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Behavioural Brain Research 237 (2013) 41– 48

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research

j ourna l ho mepage: www.elsev ier .com/ locate /bbr

esearch report

he differential effects of OX1R and OX2R selective antagonists on morphineonditioned place preference in naïve versus morphine-dependent mice

ohammad Tabaeizadeha,b, Rouzbeh Motiei-Langroudia,1, Hilda Mirbahaa,c,2, Behnaz Esmaeilid,ouya Tahsili-Fahadane, Mehrak Javadi-Paydara,c, Majid Ghaffarpourb, Ahmad Reza Dehpoura,f,∗

Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, IranIranian Center of Neurological Research, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, IranBrain and Spinal Cord Injury Repair Research Center, Tehran University of Medical Sciences, Tehran, IranDepartment of Neuroscience, University of Pittsburg, Pittsburg, PA, USADepartment of Neurology, Washington University School of Medicine, St. Louis, MO, USAExperimental Medicine Research Center, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

i g h l i g h t s

We tested the effect of SB 334867 and TCS-OX2-29 on morphine CPP.Both antagonists in naïve mice inhibited morphine CPP acquisition and expression.SB 334867 did not suppress CPP acquisition and expression in dependent mice.TCS-OX2-29 inhibited CPP acquisition and expression in dependent mice.

r t i c l e i n f o

rticle history:eceived 21 February 2012eceived in revised form 7 September 2012ccepted 11 September 2012vailable online 17 September 2012

eywords:rexinorphine conditioned place preference

B 334867

a b s t r a c t

Conditioned place preference (CPP) has been associated with orexinergic (hypocrtinergic) system activa-tion in naïve mice; however, the distinct role of different receptors of orexin in this paradigm has not beencharacterized yet. Moreover, the relationship between orexins and morphine in dependent mice maynot be equal to naïve mice and seems noteworthy to investigate. We investigated the effects of systemicadministration of orexin-1-receptor antagonist, SB 334867, and orexin-2 receptor antagonist, TCS-OX2-29 on the acquisition and expression of morphine conditioned place preference (CPP) in both naïve andmorphine-dependent mice. We tested SB 334867 in three doses (10, 20 and 30 mg/kg), TCS-OX2-29 in twodoses (5 and 10 mg/kg) and morphine with highest effective dose based on our dose–response experiment(5 mg/kg). Our results revealed that while SB 334867 suppressed CPP acquisition and expression in naïve

ependenceice

CS-OX2-29

mice, it failed to block CPP acquisition and expression in morphine dependent animals. In contrast, TCS-OX2-29 suppressed CPP acquisition and expression in both naïve and dependent mice significantly. Therewarding effect of morphine has stronger correlation with orexin-2 receptors in morphine-dependentmice while it depends on both kinds of receptors in naïve mice. This finding, if confirmed in other studies,persuades us to further investigate the role of orexin-2 receptor antagonists as potent drugs in addictiontreatment.

∗ Corresponding author at: Department of Pharmacology, School of Medicine,ehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran.el.: +98 21 66402569; fax: +98 21 66402569.

E-mail addresses: dehpoura@sina.tums.ac.ir, dehpour@yahoo.comA.R. Dehpour).

1 Now at: Department of Neurosurgery, Shahid Beheshti Medical University,ehran, Iran.2 Now at: Department of Neurology, Washington University School of Medicine,

t. Louis, MO, USA.

166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2012.09.010

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Orexins (hypocretins) are neuropeptides exclusively producedin hypothalamic neurons including dorsomedial hypothalamus(DMH), perifornical area (PFA), and lateral hypothalamus (LH)[1–4]. They constitute a peptide family with two ligand subgroups,orexin A and orexin B [5] affecting G protein-coupled orexin-1

(OX1R) and orexin-2 receptors (OX2R). Orexin A is selective fororexin 1 receptor whereas both orexin A and B act upon orexin 2receptor [4]. These receptors are differentially distributed through-out the brain [6].

