packard and Knowlton, 2002). Briefly,e observed an increased vulnerability of the striatum toigh-intensity exercise associated with a disruption of the

mplicit memory in mice that was accompanied by an al-eration of the signaling proteins involved in the neuroplas-icity of this structure.

EXPERIMENTAL PROCEDURES

nimals

xperiments were conducted using 6–8-week-old male Swissice, weighing 40–50 g at the beginning of the experiments,

upplied by the animal facility of the Universidade Federal de Santaatarina (UFSC, Florianópolis, Brazil). The animals were kept inollective cages (15 animals per cage) and maintained in a roomnder controlled temperature (23�1 °C) and 12-h light cycle (lightsn 7:00 AM), with free access to food and water. Efforts were made toinimize the number of animals used and their suffering. The pro-

edures used in the present study complied with the guidelines onnimal care of the UFSC Ethics Committee on the Use of Animals,hich follows the “Principles of laboratory animal care” from NIH.

xercise protocol

e randomly assigned two groups: untrained controls and high-ntensity exercise mice. We adapted this high-intensity exerciserotocol from high-intensity sprint interval training descriptions

ig. 1. Mice were submitted to a high-intensity program of treadmilxercise in relation to resting levels of untrained controls (A). The first tteady state (MLSS, 3 mmol/l, dashed line), meaning high-intensity effodaptations [B, C and D, * P�0.05 from untrained (Student’s t dist

xercising at twice the initial running speed (A). Data are expressed as mean �istribution). * P�0.05 from 1st day, P�0.05 from untrained control (Repeated

Troup et al., 1986; Kubukeli et al., 2002), an exercise strategyhat is intended to improve performance with short training thatary from 10–20 min. The original sprint protocol set a 2:1 ratio forork-to-recovery periods. Thus, 60-min high-intensity sprint inter-al training is performed with two 20-min bouts of exercise sepa-ated by two 10-min periods of rest. Here, we removed the restingime to avoid recovery and to reach high intensities of exercise.

hen the animals reached the stipulated maximum volume ofxercise (60 min), we lowered this volume in the following week to

ncrease the speed running. Mice were habituated to the exerciseoom for 1 h before each exercise session, carried out during theight phase of the cycle (10:00–17:00). They were intensivelyxercised with an adjustable variable-speed belt treadmill, accord-

ng to exercise protocol summarized in Fig. 1A. Blood samples15–25 �L) were collected from the tail vein for measuring lactateoncentration and then analyzed by lactate meter (Accutrendoehringer GmbH, Mannheim BW, Germany) and BM-Lactate

apes (Roche Diagnostics, Mannheim BW, Germany).

ehavioral tests

he physical training was discontinued 48 h before beginning ofhe behavioral tests. Animals were habituated to the experimentaloom for 1 h prior to the beginning of behavior tests carried outuring the light phase of the cycle (10:00–17:00 h).

ater maze

ndependent groups of animals were submitted to two differentersions of the water maze task. The apparatus was made of

(A). Blood lactate levels confirm increased lactate formation afters were characterized by blood lactate levels similar to maximal lactatecise (A). After three weeks of exercise, mice showed muscle oxidativeand lactate levels began decreasing even when the animals were

l exercisewo weekrt of exerribution)]

S.E.M (n�5–6 mice per group). ** P�0.05 from MLSS (Student’s tmeasures ANOVA followed by Newman-Keuls post-hoc test).

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lack painted fiberglass (97�60�60 cm3), and the water wasaintained at 23�2 °C. Four distant visual cues (55�55 cm2)ere placed 30 cm above the upper edge of the water tank. The

arget platform (10�10 cm2) of transparent acrylic resin was sub-erged 1–1.5 cm beneath the water surface. Starting points werearked on the outside of the pool as north (N), south (S), east (E),nd west (W). They were positioned with the lower edge 30 cmbove the upper edge of the water tank, the position of eachymbol marked the midpoint of the perimeter of a quadrantcircle�NE quadrant, square�SE quadrant, cross�SW quadrant,nd diamond�NW quadrant). If a mouse did not find the platformuring the trial period of 60 s, it was gently guided to it. A monitornd a video-recording system were installed in an adjacent room.

A group of animals was submitted to a spatial referenceemory version of the water maze as previously described (Meshit al., 2006). This consisted of three phases, performed in theollowing order: visible (two training days) followed by hiddenlatform training (four training days), and probe trial (one test day)Fig. 2B). The training trials (three trials per day) were performedith an inter-trial interval (ITI) of 20 s. The platform was absent in

he probe trial, and mice were placed into the maze for a single0-s trial. The platform position was kept fixed throughout theraining, and the starting points were randomly distributed eachay.

