R

Ewa

AHAa

Sb

1

h

•••••

a

ARRAA

KTVCCA

nT

h0

Behavioural Brain Research 271 (2014) 160–170

Contents lists available at ScienceDirect

Behavioural Brain Research

jou rn al hom epage: www.elsev ier .com/ locate /bbr

esearch report

ffects of voluntary and treadmill exercise on spontaneousithdrawal signs, cognitive deficits and alterations in

poptosis-associated proteins in morphine-dependent rats

min Mokhtari-Zaera, Shahrbanoo Ghodrati-Jaldbakhana, Abbas Ali Vafaeia,ossein Miladi-Gorji a, Maziar M. Akhavanb, Ahmad Reza Bandegia,b,li Rashidy-Poura,∗

Laboratory of Learning and Memory, Research Center and Department of Physiology, School of Medicine, Semnan University of Medical Sciences,emnan, IranSkin Research Center, Laboratory of Protein and Enzyme, ShahidBeheshti University (M.C.), Shohada-e Tajrish Hospital, Shahrdari St.,989934148 Tehran, Iran

i g h l i g h t s

Chronic morphine leads to apoptosis and impairment of cognitive functions.Voluntary and treadmill exercise enhance cognitive functions.Exercise inhibits chronic morphine-induced apoptosis.Exercise alleviates memory impairment induced by chronic morphine.Exercise could be a potential method to ameliorate the adverse effects of opiate abuse.

r t i c l e i n f o

rticle history:eceived 30 March 2014eceived in revised form 23 May 2014ccepted 27 May 2014vailable online 4 June 2014

eywords:readmill exerciseoluntary exercisehronic morphineognitive defectpoptosis

a b s t r a c t

Chronic exposure to morphine results in cognitive deficits and alterations of apoptotic proteins in favor ofcell death in the hippocampus, a brain region critically involved in learning and memory. Physical activityhas been shown to have beneficial effects on brain health. In the current work, we examined the effects ofvoluntary and treadmill exercise on spontaneous withdrawal signs, the associated cognitive defects, andchanges of apoptotic proteins in morphine-dependent rats. Morphine dependence was induced throughbi-daily administrations of morphine (10 mg/kg) for 10 days. Then, the rats were trained under twodifferent exercise protocols: mild treadmill exercise or voluntary wheel exercise for 10 days. After exercisetraining, their spatial learning and memory and aversive memory were examined by a water maze and byan inhibitory avoidance task, respectively. The expression of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 in the hippocampus were determined by immunoblotting. We found that chronicexposure to morphine impaired spatial and aversive memory and remarkably suppressed the expressionof Bcl-2, but Bax expression remained constant. Both voluntary and treadmill exercise alleviated memoryimpairment, increased the expression of Bcl-2 protein, and only the later suppressed the expression of Bax

protein in morphine-dependent animals. Moreover, both exercise protocols diminished the occurrenceof spontaneous morphine withdrawal signs. Our findings showed that exercise reduces the spontaneousmorphine-withdrawal signs, blocks the associated impairment of cognitive performance, and overcomesmorphine-induced alterations in apoptotic proteins in favor of cell death. Thus, exercise may be a usefultherapeutic strategy for cognitive and behavioral deficits in addict individuals.

∗ Corresponding author at: Research Center and Department of Physiology, Sem-an University of Medical Sciences, 15131-38111 Semnan, Iran.el.: +98 09121140221; fax: +98 2333354186.

E-mail address: Rashidy-pour@semums.ac.ir (A. Rashidy-Pour).

ttp://dx.doi.org/10.1016/j.bbr.2014.05.061166-4328/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Chronic exposure to opiates can induce cognitive deficits inhumans, and experimental animals [1–8], and also leads to changesin spine density, neurogenesis and synaptic transmission in the hip-pocampus of rats [9,10]. Prenatal morphine exposure also impairs

ral Bra

liala

o[pSriprptsBBcSctt[

otl[Cot[

mpmTcatwaa

taofvcd

2

2

i2codf

A. Mokhtari-Zaer et al. / Behaviou

earning and memory in juvenile rats [11]. Chronic morphinempairs spatial memory and hippocampal long-term potentiation,

form of synaptic plasticity that may be a cellular substrate ofearning and memory [12–14], via accumulation of extracellulardenosine acting on adenosine A1 receptors [13].

Apoptosis or programmed cell death is a physiological formf cell death that occurs in embryonic development and aging15,16]. Also, neuronal loss by an inappropriate or excessive apo-tosis has been implicated in many pathological conditions [17].everal key proteins including Bcl-2 family proteins play a pivotalole in the regulation of apoptosis. The Bcl-2 family is classifiednto anti-apoptotic proteins (Bcl-2, Bcl-XL, etc.) and pro-apoptoticroteins (BH3 only family, and Bax family), and play a crucialole in the regulation of the intrinsic (mitochondrial) apoptoticathway. This pathway involves the release of cytochrome c fromhe mitochondria into cytosol and the subsequent activation ofpecific proteases termed caspases, which promotes apoptosis.cl-2 preserves mitochondrial membrane integrity, whereas theax family contributes to the permeabilization of the outer mito-hondrial membrane, allowing efflux of cytochrome c [18–20].ome studies on experimental animals have demonstrated thathronic exposure to opioid drugs can produce apoptosis in the cen-ral nervous system (CNS) [21,22]. This apoptotic effect may leado learning and memory impairment following chronic morphine23].

Human studies suggest that exercise could have benefits forverall health and cognitive functions [24]. In rodents, both volun-ary and forced exercise can increase neurogenesis, and improveearning and memory in a variety of spatial and non-spatial tasks25–28]. Physical activity increases central BDNF in rodents [29].hanges in BDNF are apparently necessary for the effects of exercisen cognition in rodents, as blocking BDNF signaling also preventshe enhancement of cognitive function following physical training7,29,30].

We recently found that concurrent of physical exercise withorphine administration reduced the occurrence of naloxone-

recipitated withdrawal signs and blocked the ability of chronicorphine to impair spatial memory retention in rats via the BDNF-

rkB mechanism [7]. However, when extrapolating to the humanonditions, it is less likely that individuals that suffer from drugddiction will engage in physical exercise while they are underhe influence of drugs of abuse. Thus, an important question ishether exercise could blunt the deleterious effects of drugs of

buse after the exposure to these substances or after long periods ofbstinence.