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Early studies suggest a role of orexin in sleep regulation asysfunction of the orexin system is strongly associated witharcoleptic symptoms in animals [7,8]; however, further studieshowed the important role of orexin neurons in reward-relatedehaviors [4–9]. Administration of SB 334867 (OX1R antagonist)educed the acquisition and expression of morphine place prefer-nce conditioning [10,11], and orexin gene knockout mice lackedonditioned place preference (CPP) for morphine [11], with contro-ersial result in another study which demonstrated while orexinntagonist, SB 334867 attenuated conditioned place preference,rexin gene knockout mice still acquired CPP [12]. The effects ofndependent OX2R antagonists like TCS-OX2-29 or dual receptorntagonists like almorexant have not been evaluated in morphineonditioned place preference yet while distinct role of thesentagonists in other phenomenon like sleep–wakefulness cycleas been described, for example several lines of evidence supporthe pivotal role of OX2R in the regulation of sleep–wakefulness5,8,13]. An investigation comparing the selective OX2R antag-nist JNJ-10397049, selective OX1R antagonist SB-408124 andlmorexant as the dual receptors antagonist in rats demonstratedhat although both JNJ-10397049 and almorexant induced androlonged sleep time, the selective OX2 receptor antagonist had

10-fold higher potency than dual OX1/OX2 antagonist. On theontrary, SB-408124 had no effect on any tested sleep parameterndependently and even attenuated the sleep-promoting effect ofNJ-10394079 administered simultaneously [14].

Current data suggest the VTA as an essential site, which therexin acts on, for the acquisition and expression of morphine placereference in non-dependent mice [10,11].

In addition to effects of acutely administered drugs, chronicdministration of drugs also influences the orexin neural activa-ion and mRNA levels of orexin or its receptors. The effect of chronicdministration of drugs – either repeated equivalent doses or esca-ating doses – on addiction may differ from acute administration ofhem [15–17].

The role of orexinergic system in morphine-induced condition-ng while the subjects are already dependent to morphine has noteen investigated yet. Orexin might play different role in mor-hine dependent versus naïve mice due to novel neuronal pathwayevelopment in dependent animals and it can potentially changeehavioral responses, which is noteworthy to study. The major goalf the present study is to evaluate the effects of SB 334867 andCS-OX2-29 as the independent antagonists of two known orexineceptors administered in both naïve and morphine-dependentice. This can reveal possible distinct role of two receptors in

ewarding effects of morphine.

. Materials and methods

.1. Animals

We began the study with a total of 440 (including preliminary study) adult maleMRI mice (Pasteur institute of Iran, Tehran, Iran), weighing 25–30 (g). Animals wereoused 5–8 per cage in transplant Plexiglas cages in a temperature and humidityontrolled colony room under a 12/12 h light/dark cycle (lights on at 7:00 AM),ith ad libitum access to the food and water except during experiments. Subjectsere experimentally naïve and each animal was used only once. They were assigned

andomly to each treatment group consisting of 6–8 animals. Mice were allowed aeek to acclimatize to the laboratory environment before testing started and during

his period they were handled, weighed, and habituated to the drug administrationrocedure. All procedures were performed according to the institutional guidelinesor animal care and use. The protocol was approved by the Committee of Ethics ofhe Faculty of Sciences, University of Tehran.

.2. Drugs

The following drugs were used: morphine sulfate (Temad, Karaj, Iran),B 334867 (Tocris Bioscience, Ellisville, MO, USA) and TCS-OX2-29 (Tocris).orphine was prepared freshly in sterile 0.9% NaCl solution and injected sub-

utaneously (s.c) with maximized effective dose of 5 mg/kg derived from our

ain Research 237 (2013) 41– 48

preliminary dose–response study. SB 334867 (OX1R antagonist) was dissolved in10% (w/v) (2-hyrdoxypropyl)-B-cyclodexterin/10% dimethyl sulfoxide (DMSO) andwas administered intraperitoneally (i.p) at different doses of 10, 20 and 30 mg/kg.SB 334867 was injected 30 min prior to each conditioning session in experiments1 and 3, and 30 min prior to post-conditioning test session in experiments 2and 4; this time and doses have been reported to be effective in several differ-ent behavioral procedures both in mice and rats [10,18–23], in fact in rats thehighest plasma and brain levels of the drug have been achieved 30 min after injec-tion [23] but this finding cannot be necessarily attributed to the mice becauseof probable different pharmacokinetics of drugs in different species. The OX2Rantagonist, TCS-OX2-29 was dissolved in physiological saline (0.9% NaCl) andadministered intraperitoneally at doses of 5 and 10 mg/kg based on a previous study[24].