Another group of mice was submitted to a working memoryersion of the water maze using a previously described protocolPrediger et al., 2010). It consisted of four training days, duringhich the animals were placed in the tank facing the wall and were

hen allowed to swim freely to the submerged platform. The initialosition in which the animal was left in the tank was one of the fourertices of the imaginary quadrants of the tank, and this wasaried among trials in a pseudo-random way. The animal wasllowed to remain on the platform for 30 s and was then moved tohe next initial position without leaving the tank. It was removedmmediately after completing the four subsequent daily trials. Thisrocedure was used to ensure that the animals maintained theisuospatial information of the maze accessible during executionf the working memory task. On each subsequent training day, thelatform position was moved to the center of another quadrant ofhe tank in a pseudo-random way.

All experiments were recorded and the escape latency to thelatform, number of crossings, time spent in each quadrant, swim-ing speed and occupation plot were later measured using theNY-maze™ video tracking system (Stoelting Co., Wood Dale, IL,SA).

one fear conditioning

e assessed freezing behavior of mice in the tone fear condition-ng task as previously described (Knafo et al., 2009). Mice werelaced in chamber A for 2 min, and then received three pairings of

one (20 s, 80 dB, 4 kHz) that terminated with foot-shocks (2 s, 0.5A), with a 2 min inter-trial interval (Fig. 3). After the last foot-

hocks, mice remained in the chamber for 1 min and were theneturned to their home cages. For tone testing, we used a differenthamber (B) to avoid spatial memory bias, in which mice werelaced for 2 min and were then exposed to the same previous

one.

pontaneous locomotor activity

n order to assess possible effects of high-intensity exercise onocomotor activity, the animals were tested in activity chambers.he activity chambers (20�20�20 cm3, steel grid floor) werequipped with three parallel horizontal infrared (IR) beams, posi-ioned 3 cm above the floor and evenly spaced along the longitu-inal axis, and a digital counter recording photocell beam inter-uptions. The data obtained were expressed as IR bin crossings

orresponding to spontaneous movements during 10 min. a

ample collection

hree independent groups of animals were used in the behavioralxperiments to avoid bias in memory tasks. In all occasions,

mmediately after ending behavioral tests, the animals werequally allocated to neurochemical analysis: Western blot andSH-antioxidant experiments. Mice were killed by cervical dislo-ation and samples of hippocampus, striatum and prefrontal cor-ex were dissected and processed according to different method-logies described below.

lutathione-dependent antioxidant system

rain samples (n�7–8 animals per group) were homogenized inhosphate buffer 20 mM (PBS, pH 7.4, 0.3 M sucrose, 5 mMOPS, 1 mM EGTA, 0.1% BSA) and centrifuged at 5,000 g for 10in at 4 °C. The low speed supernatants (S1) were separated andsed for measuring oxidative stress parameters.

lutathione peroxidase (GPx) assay

Px activity was measured using tert-butyl-hydroperoxide as sub-trate (Wendel, 1981). The enzyme activity was measured byonitoring NADPH disappearance at 340 nm in 50 mM potassiumhosphate buffer, pH 7.0, containing 1.0 mM EDTA, 2.0 mM GSH,.2 U/mL GSH reductase, 1.0 mM azide, 0.2 mM tert-butyl-hy-roperoxide, 0.2 mM NADPH, and the supernatant containing.2–0.3 mg protein/ml. GPx activity was expressed as nmol ofADPH oxidized per minute per mg of protein, using an extinctionoefficient 6.22�106 for NADPH.

lutathione reductase (GR) assay

he enzyme activity was measured in a solution containing 50 mMotassium phosphate buffer, pH 7.0, containing 1.0 mM EDTA,.2 mM NADPH, and the supernatant containing 0.2–0.3 mgrotein/ml. The reaction was initiated by adding 1.0 mM oxidizedSH, and a change in absorbance was measured at 340 nm. GRctivity was expressed as nmol of NADPH oxidized per minute perg of protein, using an extinction coefficient 6.22�106 for NADPH

Carlberg and Mannervik, 1985).

on-enzymatic antioxidant defenses

SH was assessed as NPSH (90% GSH) levels, as previouslyescribed (Ellman, 1959), with slight modifications. NPSH lev-ls were measured in tissue homogenates after protein precip-

tation with one volume of 10% trichloroacetic acid. An aliquot ofamples was added to 800 mmol/l phosphate buffer, pH 7.4,nd 500 mmol/l DTNB (5,5=-dithio-bis-2-nitrobenzoic acid).olor development resulting from the reaction between DTNBnd thiols was read at 412 nm after 10 min. A standard curve ofeduced glutathione was used in order to calculate GSH levelsn the samples.

estern blot

he primary antibody anti-ERK1/2 and the protease inhibitor cock-ail were obtained from Sigma (St. Louis, MO, USA). The anti-hospho-CREB, anti-CREB, anti-phospho-ERK1/2, anti-AKT,nti-phospho-AKT, anti-DARPP-32, anti-phospho-DARPP-32(Thr34)nd anti-phospho-DARPP-32(Thr75) antibodies were purchasedrom Cell Signaling (Beverly, MA, USA). Acrylamide, bis-acryl-mide, �-mercaptoethanol, Hybond™ nitrocellulose, sodium do-ecyl sulfate (SDS), tris, secondary antibody (anti-rabbit IgG-orse radish peroxidase (HRP)-conjugated) and ECL detectioneagents were obtained from GE Healthcare Life Division. Allther reagents were of analytical grade.