In this study, we investigated whether both voluntary and mildreadmill exercise would reduce spontaneous withdrawal signs,meliorate the associated cognitive deficits, and return the changesf hippocampal expression of apoptotic proteins (Bcl-2 and Bax) inavor of cell survival following chronic morphine. We used botholuntary and forced exercise because different kinds of physi-al activity induce differential effects on neuronal adaptations inifferent brain regions, and cognitive functions [31,32].

. Materials and methods

.1. Animals

Adult male Wistar rats (220 ± 10 g) were individually housedn cages (50 cm × 26 cm × 25 cm) in a 12-h light/dark cycle at

2–24 ◦C, with food and water ad libitum. The experimental proto-ol was approved by the Ethical Review Board of Semnan Universityf Medical Sciences (Iran). All of the experimental trials were con-ucted in agreement with the National Institutes of Health Guideor the Care and Use of Laboratory Animals.

in Research 271 (2014) 160–170 161

2.2. Induction of morphine dependence

Morphine sulphate (Temad Company, Tehran, Iran) was dis-solved (10 mg/ml) in 0.9% saline and chronically administered viasubcutaneous injections at a volume of 1 ml/kg. These injectionswere given twice per day at 12 h intervals for 10 days. This mor-phine administration protocol is sufficient to induce dependence[7,13]. The control rats were treated similarly, except with injec-tions of saline replacing morphine.

2.3. Voluntary exercise paradigm

A detail of description of the voluntary running wheel has beengiven in our previous reports [7,33]. Briefly, each of the exercisingrats was given all day/night access to a cage equipped with a run-ning wheel (diameter = 34.5 cm, width = 9.5 cm) (Novidan.Tab, Iran)that was freely rotated against a resistance of 100 g. The sedentaryrats were confined to similar cages with no access to a wheel. Theexercising groups were exposed to exercise following the develop-ment of dependence on morphine, which took 10 days before thestart of the biochemical or behavioral experiments [7].

2.4. Treadmill exercise paradigm

The rats in the treadmill exercise group were forced to run on amotorized treadmill (Borjsanat, Iran) for 30 min once a day for 10days. The exercise load consisted of running at a speed of 5 m/minfor the first 5 min, 8 m/min for the next 5 min, and 10 m/min forthe last 20 min, with no incline. This exercise regimen is a light-intensity exercise [34].

2.5. Spontaneous withdrawal responses

Spontaneous withdrawal responses of the exercising rats andtheir sedentary control were measured during exercise once aday for 10 days. Each rat was placed in a transparent cylinder(35 cm diameter, 50 cm height) for 30 min, and their behavior wasmonitored by a rater blinded to the rats’ treatment. Withdrawalsigns were recorded and scored according to a modified versionof the Gellet–Holtzman scale [35]. Briefly, on the Gellert andHoltzman scale graded signs are assigned a weighting factor 1–4based on frequency of appearance, and checked signs receivevalues of 2–3 depending upon the particular withdrawal signnoted, but regardless of frequency of appearance. Graded signsincluding jumps, wet dog shakes, and abdominal contractionswere counted as the number of events occurring during the totaltest time. Checked signs including diarrhea, ptosis, erection orgenital grooming, teeth chattering, writhing, and irritability werecounted as positive if the sign occurred at any time during theobservation period. After completion of the observation session,the overall withdrawal severity was calculated by summing theproper weighting factor of somatic signs [7,35].

2.6. Water maze

A detailed description of the apparatus and the tracking systemhas been given in our previous reports [7,33]. In brief, the watermaze (WM) was a black circular pool (140 cm in diameter and 60 cmhigh) of 25 cm depth filled with 20 ◦C water.

2.6.1. TrainingThe WM protocol was a stringent protocol consisting of four

trials per day for 5 days. During each trial, the rat was placed into thewater from one of the four cardinal points of the compass (N, E, S,and W), which varied from trial to trial in aquasi-random order. Therat had to swim until it climbed onto the escape platform. The rats

1 ral Bra

w6ia

2

sacscormsp

2

daaow

2

rddidcech

2

eet((c

2

lsNmarpptst0was

62 A. Mokhtari-Zaer et al. / Behaviou

ere guided by hand to the platform if they failed to locate it within0 s. The rat was allowed to stay on the platform for 20 s during the

nter-trial interval. After the last trial, the animal was towel driednd returned to its home cage with no access to a running wheel.

.6.2. Probe testA spatial probe test was performed 2 days after the last acqui-

ition trial, during which the platform was removed. The rats werellowed to swim for 60 s, during which, the latency to reach pre-ise platform coordinates (platform location latency), and the timepent swimming within a zone, which had a 20 cm radius that wasentered either on the original training location (target zone) orn an equivalent location in other quadrants (opposite, left andight adjacent quadrants) were recorded. The velocity of each ani-al was also calculated. The analysis of the time spent within a

pecified radius (zone) is a sensitive measure of the WM probe testerformance, in terms of detecting group differences [36,37].

.7. Inhibitory avoidance task

The experimental apparatus was a shuttle box (UgoBasile, Spain)ivided into dark and light compartments. Both compartments had

grid floor (2 mm stainless steel rods spaced at 6 mm) connected to shock generator. An automated apparatus registered the latencyf passage from the light to the dark side of the box. The apparatusas located in the sound attenuated room.

.7.1. TrainingAll experimental rats were first habituated to the apparatus. The

at was placed in the illuminated compartment and the guillotineoor was raised 7 s later. Upon entering the dark compartment, theoor was closed and the rat was taken from the dark compartment

nto the home cage. The acquisition trial was done 30 min lateruring which the door was closed and a 50 Hz, 1 mA constanturrent shock was applied for 2 s immediately after the rat hadntered the dark compartment. The rat was removed from the darkompartment about 10 s after receiving the shock and returned tois home cage.

.7.2. Memory testingAvoidance memory of all animals was assessed by a 9 min

xtinction trial 48 h later, during which shock was never deliv-red. The rat were placed in the illuminated chamber, as in theraining session, and the latency of entering the dark chamberstep-through latency, STL), and time spent in the light chamberTLC) were recorded. Longer STL and TLC were interpreted as indi-ating better memory retention.