Dependency to morphine was achieved by consecutive escalating doses ofmorphine (30, 45, 60, 90, 120 and 120 mg/kg) injected every 12 h on 3 consec-utive days followed by CPP procedure [25]. The dependent mice also received adaily dose of morphine (10 mg/kg) to avoid withdrawal syndrome during con-ditioning and test sessions [26]. It was injected late in the evening (10 PM),while animals underwent conditioning sessions in the next day (10:00 AM–2:00PM) to make the interference of morphine maintenance dose with morphineconditioning (made by administration of 5 mg/kg morphine, just before the condi-tioning) less likely and to avoid the state-dependent retrieval on postconditioningday.

2.3. General behavioral procedure

The place preference apparatus was made of wood and consisted of twosquared-base compartments (15 cm × 15 cm × 30 cm H each). To distinguish twocompartments, visual and sensory texture cues were used: the inner surface of onecompartment was painted in black with a smooth floor; the other side was whitewith a textured floor to create equally preferred compartments. Place condition-ing was conducted using an unbiased procedure. In this design, animals did notshow a significant preference toward any compartment in the pre-conditioningtest and drug administration was randomly paired with either of the compart-ments [27]. The CPP paradigm took place in nine consecutive days, which consistedof three phases: familiarization and pre-conditioning, conditioning and post-conditioning. All trials were done between 10:00 AM and 2:00 PM. On the first(i.e. familiarization) and second (i.e. pre-conditioning) days, each mouse was placedseparately into the apparatus for 10 min with free access to both compartments.The time spent in each compartment was recorded on the pre-conditioning dayto determine any individual innate preference for either of the two compart-ments. Placement in each compartment was assigned as placement of the frontpaws and head. Animals showing strong unconditioned aversion for any of thecompartments (time spent in either of the two compartments > mean ± 2SD) wereexcluded from the experiment (totally 31 animals). This familiarization phasewas performed in all experiments including experiments 3 and 4 with dependentmice.

Conditioning phase consisted of six 40-min conditioning sessions held on sixconsecutive days. Every other day, each animal was confined to one of the differ-ently textured compartments by isolating removable sheet. The mice received thedrugs on days 1, 3 and 5 and the vehicle on days 2, 4 and 6 of the conditioningphase according to the experimental design. Treatment compartments were coun-terbalanced for all groups. The locomotor activity of each animal was measured afterreceiving the drugs or vehicles on conditioning sessions or on the post condition-ing test session by dividing the ground of each compartment into four equal-sizedsquares (7.5 cm × 7.5 cm), and recording the number of mice entrances to every oneof them by an unbiased observer as the locomotor activity index [27]. This index wasused as the physical indicator of morphine sensitization in our study based on previ-ous studies [28–30]. For each animal, conditioning locomotor activity was recordedduring each conditioning session and the mean of all amounts was calculated andapplied for comparison tests.

Post-conditioning phase was carried out on the ninth day of the trial (24 h afterthe last conditioning session) in a drug-free state except for the daily treatment ofmorphine in dependent mice, which they received 12 h before the post-conditioningtest, on the afternoon of the previous day. As in the pre-conditioning phase thepartitions were raised and the animals were placed in the apparatus for 10 minwith free access to both compartments. An observer who was unaware of mice andtreatments recorded the time spent in the compartments. Change in preference(CIP) was calculated as the times spent in the drug-paired compartment on thepost-conditioning day minus the time spent in the same compartment in the pre-conditioning day.

2.4. Experimental design

2.4.1. Preliminary dose–response studyWe tested different doses of morphine (0, 2, 5, 10 and 20 mg/kg) to achieve the

maximum conditioned place preference produced by this drug in groups of ani-mals either in naïve or dependent states. They were also NMRI mice from the sameinstitute with each group contains 6–8 mice.