Brain samples (n�5–6 animals per group) were dissected

nd mechanically homogenized in 400–500 �l of Tris–base 50

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A. S. Aguiar Jr et al. / Neuroscience 171 (2010) 1216–1227 1219

M pH 7.0, EDTA 1 mM, NaF 100 mM, PMSF 0.1 mM, Na3VO4mM, Triton X-100 1%, glycerol 10% and Protein Inhibitor Cock-

ig. 2. Effects of 9 weeks of high-intensity treadmill running in the mhe water maze. Learning phase of both versions showed no differencehat untrained mice (F) had a significant preference for the platform qatency to first crossing (I) and number of crossings (J), and average pron the probe trial. Moreover, the occupation plot of probe trial shows shere red area represents maximum occupancy and blue the minimumre expressed as mean�SEM (n�9–10 mice per group). # P�0.05 frith repeated measures followed by Newman–Keuls post-hoc test). T

his figure legend, the reader is referred to the Web version of this ar

ail (Sigma, St. Louis, MO, USA), and then incubated for 30 min in t

ce. Lysates were centrifuged (1,000�g for 10 min, at 4 °C) toliminate cellular debris, and supernatants diluted 1/1 (v/v) in

ormance in the working (A, C) and spatial (B, E) memory versions ofn groups (C, E). The probe test 24 h after the last trial on day 7 showedbut not the exercised mice (G). Concerning the target quadrant, them platform (K) were similar between exercised and sedentary animalstterns of proximity to the platform region between both groups (L, M)task, visual (D) and motor (H) functions of animals were similar. Data(Student’s t test); * P�0.05 from untrained control (Two-way ANOVAd line represents 25%. For interpretation of the references to color in

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A. S. Aguiar Jr et al. / Neuroscience 171 (2010) 1216–12271220

-mercaptoethanol 8%. Protein content was estimated at 750 nmavelength and concentration calculated by a pattern curve withovine serum albumin as described previously (Peterson, 1977).he samples (60 �g of total protein/track) were separated byDS-PAGE using 10% gels. Proteins were transferred to nitrocel-

ulose membrane using a semidry blotting apparatus (1.2 mA/cm2;.5 h). Membranes were blocked (1 h) with 5% skim milk in TBStris 10 mM, NaCl 150 mM, pH 7.5). Extracellular signal-regulatedrotein kinase ½ (ERK1/2), cAMP response element bindingCREB), dopamine- and cyclic AMP-regulated phosphoproteinDARPP)-32 and AKT total and phosphorylated forms were detectedsing specific antibodies diluted in TBS-T containing BSA (2.5%) inhe following dilutions: 1:1,000 for anti-phospho-CREB, anti-CREB,nti-AKT, anti-phospho-AKT(Ser473), anti-DARPP-32 and anti-hospho-DARPP-32 (Thr34 and Thr75); and 1:2,000 for anti-phos-ho-ERK1/2 and 1:40,000 for anti-ERK1/2. The reactions wereeveloped by ECL. All steps of blocking and incubation wereollowed by washing (5 min) the membranes thrice with TBS-T (tris0 mM, NaCl 150 mM, Tween-20 0.05%, pH 7.5). Optical densityO.D.) of the western blotting bands was quantified using Scionmage® software (Scion Corporation, Frederick, MD, USA). Phos-horylation level of each phosphoprotein was determined as aatio of O.D of the phosphorylated band/O.D of the total band

ig. 3. Effects of 9 weeks of high-intensity treadmill running in the moth groups presented similar conditioning levels (B-left, D), but intehese results suggest that high-intensity exercise impairs procedural

rained mice (C). Data are expressed as mean � S.E.M (N � 9–10 miceasures followed by Newman-Keuls post-hoc test). * P�0.05 from u

Posser et al., 2008). (

ctivities of the mitochondrial respiratory systemomplex

uadriceps muscle was homogenized in 20 volumes of 50 mMhosphate buffer (pH 7.4) containing 0.3 M sucrose, 5 mM MOPS,mM EGTA and 0.1% bovine serum albumin. The homogenatesere centrifuged at 1,000�g for 10 min at 4 °C and the pellet wasiscarded. The supernatant was centrifuged at 15,000�g in ordero concentrate mitochondria in the pellet, which was finally dis-olved in the same buffer (Latini et al., 2005). The maximal periodetween preparation of the mitochondrial fraction and measure-ent of enzyme activity was always less than 5 days. Complex-Ictivity (NADH dehydrogenase) was measured by the rate ofADH-dependent ferricyanide reduction rate at 420 nm (� � 1M�1 � cm�1) (Cassina and Radi, 1996). Activities of complex-II

succinate-2, 6-dichloroindophenol (DCIP)-oxidoreductase) andomplex-IV (cytochrome c oxidase) were measured according tohe method of Fischer et al. (1985) and Rustin et al. (1994),espectively. The methods described to measure these activitiesere slightly modified, as detailed in a previous report (Latini et al.,005). The activities of the respiratory system complexes werealculated as nmol/min/mg protein. Protein content of the mito-hondrial fraction was determined by the method of Lowry et al.