.8. Protein measurement and Western blotting

Rats were sacrificed, and the hippocampal tissues were col-ected, and were then immediately frozen at −70 ◦C. Hippocampalamples were homogenized and prepared in lysis buffer (137 mMaCl, 20 mM Tris–HCl pH 8.0, 1% NP-40, 10% glycerol, 1 mM phenyl-ethylsulfonyl fluoride, 10 �g/ml aprotinin, 1 �g/ml leupeptin,

nd 0.5 mM sodium vanadate). Tissue extracts were centrifuged toemove insoluble materials (12,500 × g for 20 min at 4 ◦C) and totalrotein concentration was determined according to the Micro BCArocedure (Pierce, Rockford, IL, USA). Equal amounts (25 �g) of pro-ein from each sample were loaded on 15% polyacrylamide gels andeparated by a standard SDS-PAGE. Protein bands were transferredo PVDF membranes and then blocked using 5% skim milk and

.1% Tween-20 in Tris-buffered saline. Membranes were incubatedith primary antibodies overnight at 4 ◦C [anti-Bax, 1:500 (sc-493);

nti Bcl-2, 1:200 (sc-492); anti-actin (sc-130065)] and then with aecondary antibody [(goat anti-rabbit IgG horseradish peroxidase

in Research 271 (2014) 160–170

conjugated antibody, 1:2000 (sc-2004)] for 1 h at room temper-ature. Immunocomplexes were visualized by chemiluminescenceusing the ECL kit (Pierce, Rockford, IL, USA) according to the manu-facturer’s instructions. The results for target proteins, Bax, and Bcl-2were quantified by densitometric analysis (using Gel-Pro analyzerimaging software). The results for the Bax-to-Bcl-2 ratio were alsocalculated.

2.9. Measurement of serum corticosterone levels

Immediately after the last exercise session (between 9 and11 am), the rats were decapitated, trunk blood was collectedin tubes with EDTA and centrifuged (3000 × g, 20 min) and theplasma was stored at −70 ◦C until used for the corticosterone assay.Serum corticosterone levels were determined in duplicates usingenzyme-linked immunosorbent assay (ELISA) kit (Abcam, England)following the manufacturer’s instructions.

2.10. Statistical analysis

The withdrawal data were not distributed normally and wereanalyzed using non-parametric Kruskal–Wallis analysis followedby the Mann–Whitney test. Other data exhibited a normal distri-bution and were analyzed by mixed- and between factor analyses ofvariance (ANOVAs) followed by the Tukey’s test. Differences wereconsidered significant if the P value was less than 0.05.

3. Results

3.1. Effects of voluntary and treadmill exercise on spontaneouswithdrawal signs in rats

To determine the effects of voluntary and forced exercise onthe severity of morphine dependence, the global severity of thespontaneous withdrawal responses was measured daily in thesedentary and exercising morphine-treated rats during 10 days ofphysical activity. Rats (n = 7–8 rats per group) were divided intothe saline-sedentary (Sal/Sed), the morphine-sedentary (Mor/Sed),the morphine-voluntary exercise (Mor/VE), and the morphine-treadmill exercise (Mor/TE) groups. The global severity of themorphine withdrawal responses was measured as previouslydescribed (Fig. 1, Experiment 1).

Fig. 2 shows the running distance for the voluntary exercisegroups. A two-way ANOVA with repeated measure (day) for theaverage running distance (m) during 10 days of voluntary exerciserevealed the absence of a significant effect of groups (F1,12 = 2.19,P = 0.14), a significant effect of days (F9,120 = 14.88, P = 0.0001),and no significant interaction between both factors (F9,120 = 0.59,P = 0.80). In general, the running distance is increased significantlyin both groups as exercise days progressed. Moreover, morphinedependence did not affect voluntary running distance.

The results of the spontaneous withdrawal responses are shownin Fig. 3. Analysis of data indicated significant differences amonggroups in day 1 (H3 = 17.94, P = 0.0001), day 2 (H3 = 15.82, P = 0.001),day 3 (H3 = 16.74, P = 0.001), day 4 (H3 = 20.584, P = 0.0001), day 5(H3 = 14.96, P = 0.002), and day 6 (H3 = 15.00, P = 0.0002). The two-tailed Mann–Whitney U non-parametric test indicated a significantdifference between the Sal/Sed group with other three groups ondays 1–6 (all, P < 0.05). There was a significant difference betweenthe Mor/Sed and Mor/VE groups on days 4–6 (all, P < 0.05), andbetween the Mor/Sed and Mor/TE groups on day 6 (P < 0.05).

3.2. Effects of voluntary and forced treadmill exercise on memory

deficits in morphine-dependent rats

To determine the effect of voluntary and forced exercise onmemory deficits induced by chronic morphine, rats (n = 10 rats per

A. Mokhtari-Zaer et al. / Behavioural Brain Research 271 (2014) 160–170 163

ents

gMo(

3

wcMa

Fig. 1. Timelines of experim

roup) were divided into the Sal/Sed, the Sal/VE, the Sal/TE, theor/Sed, the Mor/VE, the Mor/TE groups. The learning and mem-

ry capabilities of all groups were tested as previously describedFig. 1, Experiment 2).

.3. Spatial learning

The pattern of voluntary running for voluntary exercise groups

as similar to that shown in Fig. 2 (data not shown). An ANOVA

arried out on swim speed revealed no differences between groups.oreover, data related to the distance swam to reach the platform

nd latency to reach the platform followed similar patterns; thus,

(see Section 2 for details).

we only present latency data. Latency data during the 5 days oftraining in the WM are illustrated in Fig. 4. A three-way ANOVA(morphine treatment × exercise × training days) were used to ana-lyze the escape latencies during training. All groups learned tolocate the platform during the five successive days of training, asindicated by decreasing escape latencies as training progressed(F4,270 = 44.62, P < 0.0001). The effect of exercise was significant(F2,270 = 16.81, P < 0.0001): the exercising groups exhibited signif-

icantly shorter escape latencies on days 3 and 4 the WM trainingthan those of the sedentary control groups (Ps ranging from <0.05to <0.0001). The main effect of morphine treatment was not signif-icant (F1,270 = 1.48, P = 0.222). There were no significant interaction

164 A. Mokhtari-Zaer et al. / Behavioural Brain Research 271 (2014) 160–170

d in m

bPmm

3

AsocbtttfsPaM

mtbS

Fsae

Fig. 2. The average of running distance (expresse

etween morphine treatment and exercise and days (F8,270 = 0.756, = 0.642). These findings indicate that both voluntary and tread-ill exercise enhanced the learning rate in both control and chronicorphine groups.