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.4.2. Experiment 1: effects of SB 334867 and TCS-OX2-29 on acquisition of placereference per se and induced by morphine in naive mice

In this experiment 3 groups of animals received SB 334867 in 3 differentoses including 10, 20 and 30 mg/kg, 2 groups received TCS-OX2-29 and one groupeceived saline, all of them 30 min prior to the administration of morphine. On thether hand, equal control groups of animals received saline in turn of morphine tonvestigate the pure effect of SB 334867 or TCS-OX2-29 on place preference.

.4.3. Experiment 2: effects of SB 334867 and TCS-OX2-29 on expression of placereference per se and induced by morphine in naive mice

This experiment was carried out to see the effect of orexin antagonists onhe expression of morphine-conditioned preference acquired formerly by mice.he animals underwent CPP paradigm as described above, except that theyeceived SB 334867, TCS-OX2-29 or vehicle 30 min prior to the post-conditioningest.

.4.4. Experiment 3: effects of SB 334867 and TCS-OX2-29 on acquisition of placereference per se and induced by morphine in dependent mice

In this experiment, animals first underwent a protocol to become depend-nt to morphine (as described above) before the familiarization phase of the CPP.ependent mice were also treated with a daily dose of 10 mg/kg on familiarization,onditioning and test days to avoid withdrawal [26]. The signs of withdrawal includ-ng jumping, wet dog shakes, diarrhea, ptosis, body tremor, piloerection and weightoss were recorded for each animal every day, together with recording locomotorctivation during both conditioning and test sessions and the animals with with-rawal signs were excluded from the study in a total count of 34 animals. All otherteps were similar to experiment 1.

.4.5. Experiment 4: effects of SB 334867 and TCS-OX2-29 on expression of placereference per se and induced by morphine in dependent mice

In this experiment, animals first underwent a protocol to become dependent toorphine before the familiarization phase of the CPP and then were treated dailyith morphine (as described above). All other steps were similar to experiment 2.

.5. Data analysis

All results are presented as mean ± SEM. The effect of each OXR antagonistsn the change in preference (CIP) of morphine treated animals or vehicle treatednimals were assessed by two-way analysis of variance (ANOVA), separately. One-ay ANOVA was also applied to assess the independent effects of morphine or

ntagonists on CIP and locomotor activity in each experiment. Post hoc analysisTukey–Kramer) was performed to determine the effects of various doses. P valuesess than 0.05 were considered as significant.

. Results

.1. Preliminary dose–response study

A significant dose-dependent effect of morphine on condi-ioning was found (one-way ANOVA, F4,42 = 5.953, P < 0.001). Postoc analyses revealed that the doses of 2, 5 and 10 mg/kg

nduced significant place preference (P < 0.01, P < 0.001, P < 0.01espectively) compared to saline treatment. The maximumesponse was observed with 5 mg/kg of morphine. The largerose, 20 mg/kg of morphine failed to produce CPP (P > 0.05)Fig. 1).

.2. Effect of SB 334867 or TCS-OX2-29 on acquisition oforphine CPP in naïve mice

A significant interaction between morphine treatment andX1R antagonist pretreatment on the acquisition of morphinePP was indicated applying two-way ANOVA (factor morphine1,16 = 27.340, P < 0.001; factor SB 334867 F3,25 = 0.843, P > 0.05; fac-or morphine × SB 334867 F3,50 = 3.349, P < 0.05) in naïve mice.urther analysis with Tukey–Kramer’s multiple comparison testsn the first experimental group indicated that morphine condi-ioned animals which were not pretreated with SB 334867 showed

significant preference to the drug-paired chamber in compari-

on to all SB 334867 pretreated groups (P < 0.05, P < 0.001, P < 0.05or 10 mg/kg, 20 mg/kg and 30 mg/kg respectively) and salineonditioned animals (P < 0.01). Post hoc analysis also revealedhat animals receiving SB 334867, which was not followed

ain Research 237 (2013) 41– 48 43

by morphine, did not show any significant preference towardeach of the compartments in comparison to saline treated mice(P > 0.05 for 10 mg/kg, 20 mg/kg and 30 mg/kg of OX1R antagonist)(Fig. 2.1).