ormance in the tone fear conditioning and activity chamber tests (A).ined mice showed lower tone-induced freezing retrieval (B-right, E).processing. There were no locomotor changes in the high-intensity

up). # P�0.05 from untrained control (Two-way ANOVA with repeatedcontrol (Student’s t test).

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A. S. Aguiar Jr et al. / Neuroscience 171 (2010) 1216–1227 1221

tatistical analysis

ata are expressed as mean�SEM. Unpaired Student’s t-test andepeated measures analysis of variance (ANOVA) were appliedhen appropriate and informed in the results section and figure

egends. The accepted level of significance for the tests was�0.05. All tests were performed using the STATISTICA® soft-are package (StatSoft Inc., Tulsa, OK, USA).

RESULTS

ffects of high-intensity exercise on skeletal muscleitochondrial metabolism

aximal lactate steady state (MLSS, 3 mmol/l, Fig. 1A,ashed line) represents equilibrium between lactate trans-ort and its removal from the blood was used as a refer-nce value (Ferreira et al., 2007). In the present study,ice started running above (t4,0.05�4.38, P�0.05, Fig. 1A)

he MLSS range in the first 2 weeks of training, meaningigh-intensity levels of exercise with predominant musclelycolytic metabolism and pyruvate formation (Brooks andercier, 1994). These findings suggest an exercise inten-

ity above which the rate of glycolysis exceeds the rate ofitochondrial pyruvate utilization, causing net lactate ac-

umulation (Ferreira et al., 2007). Lactate levels de-reased with training repetition (F9,36�15.96, P�0.05, Fig.A) when compared to the first 2 weeks of physical trainingP�0.05), indicating exercise-induced increase in musclexidative metabolism (Brooks and Mercier, 1994) associ-ted with greater pyruvate conversion by citrate synthaseFig. 1B). As expected, lactate levels in untrained ani-als remained unchanged during all the evaluated pe-

iod (Fig. 1A).We confirmed it through enzyme activity in mitochon-

ria isolated from skeletal muscle (Fig. 1B–D). Exercisencreased citrate synthase (CS, t10,0.05�3.38, P�0.05,ig. 1B), mitochondrial complex I (t4,0.05�3.49, P�0.05,ig. 1C left) and complex IV (t4,0.05�4.73, P�0.05, Fig. 1Cight) but not complex II (t4,0.05�1.22, P�0.28, Fig. 1Center) activities in high-intensity exercised animals in re-ation to untrained mice. In addition, the complex IV/CSate was also significantly higher (about 36%) in trainedice (t4,0.05�13.38, P�0.05, Fig. 2D right), while the com-lex I/CS (t4,0.05�0.99, P�0.41, Fig. 2D left) and II/CSt4,0.05�0.01, P�0.98, Fig. 2C center) rates remained un-hanged. This increased muscle oxidative metabolism ledo significantly reduced body weight in trained mice (dataot shown).

ffects of high-intensity exercise on cognitiveerformance of mice

e tested independent groups of mice in two differentersions of the water maze (spatial reference memory andorking memory). Two-way ANOVA with repeated mea-ures indicated a significant effect for the repetition factorn the training sessions of the two water maze versionsworking memory: F3,84�11.05, P�0.05, Fig. 2C; spatialemory: F3,252�8.24, P�0.05, Fig. 2E), but not for thexercise factor (working memory: F �0.21, P�0.64,

1,28

ig. 2C; spatial memory: F2,84�0.28, P�0.74, Fig. 2E), c

ndicating that high-intensity exercised and untrained ani-als presented a similar learning performance in bothxperiments. Indeed, high-intensity exercise did not alterhe control variables of the experiment, that is the visiblelatform phase (F1,58�1.72, P�0.19, Fig. 2D) and thewimming speed (Wilks � F1,58�0.68, P�0.09, Fig. 2H),ndicating no significant differences between groups inisual and motor performance.

On day 7, the platform was removed for a 60 s proberial 24 h after the last trial to test recall (Fig. 2B). Analysisf latency to find the platform region (t17,0.05�1.01,�0.32, Fig. 2I), the number of platform crossings

t15,0.05�0.19, P�0.85, Fig. 2J), and the average distancerom the platform region (t18,0.05�0.19, P�0.84, Fig. 2K)howed similar performance between both groups. Un-rained controls spent more time swimming in the targetone (Fig. 2F, G, white column) than in all other threeuadrants (F3,36�11.95, P�0.05, Fig. 2F). Moreover, theime in the correct quadrant was significantly higher thanhe random level of choice of 25% (Fig. 2F, G, dashed line)or each quadrant (t9,0.05�4.49, P�0.05, Fig. 2F). On thether hand, exercised mice showed significant bias for thearget zone (F3,32�5.05, P�0.05, Fig. 2G).