.4. Spatial memory

The data for the memory retention test are shown in Fig. 5. two-way ANOVA on the platform location latency (Fig. 5A)howed significant effects of exercise (F2,54 = 5.59, P < 0.01), andf morphine treatment (F1,54 = 4.02, P < 0.05), and a signifi-ant interaction between both factors (F2,54 = 3.47, P < 0.05). Theetween group comparisons indicated that the platform loca-ion latency of the Mor/Sed group was significantly longerhan that of the Sal/Sed group (P < 0.05, Fig. 5A), indicatinghat chronic morphine impairs memory retention. The plat-orm location latency of the Sal/VE and Sal/TE groups wasignificantly shorter than that of the Sal/Sed group (P < 0.01 and

< 0.05, respectively). The platform location latency of the Mor/VEnd Mor/TE groups was significantly shorter than that of theor/Sed group (P < 0.05 and P < 0.01, respectively).A three-way ANOVA with quadrant (Fig. 5B) as repeated

easures showed a significant interaction between morphinereatment and exercise and zones (F3,216 = 134.52, P < 0.0001). Theetween-group comparisons indicated that both the Sal/VE andal/TE groups spent significantly more time in the target zone than

ig. 3. Effect of voluntary and treadmill exercise on the spontaneous withdrawal signs ineverity was calculated using the proper weighting factor. Both voluntary and treadmill exre expressed as the median ± inter-quartile range. *P < 0.05 compared with the Sal/Sed gxercise, TE: treadmill exercise.

eter per day) in the voluntary exercising groups.

the Sal/Sed (P < 0.01) and Mor/Sed groups (P < 0.001). The Mor/Sedgroup spent more time in the opposite zone than the Sal/Sed group(P < 0.05).

To control for differences in WM performance, we also recordedeach animal’s swimming speed. We found no difference (F5,54 = 0.6,P = 0.55) in the swimming speeds of all six groups: the Sal/Sedgroup (33.33 cm/s), the Sal/VE group (33.17 cm/s), the Sal/TE(30.91 cm/s), the Mor/Sed group (34.22 cm/s), and the Mor/VEgroup (35.89 cm/s), and the Mor/TE group (35.76 cm/s).

3.5. Retention memory in an inhibitory avoidance task

Memory retention data of inhibitory avoidance task are illus-trated in Fig. 6. A two-way ANOVA on STL data (Fig. 6A) indicated asignificant effect of exercise (F2,54 = 14.75, P < 0.0001), a significanteffect of chronic morphine (F2,54 = 2.79, P = 0.1) and no significantinteraction between both factors (F2,54 = 0.21, P = 0.80). Post hoccomparisons showed that STL of the Sal/VE and Sal/TE groups wassignificantly longer than the Sal/Sed (both, P < 0.01). The STL of theMor/Sed group was significantly shorter than the Sal/Sed (P < 0.05).The STL of Mor/VE, and Mor/TE groups was significantly longer thanthe Mor/Sed (both, P < 0.01).

ANOVA on TLC data (Fig. 6B) also showed a significant effectof exercise (F2,54 = 14.25, P < 0.0001), a significant effect of chronicmorphine (F2,54 = 9.24, P < 0.01) and no significant interactionbetween both factors (F2,56 = 0.98, P = 0.33). Post hoc comparisons

morphine-dependent rats. The Gellert–Holtzman score of the overall withdrawalercise significantly decreased the occurrence of spontaneous withdrawal signs. Dataroup; /= P < 0.05 compared with the Mor/Sed group. Mor: morphine, VE: voluntary

A. Mokhtari-Zaer et al. / Behavioural Brain Research 271 (2014) 160–170 165

Fig. 4. Effects of voluntary and treadmill exercise on spatial learning in the water maze. Both types of exercise improved the learning abilities of morphine-dependent ratsin a similar manner to rats that were exercising daily and were given saline. The rats that were exercising took significantly less time to learn the location of the platformc exprew oup.

siMTt

3am

oa(SEw(

wow9aoPb

ompared to the sedentary control groups on days 3 and 4 of training. The data are

ith the Sal/Sed group. (b) P < 0.5, and (d) P < 0.01 as compared with the Mor/Sed gr

howed that the TLC of the Sal/VE and Sal/TE groups was signif-cantly longer than the Sal/Sed (both, P < 0.001). The TLC of the

or/Sed group was significantly shorter than the Sal/Sed (P < 0.05).he TLC of the Mor/VE, and Mor/TE groups was significantly longerhan the Mor/Sed group (both, P < 0.001).

.6. Effects of voluntary and forced exercise on hippocampalpoptotic proteins and serum corticosterone levels inorphine-dependent rats

To determine the effect of voluntary and forced exercisen serum corticosterone levels and hippocampal expression ofpoptotic-associated proteins induced by chronic morphine, ratsn = 7 rats per group) were divided into the Sal/Sed, the Sal/VE, theal/TE, the Mor/Sed, the Mor/VE, and the Mor/TE groups (Fig. 1,xperiment 3). To verify apoptosis induced by chronic morphine,e determined the relative expression of Bax, and Bcl-2 proteins

Fig. 7A). The ratio of Bax to Bcl-2 was also calculated.The pattern of voluntary running for voluntary exercise groups

as similar to that shown in Fig. 2 (data not shown). When the levelf Bax protein in the Sal/Sed group was set at 100, the level of Baxas 94.16 ± 0.51 in the Sal/VE group, 93.7 ± 0.27 in the Sal/TE group,

9.43 ± 0.43 in the Mor/Sed group, 97.12 ± 0.4 in the Mor/VE group,

nd 96.92 ± 0.32 in the Mor/TE group (Fig. 7B). A two-way ANOVAn Bax data showed significant effects of treatment (F1,36 = 67.20,

< 0.0001), no significant effects of exercise (F2,36 = 1.78, P = 0.18),ut a significant interaction between both factors (F2,36 = 13.57,

ssed as the mean ± SEM. (a1) P < 0.001, (c) P < 0.01, and (a2) P < 0.0001, as compared

P < 0.0001). The between-group comparisons indicated that chronicmorphine did not change the expression of Bax protein. Both VE andTE significantly (P < 0.01) decreased the expression of Bax proteinin saline-treated rats. Only TE decreased the expression of Bax inmorphine treated groups (P < 0.05).