Animals, which received morphine before conditioning sessionsincluding those which were pretreated by SB 334867 showed sig-nificant, enhanced locomotor activity compared to saline treatedmice (one-way ANOVA; F4,32 = 10.659, P < 0.001); however, none ofdoses of SB 334867 suppressed this enhanced locomotion signif-icantly when they were administered before morphine (post hocanalysis indicated P > 0.05 for 10, 20 and 30 mg/kg).

A significant interaction between morphine treatment andOX2R antagonist pretreatment on the acquisition of morphineCPP was indicated applying two-way ANOVA (factor morphineF1,16 = 27.340, P < 0.001; factor TCS-OX2-29 F2,24 = 0.895, P > 0.05;factor morphine × SB 334867 F2,48 = 8.312, P < 0.001) in naïve mice.Further analysis with Tukey–Kramer’s multiple comparison tests inthe first experimental group indicated that morphine conditionedanimals which were not pretreated with TCS-OX2-29 showed asignificant preference to the drug-paired chamber in compari-son to all TCS-OX2-29 pretreated groups (P < 0.001 for both 5 and10 mg/kg) and saline conditioned animals (P < 0.001). Post hoc anal-ysis also revealed that animals receiving TCS-OX2-29, which wasnot followed by morphine, did not show any significant preferencetoward each of the compartments in comparison to saline condi-tioned mice (P > 0.05 for 5 mg/kg and 10 mg/kg of OX2R antagonist)(Fig. 2.2).

Animals, which received morphine before conditioning sessionsincluding those pretreated by TCS-OX2-29 showed significant,enhanced locomotor activity compared to saline treated mice (one-way ANOVA; F3,32 = 25.360, P < 0.001); however, none of doses ofTCS-OX2-29 suppressed this enhanced locomotion significantlywhen they were administered before morphine, compared to ani-mals which were treated only with morphine (post hoc analysisindicated P > 0.05 for 5 and 10 mg/kg).

Morphine-conditioned animals showed enhanced locomotionduring the post-conditioning test compared to SB 334867 pre-treated group (one-way ANOVA F3,24 = 5.641, P < 0.01, post hocanalysis P < 0.05 for 10 mg/kg, P < 0.05 for 20 mg/kg and P < 0.032for 30 mg/kg), TCS-OX2-29 pretreated group (one-way ANOVAF2,24 = 7.041, P < 0.01, post hoc analysis P < 0.05, P < 0.01 respec-tively for 5 mg/kg and 10 mg/kg) and saline control group (one-wayANOVA, F1,16 = 11.029, P < 0.01).

3.3. Effect of SB 334867 or TCS-OX2-29 on the expression ofmorphine CPP in naïve mice

A significant interaction between morphine treatment andOX1R antagonist administration, received on the post-conditioningtest date, on the expression of morphine CPP was indicatedby two-way ANOVA (factor morphine, F1,16 = 17.745, P < 0.001;factor SB 334867, F3,25 = 0.497, P > 0.05; factor morphine × SB334867, F3,52 = 2.910, P < 0.05) in naïve mice. Further analysis withTukey–Kramer’s multiple comparison tests in the second experi-mental group indicated that morphine conditioned animals whichdid not receive SB 334867 on the post-conditioning test day showeda significant preference to the drug-paired chamber compared to allSB 334867 treated groups (P < 0.05, P < 0.001, P < 0.05 for 10 mg/kg,20 mg/kg and 30 mg/kg, respectively) and saline treated animals(P < 0.01) (Fig. 3.1).

A significant interaction between morphine treatment andOX2R antagonist administration, received on the post-conditioning

test date, on the expression of morphine CPP was indicatedby two-way ANOVA (factor morphine, F1,16 = 17.745, P < 0.001;factor TCS-OX2-29, F2,23 = 0.174, P > 0.05; factor morphine × TCS-OX2-29, F2,48 = 7.596, P < 0.001) in naïve mice. Further analysis

44 M. Tabaeizadeh et al. / Behavioural Brain Research 237 (2013) 41– 48

Fig. 1. The dose–response experiment to find the highest effective dose of morphine in producing CPP. Different doses (0, 2, 5, 10 and 20 mg/kg) were tested. All data areshown as mean ± SEM of change in preference (CIP). Significant differences are illustrated as * P < 0.05 and ** P < 0.001.