In addition, we assessed striatal functions of mice us-ng the tone fear conditioning, an associative learning taskhat relies on the paired-associates learning between aone and electrical footshocks (Fig. 3A). In contrast to theontextual fear conditioning, tone fear conditioning indi-ectly requires the amygdala-dorsal striatum pathwayFerreira et al., 2008) and the retrieval after one day isippocampus-independent (Kitamura et al., 2009). Un-

rained and high-intensity exercised mice conditionedell to repeated tone-shock training (repetition F4,52�8.75, P�0.05; Fig. 3B-left; condition F1,13�2.0, P�0.17).owever, high-intensity trained mice presented worseemory retrieval in response to the tone than untrainedice (repetition F4,52�75.24, P�0.05; Fig. 3B-right; con-

ition F1,13�5.62, P�0.05). These results suggest thatigh-intensity exercise impairs procedural memory pro-essing. A possible motor dysfunction induced by high-ntensity exercise was controlled in the activity chamber.his is relevant because basal ganglia-dependent pro-essing learning and memory depend on unaltered mo-or drive (Packard and Knowlton, 2002). There were noocomotor changes in the trained mice (t17,0.05�1.32,�0.2, Fig. 3A, C).

ffects of high-intensity exercise on GSH-antioxidantystem in the striatum, hippocampus and prefrontalortex of mice

he superoxide anion (O2•) is dismuted by superoxide

ismutase (SOD) in hydrogen peroxide (H2O2), which isatalyzed by catalase (CAT) and GSH peroxidase (GPx) to

2O and O2. GPx oxidizes GSH in this process, which ishen converted back to its reduced form by glutathioneeductase (GR). Previously, we reported increased SODctivity in the hippocampus and striatum of mice afterigh-intensity treadmill exercise, while CAT activity in-

reased only in the striatum (Aguiar et al., 2008c). Here,

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A. S. Aguiar Jr et al. / Neuroscience 171 (2010) 1216–12271222

e found increased GPx activity in the prefrontal cortext18,0.05�4.02, P�0.05, Fig. 4A) and hippocampust18,0.05�3.05, P�0.05, Fig. 4A) of trained mice, whiletriatal GPx (t16,0.05�0.01, P�0.98, Fig. 5A) and GR ac-ivity (prefrontal cortex t18,0.05�0.04, P�0.96; hippocam-us t18,0.05�1.6, P�0.12, striatum t16,0.05�0.03, P�0.97;ata not shown) remained unchanged. In addition, webserved that GSH decreased in the hippocampust18,0.05�2.41, P�0.05, Fig. 4B) while it accumulated in therefrontal cortex (t17,0.05�2.18, P�0.05, Fig. 4B) and stri-tum (t18,0.05�2.51, P�0.05, Fig. 4B).

ffects of high-intensity exercise on intracellularignaling in the striatum, hippocampus andrefrontal cortex of mice

e examined phosphorylated (p-) and total (t-) forms ofRK1/2, AKT, CREB and DARPP-32 in striatum, hip-ocampus and prefrontal cortex of mice after 9 weeks ofigh-intensity treadmill exercise. We observed decreasedhosphorylation of ERK1/2 in the striatum of high-intensityrained mice in relation to untrained controls (p-ERK1

5,0.05�5.79, P�0.05; p-ERK2 t5,0.05�8.44, P�0.05; Fig.C). In contrast, ERK1/2 phosphorylation in the hippocam-us (p-ERK1 t5,0.05�1.42, P�0.18; p-ERK2 t5,0.05�0.58,�0.57; Fig. 4C) and prefrontal cortex (p-ERK1 t5,0.05�.44, P�0.66; p-ERK2 t5,0.05�0.39, P�0.69; Fig. 4C) didot change by high-intensity exercise. AKT phosphoryla-ion (Ser-473) decreased in the prefrontal cortext5,0.05�11.81, P�0.05; Fig. 4D), but did not change inhe hippocampus or striatum. CREB phosphorylationSer-133) (Fig. 4E) was decreased in the prefrontal cor-ex (t4,0.05�9.53, P�0.05) and striatum (t3,0.05�2.49,�0.05), but was increased in the hippocampus

t4,0.05�4.54, P�0.05) after 9 weeks of high-intensity ex-rcise. Phosphorylation level at Thr34 and Thr75 ofARPP-32 was investigated in the striatum of mice, due to

unctional impairment in this structure. Results obtainedhow that p-DARPP-32-Thr-34 was unchanged (t5,0.05�.76, P�0.1; Fig. 4F), but that there was a significant

ncrease in phosphorylation at Thr-75 of DARPP-32t5,0.05�4.94, P�0.05; Fig. 4F).