When the level of Bcl-2 protein in the Sal/Sed group was setat 100, the level of Bcl-2 was 117.96 ± 0.97 in the Sal/VE group,116.1 ± 1.12 in the Sal/TE group, 82.45 ± 3.83 in the Mor/Sed group,100.64 ± 3.83 in the Mor/VE group, and 91.62 ± 3.94 in the Mor/TEgroup (Fig. 7C). A two-way ANOVA on Bcl-2 data showed sig-nificant effects of treatment (F1,36 = 52.34, P < 0.0001), exercise(F2,36 = 15.36, P < 0.0001), and no significant interaction betweenboth factors (F2,36 = 0.73, P = 0.49). The between-group comparisonsindicated that chronic morphine decreased the expression of Bcl-2protein (P < 0.01). Both VE and TE significantly (P < 0.01) increasedthe expression of Bcl-2 protein in both the saline and morphinetreated groups.

When the level of Bax to Bcl-2 ratio in the Sal/Sed groupwas set at 100, this level was 78.84 ± 1.11 in the Sal/VE group,79.75 ± 0.84 in the Sal/TE group, 120.20 ± 4.97 in the Mor/Sedgroup, 96.12 ± 3.53 in the Mor/VE group, and 105.54 ± 4.15 inthe Mor/TE group (Fig. 7D). A two-way ANOVA on Bax to Bcl-2ratio data showed significant effects of treatment (F1,36 = 24.38,

P < 0.0001), exercise (F2,36 = 12.79, P < 0.0001), and no significantinteraction between both factors (F2,36 = 1.47, P = 0.49). Chronicmorphine increased significantly the Bax to Bcl-2 ratio as comparedwith control group (P < 0.01). Both voluntary and treadmill exercise

166 A. Mokhtari-Zaer et al. / Behavioural Brain Research 271 (2014) 160–170

Fig. 5. Effects of voluntary and forced exercise on spatial memory retention in the water maze. (A) The mean latency to reach the platform location. (B) The mean time spent(%) in within a zone, which had a radius of 20 cm and was centered either on the original training location (the target zone) or on an equivalent location in other quadrants.E ats tom oup. #

t

ss

gmaP

4

rcpbca

4w

md

xercise improved memory retention, as evidenced by the fact that the exercising rore time in the target zone (B). *P < 0.05. **P < 0.01 as compared with the Sal/Sed gr

he Mor/Sed group.

ignificantly (P < 0.01) decreased the Bax to Bcl-2 ratio in both thealine and morphine treated groups.

Fig. 8 shows serum corticosterone levels of the six experimentalroups. A two way-ANOVA showed no significant effects of treat-ent (F1,36 = 0.324, P = 0.573), or exercise (F2,36 = 0.265, P = 0.77),

nd no significant interaction between both factors (F2,36 = 0.063, = 0.939).

. Discussion

The main findings of the present study are that exerciseeduces spontaneous morphine-withdrawal and blocks the asso-iated impairment of cognitive performance. In parallel, exerciserevents the hippocampal increase of the ratio Bax/Bcl-2 inducedy morphine dependence. These findings suggest that exerciseould be a useful therapy to prevent cognitive deficits and neuronalpoptosis in addict individuals.

.1. Voluntary and treadmill exercise reduce spontaneousithdrawal signs

We found that both voluntary and forced exercise after chronicorphine exposure decrease the occurrence of spontaneous with-

rawal signs in morphine-dependent rats. Interestingly, the effects

ok significantly less time to reach the platform location (A), and spent significantlyP < 0.05 as compared with the Sal/Sed group.+ P < 0.05, ++ P < 0.01 as compared with

of voluntary exercise was appeared 4 days, while that of tread-mill was seen 6 days following exercise, indicating a more effectiveeffect of voluntary exercise than forced exercise. These findingsare in accordance with our previous studies showing that volun-tary exercise during morphine dependence development reducesdependence severity [7]. Additionally, it is important to note thatexercise exerts a mild effect in reducing withdrawal signs, whilethe abstinence period is the main factor responsible for the with-drawal score reduction in all groups. Presently, it is not clear howexercise during the abstinence period can reduce the occurrence ofspontaneous withdrawal signs. Exercise has been shown to reduceself-administration of morphine [38], the craving for morphine[39], and the potency of morphine [40]. Additionally, long-termexercise (6 weeks) decreases sensitivity of the anti-nociceptiveeffects of morphine and other mu opioids, which is mediated bythe chronic release of endogenous opioid peptides and functionalchanges in the opioid receptor system [40,41]. Finally, voluntaryexercise can induce a rewarding state that continues followingexercise cession, and cause plastic changes in the dopaminergicreward pathway of the mesolimbic system [42]. Consequently,

these changes in potency and sensitivity of morphine and neu-roplastic modifications in the reward pathway may contributeto the attenuation of spontaneous withdrawal signs followingexercise.

A. Mokhtari-Zaer et al. / Behavioural Brain Research 271 (2014) 160–170 167

Fig. 6. Effects of voluntary and forced exercise on memory retention of an inhibitory avoidance task. (A) The step-through latency (STL). (B) The time spent in the lightc t thatw comp

4a

iwt[wostgoeweiimat

rfe

ompartment (TLC). Exercise improved memory retention, as evidenced by the facith the Sal/Sed group; #P < 0.05 as compared with the Sal/Sed group; ++ P < 0.01 as

.2. Chronic morphine impairs memory retention and inducespoptosis

We found that chronic morphine impairs memory retentionn both spatial and aversive tasks. These findings are consistent

ith previous studies showing prenatal as well as postna-al administration of morphine can impair cognitive functions3,6–8,11,20,23,37]. This deficit is unlikely because of spontaneousithdrawal, given that no significant difference was found in signs

f withdrawal between the Sal/Sed and Mor/Sed groups whenomatic signs were assessed 7 days after the last morphine injec-ion (Fig. 3). This suggests that morphine withdrawal is not stilloing at the time of training and testing. Thus, the cognitive deficitsbserved in the dependent rats are related to past exposure toither opiates and/or opiate withdrawal but not to direct opiateithdrawal. Although the mechanism that underlies the impairing

ffects of morphine remains unknown, long-term opiate use maynduce maladaptive plasticity in brain structures involved in learn-ng and memory, such as the hippocampus. Chronic exposure to

orphine can interact with hippocampal synaptic transmission,nd cause cognitive deficits in several hippocampal-dependentasks [4,8,11,43].