Fig. 2. The interaction of morphine and orexin antagonists on acquisition of CPP in naïve mice. (1) SB 334867 (OX1R antagonist) different doses (10, 20 and 30 mg/kg) orvehicle (saline) were administered 30 min prior to morphine (5 mg/kg) or vehicle (saline) before each session of conditioning. SB 334867 pretreated morphine-conditionedanimals are compared to vehicle pretreated morphine-conditioned animals, whereas SB 334867 pretreated animals not conditioned with morphine are compared to saline-treated group. (2) TCS-OX2-29 (OX2R antagonist) different doses (5 and 10 mg/kg) or vehicles (saline) were administered 30 min prior to morphine (5 mg/kg) or vehicle( nditiow ared t(

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saline) before each session of conditioning. TCS-OX2-29 pretreated morphine-cohereas TCS-OX2-29 pretreated animals not conditioned with morphine are comp

CIP). Significant differences are illustrated as *P < 0.05 and **P < 0.001.

ith Tukey–Kramer’s multiple comparison tests in the secondxperimental group indicated that morphine conditioned animalshich did not receive TCS-OX2-29 on the post-conditioning test

ay showed a significant preference to the drug-paired chamberompared to all TCS-OX2-29 treated groups (P < 0.001, P < 0.01 for

and 10 mg/kg respectively) and saline treated animals (P < 0.001)Fig. 3.2).

ig. 3. The interaction of morphine and orexin antagonists on expression of CPP in naïvehicle (saline) were administered 30 min prior to post-conditioning test. The data are sonditioned animals are compared to morphine-conditioned vehicle treated animals, wo saline-treated group. (2) TCS-OX2-29 (OX2R antagonist) different doses (5 and 10 mgCS-OX2-29 treated morphine-conditioned animals are compared to vehicle treated morith morphine are compared to saline-treated group. All data are shown as mean ± SEM

*P < 0.001.

ned animals are compared to vehicle pretreated morphine-conditioned animals,o saline-treated group. All data are shown as mean ± SEM of change in preference

Considering all SB 334867 treated groups and their controls,animals, which received morphine before conditioning, showedenhanced locomotor activity during conditioning sessions (one-

way ANOVA, F1,52 = 34.156, P < 0.001).

Morphine-conditioned animals showed enhanced locomotionin the post-conditioning test compared to SB 334867 treated group(one-way ANOVA F3,26 = 4.746, P < 0.010, post hoc analysis P < 0.041

e mice. (1) SB 334867 (OX1R antagonist) different doses (10, 20 and 30 mg/kg) orhown as mean ± SEM of change in preference (CIP). SB 334867 treated morphine-hereas SB 334867 treated animals not conditioned with morphine are compared/kg) or vehicles (saline) were administered 30 min prior to post-conditioning test.phine-conditioned animals; whereas TCS-OX2-29 treated animals not conditionedof change in preference (CIP). Significant differences are illustrated as *P < 0.05 and

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or 10 and 30 mg/kg and P < 0.05 for 20 mg/kg), TCS-OX2-29 treatedroup (one-way ANOVA F2,24 = 6.108, P < 0.01, post hoc analysis

< 0.05 for 5 mg/kg and P < 0.05 for 10 mg/kg) and saline controlroup (one-way ANOVA, F1,16 = 7.039, P < 0.05).

Considering all TCS-OX2-29 treated groups and their controls,nimals, which received morphine before conditioning, showednhanced locomotor activity during conditioning sessions (one-ay ANOVA, F1,48 = 77.209, P < 0.001).