DISCUSSION

rain oxygen consumption (Herholz et al., 1987; Ide andecher, 2000) and ROS production (Radak et al., 2006;guiar Jr and Pinho, 2010) increase during exercise. How-ver, regular exercise modulates cerebral redox signalinghat supports neuroplasticity responses due to repeatedxidative challenge (Radak et al., 2001b; Molteni et al.,002; Aguiar Jr and Pinho, 2010). Under extreme condi-ions, such as in high-intensity physical exercise, ROSroduction may be more strongly and persistently in-reased, and the antioxidant response may not be suffi-ient to reset the system to the original level of brain redoxomeostasis (Rosa et al., 2007; Aguiar et al., 2008b; Agu-

ar Jr and Pinho, 2010). Here, we demonstrated high-ntensity exercise-induced antioxidant imbalance and cel-

ular signaling disturbing (Fig. 4) associated with learning t

nd memory dysfunction (Fig. 3). Fig. 5 shows a schematiciagram that summarizes these novel findings in prefrontalortex and striatum, since they were more sensitive tohese alterations.

Moreover, it is generally accepted that there are mul-iple memory systems. The hippocampal system pro-esses spatial-temporal memories involving relationsmong environmental cues (e.g. episodic memory in hu-ans), while the basal ganglia system is involved in im-licit learning (e.g. procedural memory), in which a singletimulus is repeatedly associated with a response (Pack-rd and Knowlton, 2002). Besides decreased quadrantccupancy, we found no significant spatial memoryhanges following high-intensity physical exercise in miceFig. 2). However, it must be conceded that the effect suchntense exercise on hippocampus function remains contro-ersial. There are descriptions of spatial memory disrup-ion (Blustein et al., 2006) and object recognition impair-ent (Garcia-Capdevila et al., 2009) after high levels of

unning exercise. In contrast, rats over-trained in anotherxercise paradigm—the swimming exercise—presented

ncreased inhibitory passive avoidance performanceOgonovszky et al., 2005). However, it seems that thentioxidant imbalance and cellular signaling impairmentbserved in high-intensity trained animals resulted in cog-itive impairment dependent on basal ganglia (Figs. 3–5).hese results are supported by recent reports showingecreased working memory performance of Specialorces soldiers after high-intensity training (Grebot et al.,003; Taverniers et al., 2010).

The impairment of CREB phosphorylation within therefrontal cortex and striatum after high-intensity exerciseight underlie the impaired working memory observedere. Exercise-induced ROS overproduction is a neurobi-logical paradox, since it may lead to detrimental effects,uch as energy metabolism shift (Nybo and Secher, 2004),xidation damage (Davies et al., 1982; Aguiar Jr andinho, 2010) and thermodynamic stress (Nybo andecher, 2004), as well as to neural benefits, includingpregulation of growth factors (Gomez-Pinilla et al., 2002;guiar et al., 2008a) and stimulation of neurogenesis

Schmidt-Hieber et al., 2004; Figueiredo et al., 2010), den-ritic branching (Gazula et al., 2004) and synaptogenesisBlack et al., 1990). Moreover, activation of intracellularignaling proteins associated with neuronal plasticity, for

nstance CREB, AKT and ERK1/2, has been reportedVaynman et al., 2004; Chen and Russo-Neustadt, 2005,009). In addition, we recently demonstrated that levels ofrain-derived neurotrophic factor (BDNF)—a CREB-regu-

ated target gene—was increased in the hippocampusAguiar et al., 2008a), but not in the prefrontal cortexAguiar et al., 2008b) and striatum (Aguiar et al., 2008c) ofice after high-intensity exercise (Fig. 5). Here we have

ound that phosphorylation of the Ser-133 site—a funda-ental step for CREB activation—was stimulated in theippocampus by high-intensity exercise. The robust in-rease in CREB phosphorylation observed in this studyould be explained by the fact that CREB is a well-known

arget for many protein kinases, including cyclic AMP-

Fal(rrpb

A. S. Aguiar Jr et al. / Neuroscience 171 (2010) 1216–1227 1223

ig. 4. Effects of 9 weeks of high-intensity treadmill running in the oxidant and intracellular signaling responses in the prefrontal cortex, hippocampusnd striatum of mice. The hippocampus and prefrontal cortex showed increased GPX activity (A), while NPSH levels were higher in the cortex and

ower in the striatum (B). The striatum presented unchanged GPx (A) activity and accumulation of NPSH (B). * P�0.05 from untrained controlStudent’s t test). Moreover, we found decreased p-ERK1/2 levels in the striatum (C, right), p-AKT in the prefrontal cortex (D, left), and p-CREB in allegions (E). DARPP-32-Thr-75 phosphorylated in the striatum (F, right). Dashed line represents 100% of untrained values and columns represent theesults of trained mice. Data are expressed as mean�SEM (n�6–8 mice per group). * P�0.05 from 100% untrained control (Student’s t test). P,hosphorylation; ERK, extracellular signal-regulated kinases; AKT, phosphatidylinositol 30-kinase (PI-3K)-AKT; CREB, cAMP response element

inding; Thr, threonine; DARPP-32, dopamine- and cyclic AMP-regulated phosphoprotein.