Past studies have shown that chronic morphine induces up-egulation of the pro-apoptotic FasL, Fas, and Bad and the activeragments of caspases-8 and 3 in mice and rat brains [21]. Thenhanced apoptosis by chronic contact to opioid drugs may

the exercising rats showed longer STL and TLC. In A and B: **P < 0.01 as comparedared with the Mor/Sed group.

interfere with memory and learning [23]. Prenatal morphine expo-sure has been also demonstrated to enhance the level of neuroblastapoptosis and delays the neural tube closure in developing CNS[44]. This prenatal imbalanced apoptosis may continue even afterdelivery, as in a recent study the memory deficits resulted from pre-natal morphine exposure is attributed to the increased neuronalapoptosis in the hippocampus of young offspring [43]. We foundthat chronic exposure to morphine induced down-regulation of theanti-apoptotic protein Bcl-2 in the hippocampus, but Bax expres-sion remained constant, resulting in a high Bax/Bcl-2 ratio. Thesealterations, in turn, shifted the balance between pro-apoptotic andanti-apoptotic factors in favor of cell death. A previous study hasshown that chronic-morphine treated mice and abstinent mice(one day after the last morphine injection) exhibited scatteredapoptotic neurons and astrocytes in the brain. This neurotoxiceffect was accompanied by up-regulation of the pro-apoptoticproteins and the active fragments of caspases in cortical and hip-pocampal lysates. Abstinence from chronic morphine also resultedin a reduced expression of the anti-apoptotic protein Bcl-2 inmice [45]. This finding is in agreement with the present resultsshowing similar changes in Bcl-2 expression in the abstinentrats (11 days after the last injection of morphine). Chronic mor-

phine may enhance apoptosis through the sustained activationof opioid receptors as the opioid receptor antagonist naloxonecompletely prevented morphine-induced changes in the expres-sion of apoptosis-associated protein in the brain [21]. Thus, the

168 A. Mokhtari-Zaer et al. / Behavioural Brain Research 271 (2014) 160–170

F -2 in

e n. ##Pg exerc

ihanmwTsosasfi

ig. 7. Effects of voluntary and treadmill exercise on the expression of Bax and Bclxpression of Bax and Bcl-2 proteins, respectively. (D) Ratio of Bax to Bcl-2 expressioroup; + P < 0.05, and ++ P < 0.01 as compared with the Mor/Sed group. TE: treadmill

nduction of deviant apoptosis in specific regions of brain such asippocampus may be a major consequence of the neuronal dam-ge induced by opiate drugs after long-term exposures [46]. Thiseuronal damage may lead to cognitive deficits and anxiety andood disorders that seen in drug abusers [1,2]. In the present study,e did not identify/quantify apoptotic cells in the hippocampus.

hus, further study such as the quantification of neuronal apopto-is following chronic morphine will be required to determine theverall significance of morphine induced reduction of Bcl-2 expres-ion. Although these data from animal studies may indicate the

bnormal activation of apoptotic pathways following chronic expo-ure to opiates, a study performed on postmortem human brainrom heroin or methadone abusers revealed that the extrinsic andntrinsic apoptotic pathways are not abnormally activated in the

Fig. 8. Effects of voluntary and treadmill exercise on serum corticostero

the hippocampus. (A) Representative immunoblotting micrographs. (B, C) Relative < 0.01 as compared with the Sal/Sed group; **P < 0.01 as compared with the Sal/Sedise, VE: voluntary exercise, SED: sedentary.

prefrontal cortex, suggesting the differential effects of long-termuse of opiates on apoptotic pathways in humans and experimentalanimals [47].

4.3. Voluntary and treadmill exercise alleviate cognitive deficitsand the enhanced-apoptosis induced by chronic morphine

We found that both voluntary and treadmill exercise enhancecognitive functions in spatial and aversive tasks, confirming andextending previous studies showing the beneficial effects of differ-

ent types of exercise on learning and memory in a variety of tasks[3,5,7,18,19]. These beneficial effects of exercise on memory andneuroplasticity are mediated by the BDNF-TrkB pathway in dis-tinct brain regions such as the hippocampus, as blockade of this

ne levels. No significant differences were found between groups.

ral Bra

pa

rotfmittdm[

cdidatsftw

eictsahottAtgc

C

oad

A

F(wM

R

[

[

[

[

[

[[[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

A. Mokhtari-Zaer et al. / Behaviou

athway abolished the enhancing effects of exercise on learningnd memory [7,29,48,49].

We found that both types of exercise enhanced the up-egulation of the anti-apoptotic protein Bcl-2 and down-regulationf the pro-apoptotic protein Bax in the hippocampus, which shiftedhe balance between pro-apoptotic and anti-apoptotic factors inavor of cell survival. These effects were seen in both the saline and

orphine-treated animals, suggesting that exercise has a positivenfluence on cell survival in normal and patho-physiological condi-ions. In accordance with these findings, other studies have shownhat both types of exercise suppressed neuronal apoptotic celleath in the hippocampus probably via BDNF- and NGF-mediatedechanisms and subsequently, improve learning and memory

28,49–51].Previous studies have demonstrated that different types of exer-

ise induce neuroplasticity changes and neuronal adaptations inifferent brain regions, and thus exert differential effects on var-

ous forms of learning and memory [31,32]. The exercise-inducedifferent intensity of stress and hence glucocorticoid levels could ben underlying modulator for their differential effects on brain func-ions. We found that both exercise did not significantly increasederum corticosterone levels, but exerted similar effects on cognitiveunctions and apoptosis-related proteins. This suggests that bothypes of excises regime did not induce a significant stress responsehich may interfere with their beneficial effects on brain.