dMkpic

bippc

FsaiieoaG(Agv

A. S. Aguiar Jr et al. / Neuroscience 171 (2010) 1216–12271224

ependent protein kinase (PKA), protein kinase C (PKC),APKs via Rsk and Ca2�/calmodulin-dependent protein

inase (CaMK) II and IV. Since these kinases are in theathway of different transduction systems, CREB plays an

mportant role in integrating many signals into neuronal

ig. 5. High-intensity exercise increases overall brain oxygen consumtriatum and prefrontal cortex of trained mice. In the same brain regionnd the (*) the data obtained in the present study. Scheme (A) High-int

nduces implicit memory impairment. It probably occurs due to decreasnhibition by p-DARPP-32-Thr-75 (*) in the striatum of trained mice. Tot al., 2008c) in this region even after oxidative damage (Aguiar et al.f the BDNF-dependent receptor is impaired in the prefrontal cortex and p-CREB (*) observed in this region, associated with previousSH-dependent antioxidant system [GSH (*) and GPx (*)] is vulnerable

Aguiar et al., 2008b). PI3K, phosphatidylinositol 3-kinase; CREB,MP-regulated phosphoprotein; PKA, protein kinase A; ERK1/2, extrlutathione; p, phosphorylation; Thr, threonine. For interpretation of tersion of this article.

ells (Countryman and Gold, 2007). Therefore, it is possi- p

le to postulate that high-intensity exercise induces BDNFn hippocampus (Aguiar et al., 2008a) via CREB activation,roviding further evidences regarding increased hip-ocampal neuroplasticity even after high-intensity exer-ise-induced oxidative challenge. On the other hand, in the

ice leading to oxidative stress and poor antioxidant adaptations in thesignaling was disrupted. The number represents our previous findingsercise reduces the efficacy of dopamine signaling in basal ganglia andEB (*) through cooperation of decreased ERK1/2 activity (*) and PKAse findings might explain the unchanged striatal BDNF levels (Aguiarinduced by high-intensity exercise. Scheme (B) Autophosphorylationintensity exercise. We hypothesized it is due to decreased p-AKT (*)of decreased BDNF levels (Aguiar et al., 2008b). Moreover, the

intensity exercise and culminates in cortical mitochondrial dysfunctionsponse element binding protein; DARPP-32, dopamine- and cyclicsignal-regulated protein kinase; GPx, glutathione peroxidase; GSH,nces to color in this figure legend, the reader is referred to the Web

ption in ms, CREBensity exed p-CR

gether the, 2008a)fter high-findingsto high-

cAMP reacellularhe refere

refrontal cortex and striatum, where the BDNF level was

u2t

ipeea(p(atfetE2CitthtpBr2

rtlemtDitcpdaTsntscrtfitpcsilb

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stpbcFrtGtbwed2h(sHR1inGPittdicwt

Adprwb

AseefnBmAF(C

A. S. Aguiar Jr et al. / Neuroscience 171 (2010) 1216–1227 1225

nchanged by high-intensity exercise (Aguiar et al.,008b,c), we observed a decrease in CREB phosphoryla-

ion after high-intensity exercise (Fig. 5).Furthermore, we observed a high-intensity exercise-

nduced reduction in striatal ERK1/2 and cortical AKThosphorylation, the opposite reported after light-intensityxercise (Shen et al., 2001). The literature is scarce toxplain this observation. ERK1/2 signaling is regulated bydiversity of stimuli and may control synaptic plasticity

Thomas and Huganir, 2004). ERK1/2 has more than 70ossible targets in the cells, including CREB, Elk1Thomas and Huganir, 2004) and Nrf2, and it activatesntioxidant response element (ARE)-dependent transcrip-ion of antioxidant defense enzymes, such as GSH-t-trans-erase, GSH peroxidase and heme oxygenase-1 (Vauzourt al., 2010). The oxidative modulation of ERK1/2 is con-roversial. It has been shown in in vitro activation ofRK1/2 by H2O2 (Crossthwaite et al., 2002; Posser et al.,008) or during oxidative neuronal injury (for review, seehu et al., 2004). However, there are also reports of H2O2-

nduced decrease in ERK activity (Crivello et al., 2007). Inhe present study, ERK1/2 phosphorylation was reduced inhe striatum without changes in the prefrontal cortex andippocampus. Moreover, the prefrontal cortex of high-in-ensity exercised mice also presented lower levels of AKThosphorylation (as summarized in Fig. 5), which is aDNF-regulated kinase involved in neuronal survival, neu-

oplasticity and memory processes (Crossthwaite et al.,002.