In conclusion, we observed that both voluntary and treadmillxercise increased cognitive functions and shifted the alterationsn apoptosis-related proteins in favor of cell survival in normalonditions. Chronic exposure to morphine impaired cognitive func-ions and remarkably suppressed the expression of Bcl-2 with noignificant changes in Bax expression, shifting the balance of pro-poptotic and anti-apoptotic proteins in favor of cell death in theippocampus and consequently, impairment of learning and mem-ry. Conversely, both voluntary and treadmill exercise were ableo alleviate memory impairment and return the changes of apop-otic proteins in favor of cell survival in morphine-dependent rats.lthough specific extrapolation of the results of rodent experiments

o the human condition appears difficult, our results could sug-est the application of exercise as a useful therapeutic strategy forognitive and behavioral deficits in addict individuals.

onflict of interest statement

We attest that we have herein disclosed any and all financial orther relationships that could be construed as a conflict of interestnd that all sources of financial support for this study have beenisclosed.

cknowledgments

This work was supported by grants from Iran National Scienceoundation (91001448) and Semnan University of Medical SciencesSemnan, Iran). In addition, Mr. Amin Mokhtarizaer carried out thisork in partial project fulfillment of the requirements to obtain theaster of Science degree in physiology.

eferences

[1] Davis P, Liddiard H, McMillan T. Neuropsychological deficits and opiate abuse.Drug Alcohol Depen 2002;67:105–8.

[2] Robbins TW, Everitt BJ. Drug addiction: bad habits add up. Nature1999;398:567–70.

[3] Dougherty K, Walsh T, Bailey S, Schlussman S, Grasing K. Acquisition of a Mor-

ris water maze task is impaired during early but not late withdrawal frommorphine. Pharmacol Biochem Behav 1996;55:227–35.

[4] Li Z, Wu C, Pei G, Xu N. Reversal of morphine-induced memory impairment inmice by withdrawal in Morris water maze: possible involvement of cholinergicsystem. Pharmacol Biochem Behav 2001;68:507–13.

[

in Research 271 (2014) 160–170 169

[5] Spain JW, Newsom GC. Chronic opioids impair acquisition of both radial mazeand Y-maze choice escape. Psychopharmacology 1991;105:101–6.

[6] Miladi Gorji H, Rashidy-Pour A, Fathollahi Y. Effects of morphine dependenceon the performance of rats in reference and working versions of the water maze.Physiol Behav 2008;93:622–7.

[7] Miladi-Gorji H, Rashidy-Pour A, Fathollahi Y, Akhavan MM, Semnanian S, SafariM. Voluntary exercise ameliorates cognitive deficits in morphine dependentrats: the role of hippocampal brain-derived neurotrophic factor. NeurobiolLearn Mem 2011;96:479–91.

[8] Lu G, Zhou Q-X, Kang S, Li Q-L, Zhao L-C, Chen J-D, et al. Chronic morphinetreatment impaired hippocampal long-term potentiation and spatial memoryvia accumulation of extracellular adenosine acting on adenosine A1 receptors.J Neurosci 2010;30:5058–70.

[9] Eisch AJ, Barrot M, Schad CA, Self DW, Nestler EJ. Opiates inhibit neurogenesisin the adult rat hippocampus. Proc Natl Acad Sci 2000;97:7579–84.

10] Mandyam CD, Norris RD, Eisch AJ. Chronic morphine induces premature mito-sis of proliferating cells in the adult mouse subgranular zone. J Neurosci Res2004;76:783–94.

11] Niu L, Cao B, Zhu H, Mei B, Wang M, Yang Y, et al. Impaired in vivo synaptic plas-ticity in dentate gyrus and spatial memory in juvenile rats induced by prenatalmorphine exposure. Hippocampus 2009;19:649–57.

12] Bao G, Kang L, Li H, Li Y, Pu L, Xia P, et al. Morphine and heroin differentiallymodulate in vivo hippocampal LTP in opiate-dependent rat. Neuropsychophar-macology 2007;32:1738–49.

13] Pu L, Bao G-B, Xu N-J, Ma L, Pei G. Hippocampal long-term potentiation isreduced by chronic opiate treatment and can be restored by re-exposure toopiates. J Neurosci 2002;22:1914–21.

14] Salmanzadeh F, Fathollahi Y, Semnanian S, Shafizadeh M. Dependence on mor-phine impairs the induction of long-term potentiation in the CA1 region of rathippocampal slices. Brain Res 2003;965:108–13.

15] Vaux DL, Korsmeyer SJ. Cell death in development. Cell 1999;96:245–54.16] Meier P, Finch A, Evan G. Apoptosis in development. Nature 2000;407:796–801.17] Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol

2007;35:495–516.18] Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the

mitochondria in apoptosis. Genes Dev 1999;13:1899–911.19] Zong W-X, Lindsten T, Ross AJ, MacGregor GR, Thompson CB. BH3-only proteins

that bind pro-survival Bcl-2 family members fail to induce apoptosis in theabsence of Bax and Bak. Genes Dev 2001;15:1481–6.

20] Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the releaseof apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature1999;399:483–7.

21] Boronat MA, García-Fuster MJ, García-Sevilla JA. Chronic morphine induces up-regulation of the pro-apoptotic Fas receptor and down-regulation of the anti-apoptotic Bcl-2 oncoprotein in rat brain. Br J Pharmacol 2001;134:1263–70.

22] Hu S, Sheng WS, Lokensgard JR, Peterson PK. Morphine induces apoptosis ofhuman microglia and neurons. Neuropharmacology 2002;42:829–36.

23] Tramullas M, Martínez-Cué C, Hurlé MA. Chronic administration of heroin tomice produces up-regulation of brain apoptosis-related proteins and impairsspatial learning and memory. Neuropharmacology 2008;54:640–52.

24] Kramer AF, Erickson KI, Colcombe SJ. Exercise, cognition, and the aging brain. JAppl Physiol 2006;101:1237–42.

25] Van der Borght K, Havekes R, Bos T, Eggen BJ, Van der Zee EA. Exercise improvesmemory acquisition and retrieval in the Y-maze task: relationship with hip-pocampal neurogenesis. Behav Neurosci 2007;121:324.