To better understand the impaired retrieval freezingesponses of trained mice, we analyzed the involvement ofhe DARPP-32 protein, since it is the integrator of intracel-ular signaling pathways of basal ganglia. DARPP-32 isnriched in almost all striatal medium spiny neurons and isodulated by dopaminergic and glutamatergic inputs in

his area (for review see Svenningsson et al., 2004).ARPP-32 is phosphorylated at Thr-34 by PKA, resulting

n its conversion into a potent inhibitor of protein phospha-ase-1 (PP-1). Differently, Thr-75 phosphorylation by cy-lin-dependent kinase 5 (Cdk5) converts DARPP-32 into aotent PKA inhibitor and thereby reduces the efficacy ofopamine signaling (Svenningsson et al., 2004; Nishi etl., 2005). Here, we found increased striatal p-DARPP-32-hr-75 in high-intensity trained mice. Therefore, it is pos-ible to suppose that PKA activity was decreased (Sven-ingsson et al., 2004), thereby suppressing phosphoryla-ion of downstream target proteins involved in the control oftriatal medium spiny neurons, such as CREB. This effectould undermine an important mechanism involved in theesponses produced by activation of dopamine D1 recep-ors (Gould and Manji, 2005). Fig. 5 illustrates the mainndings of the present study, which allow us to postulatehat a high-intensity exercise program increases striatalhosphorylation in DARPP-32-Thr75, which could de-rease PKA-dependent CREB phosphorylation, impairingtriatal-dependent implicit memory. Moreover, a decreasen ERK1/2 activity cooperates with this CREB phosphory-ation decline. Additionally, PP1 activity is not counteracted

y P-Thr34 of DARPP-32, since this site was not altered by r

igh-intensity exercise. Taken together, the signaling im-alance observed in high-intensity trained mice may be

nvolved in the disruption of neural plasticity and behavioraleficits observed in the striatum.

Other possible mechanism associated with the ob-erved striatal cognitive and neural plasticity impairment ishe GSH-antioxidant system. Reduced GSH plays an im-ortant role in cellular protection against oxidative insults,ecause GPx has an absolute requirement for it as ao-substrate in H2O2-detoxifying GPx reactions (Brigelius-lohe, 1999). We verified this GPx-dependent antioxidantesponse in the prefrontal cortex and hippocampus ofrained mice. However, the striatum presented unchangedPx activity. Consequently, it is possible to postulate that

he striatal oxidant-antioxidant balance system is vulnera-le to high-intensity exercise. This striatal response of GPxas also observed in rodents even after low-intensity ex-rcise (Somani et al., 1995, 1996) or treatment with oxi-ants (Hung and Lee, 1998; Rodriguez-Martinez et al.,000). In this way, our laboratory previously reported thatigh-intensity exercise increased striatal SOD activityAguiar et al., 2008c). This efficient SOD-induced O2

•

cavenging results in H2O2 accumulation, due to inefficient

2O2 removal by GPx, and the formation of the most toxicOS in vivo: the hydroxyl radical OH• (Hung and Lee,998; Droge, 2002). Paradoxically, GSH levels increased

n the striatum and prefrontal cortex of trained mice. This isot possible due to regeneration of GSH from GSSG byR, because GR was unchanged in the tissues studied.robably, these increased GSH levels may be due to an

ncreased activity of �-glutamyl transpeptidase or �-glu-amyl cysteine synthase, a possibility that should be inves-igated in future studies. Our findings provide further evi-ences that the striatum is vulnerable to oxidative damage

nduced by high-intensity exercise, with a potential asso-iation with synaptic signaling impairment in this area,hich culminated in the cognitive dysfunction presented by

he trained animals.

CONCLUSION

ltogether, the results indicate that high-intensity exerciseisturbs the ERK1/2 pathway in the striatum, the PI3K/AKTathway in the prefrontal cortex and CREB in all brainegions studied. These cellular signaling disturbancesere associated with poor antioxidant response in theasal ganglia and with implicit memory impairment.

cknowledgments—The authors declare that they have no per-onal and competing financial interests. We gratefully acknowl-dge suggestions from Dr. Fabrício Pamplona (Universidade Fed-ral de Santa Catarina, Brazil) and statistical analysis supportrom Dr. Álvaro José Back (Universidade do Extremo Sul Catari-ense). We also acknowledge financial support received from therazilian funding agencies Conselho Nacional de Desenvolvi-ento Científico e Tecnológico (CNPq), the Coordenacão deperfeiçoamento de Pessoal de Nível Superior (CAPES), andundação de Apoio à Pesquisa do Estado de Santa CatarinaFAPESC). A.S.A-Jr and D.R. are supported by scholarships fromNPq. R.W., A.L., R.B.L., R.A.P and R.D.S.P. are supported by

esearch fellowships from CNPq.

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(Accepted 23 September 2010)(Available online 1 October 2010)