26] van Praag H, Christie BR, Sejnowski TJ, Gage FH. Running enhances neu-rogenesis, learning, and long-term potentiation in mice. Proc Natl Acad Sci1999;96:13427–31.

27] Albeck DS, Sano K, Prewitt GE, Dalton L. Mild forced treadmill exercise enhancesspatial learning in the aged rat. Behav Brain Res 2006;168:345–8.

28] Wu CW, Chen YC, Yu L, Chen Hi, Jen CJ, et al. Treadmill exercise counteractsthe suppressive effects of peripheral lipopolysaccharide on hippocampal neu-rogenesis and learning and memory. J Neurochem 2007;103:2471–81.

29] Vaynman S, Ying Z, Gomez-Pinilla F. Hippocampal BDNF mediates theefficacy of exercise on synaptic plasticity and cognition. Eur J Neurosci2004;20:2580–90.

30] Bekinschtein P, Oomen CA, Saksida LM, Bussey TJ. Effects of environmentalenrichment and voluntary exercise on neurogenesis, learning and memory,and pattern separation: BDNF as a critical variable. Semin Cell Dev Biol2011;22:536–42.

31] Liu YF, Chen Hi, Wu CL, Kuo YM, Yu L, Huang AM, et al. Differential effects oftreadmill running and wheel running on spatial or aversive learning and mem-ory: roles of amygdalar brain-derived neurotrophic factor and synaptotagminI. J Physiol 2009;587:3221–31.

32] Lin T-W, Chen S-J, Huang T-Y, Chang C-Y, Chuang J-I, Wu F-S, et al. Differ-ent types of exercise induce differential effects on neuronal adaptations andmemory performance. Neurobiol Learn Mem 2012;97:140–7.

33] Ebrahimi S, Rashidy-Pour A, Vafaei AA, Akhavan MM. Central �-adrenergicreceptors play an important role in the enhancing effect of voluntary exerciseon learning and memory in rat. Behav Brain Res 2010;208:189–93.

34] Kim Y-P, Kim H-B, Jang M-H, Lim B-V, Kim Y-J, Kim H, et al. Magnitude-and

time-dependence of the effect of treadmill exercise on cell proliferation in thedentate gyrus of rats. Int J Sports Med 2003;24:114–7.

35] Gellert VF, Holtzman SG. Development and maintenance of morphine toleranceand dependence in the rat by scheduled access to morphine drinking solutions.J Pharmacol Exp Ther 1978;205:536–46.

1 ral Bra

[

[

[

[

[

[

[

[

[

[

[[

[

[

[

70 A. Mokhtari-Zaer et al. / Behaviou

36] Gallagher M, Burwell R, Burchinal MR. Severity of spatial learning impairmentin aging: development of a learning index for performance in the Morris watermaze. Behav Neurosci 1993;107:618.

37] Maei HR, Zaslavsky K, Teixeira CM, Frankland PW. What is the most sensi-tive measure of water maze probe test performance? Front Integ Neurosci2009;3:4–12.

38] Hosseini M, Alaei HA, Naderi A, Sharifi MR, Zahed R. Treadmill exercise reducesself-administration of morphine in male rats. Pathophysiology 2009;16:3–7.

39] Smith MA, McClean JM, Bryant PA. Sensitivity to the effects of a kappa opioid inrats with free access to exercise wheels: differential effects across behavioralmeasures. Pharmacol Biochem Behav 2004;77:49–57.

40] Smith MA, Lyle MA. Chronic exercise decreases sensitivity to mu opioidsin female rats: correlation with exercise output. Pharmacol Biochem Behav2006;85:12–22.

41] Smith MA, Yancey DL. Sensitivity to the effects of opioids in rats with free accessto exercise wheels: �-opioid tolerance and physical dependence. Psychophar-macology 2003;168:426–34.

42] Greenwood BN, Foley TE, Le TV, Strong PV, Loughridge AB, Day HE, et al. Long-term voluntary wheel running is rewarding and produces plasticity in the

mesolimbic reward pathway. Behav Brain Res 2011;217:354–62.

43] Yang SN, Huang LT, Wang CL, Chen WF, Yang CH, Lin SZ, et al. Prenatal adminis-tration of morphine decreases CREBSerine-133 phosphorylation and synapticplasticity range mediated by glutamatergic transmission in the hippocampalCA1 area of cognitive-deficient rat offspring. Hippocampus 2003;13:915–21.

[

in Research 271 (2014) 160–170

44] Nasiraei-Moghadam S, Sahraei H, Bahadoran H, Sadooghi M, Salimi SH, KakaGR, et al. Effects of maternal oral morphine consumption on neural tube devel-opment in Wistar rats. Dev Brain Res 2005;159:12–7.

45] Emeterio EPS, Tramullas M, Hurlé MA. Modulation of apoptosis in the mousebrain after morphine treatments and morphine withdrawal. J Neurosci Res2006;83:1352–61.

46] Nestler EJ. Under siege: the brain on opiates. Neuron 1996;16:897–900.47] García-Fuster MJ, Ramos-Miguel A, Rivero G, La Harpe R, Meana JJ, García-

Sevilla JA. Regulation of the extrinsic and intrinsic apoptotic pathways in theprefrontal cortex of short- and long-term human opiate abusers. Neuroscience2008;157:105–19.

48] Gómez-Pinilla F, Ying Z, Roy RR, Molteni R, Edgerton VR. Voluntary exerciseinduces a BDNF-mediated mechanism that promotes neuroplasticity. J Neuro-physiol 2002;88:2187–95.

49] Kim S-E, Ko I-G, Kim B-K, Shin M-S, Cho S, Kim C-J, et al. Treadmill exerciseprevents aging-induced failure of memory through an increase in neuro-genesis and suppression of apoptosis in rat hippocampus. Exp Gerontol2010;45:357–65.

50] Itoh T, Imano M, Nishida S, Tsubaki M, Hashimoto S, Ito A, et al. Exercise inhibitsneuronal apoptosis and improves cerebral function following rat traumatic

brain injury. J Neural Transm 2011;118:1263–72.

51] Chae C-H, Kim H-T. Forced, moderate-intensity treadmill exercise suppressesapoptosis by increasing the level of NGF and stimulating phosphatidylinositol3-kinase signaling in the hippocampus of induced aging rats. Neurochem Int2009;55:208–13.