.4. Effect of SB 334867 on the acquisition of morphine CPP inependent mice

There was no significant interaction between morphine treat-ent and OX1R antagonist pretreatment on the acquisition oforphine CPP, indicated by two-way ANOVA (factor morphine,

1,11 = 30.459, P < 0.001; factor SB 334867, F3,24 = 0.332, P > 0.05,actor morphine × SB 334867, F3,49 = 0.136, P > 0.05) in morphineependent mice. Further analysis with Tukey–Kramer’s multi-le comparison tests in the third experimental group indicatedhat morphine conditioned animals which were not pretreatedith SB 334867 did not show a significant difference in theirreference to the drug-paired chamber in comparison to allB 334867 pretreated groups (P > 0.05 in 10 mg/kg, 20 mg/kgnd 30 mg/kg), however all of morphine-conditioned animalsncluding those pretreated with SB 334867 displayed significantreference to the drug-paired compartment compared to salineonditioned animals (P < 0.01). Post hoc analysis also revealedhat animals receiving SB 334867, which was not followedy morphine, did not show any significant preference towardach of the compartments in comparison to saline-treated miceP > 0.05 for 10 mg/kg, 20 mg/kg and 30 mg/kg of SB 334867)Fig. 4.1).

Locomotor activity was enhanced during conditioning sessionsn groups of animals, which received morphine compared to salinereated group (one-way ANOVA, F4,30 = 14.114, P < 0.001) but SB34867 did not suppress this enhancement when administeredefore morphine. Post hoc analysis with Tukey test indicated

> 0.05 for 10, 20 and 30 mg/kg of SB 334867.A significant interaction between morphine treatment and

X2R antagonist pretreatment on the acquisition of morphinePP was indicated applying two-way ANOVA (factor morphine1,11 = 30.459, P < 0.001; factor SB 334867 F2,21 = 0.040, P > 0.05; fac-or morphine × SB 334867 F2,43 = 17.008, P < 0.001) in dependent

ice. Further analysis with Tukey–Kramer’s multiple comparisonests in the third experimental group indicated that mor-hine conditioned animals which were not pretreated withCS-OX2-29 showed a significant preference to the drug-pairedhamber in comparison to all SB 334867 pretreated groupsP < 0.001 for both 5 and 10 mg/kg) and saline treated con-rol animals (P < 0.001). Post hoc analysis also revealed thatnimals receiving TCS-OX2-29, which was not followed by mor-hine, did not show any significant preference toward eachf the compartments in comparison to saline treated controlice (P > 0.05 for 5 mg/kg and 10 mg/kg of OX2R antagonist)

Fig. 4.2).Animals, which received morphine, showed enhanced loco-

otor activity in comparison to saline treated animals (one-wayNOVA, F3,27 = 25.660, P < 0.001), however TCS-OX2-29 did not sup-ress this enhancement when administered prior to morphinepost hoc analysis indicated P > 0.05 for 5 mg/kg and 10 mg/kg ofCS-OX2-29).

Morphine-conditioned animals did not show significant differ-

nt lomomotor activity in the post-conditioning test comparedo SB 334867 pretreated group (one-way ANOVA F3,24 = 0.625,

> 0.05, post hoc analysis P > 0.05 for 10 mg/kg, 20 mg/kgnd 30 mg/kg), however they displayed enhanced locomotion

ain Research 237 (2013) 41– 48 45

compared to TCS-OX2-29 pretreated group (one-way ANOVAF2,21 = 5.563, P < 0.05, post hoc analysis P < 0.05 for 5 mg/kg andP < 0.019 for 10 mg/kg) and saline control group (one-way ANOVA,F1,11 = 10.938, P < 0.01).

3.5. Effect of SB 334867 on the expression of morphine CPP independent mice

There was no significant interaction between morphinetreatment and OX1R antagonist treatment, received on the post-conditioning test date, on the expression of morphine CPP,indicated by two-way ANOVA (factor morphine, F1,12 = 48.255,P < 0.001; factor SB 334867, F3,24 = 0.377, P > 0.05; factor mor-phine × SB 334867, F3,49 = 0.224, P > 0.05) in dependent mice.Further analysis with Tukey–Kramer’s multiple comparison testsin the fourth experimental group indicated that morphine con-ditioned animals which did not received SB 334867 on thepost-conditioning test day did not show a significant prefer-ence to the drug-paired chamber compared to all SB 334867treated groups (P > 0.05 for 10 mg/kg, 20 mg/kg and 30 mg/kg),however all morphine-conditioned animals including those receiv-ing SB 334867 at post conditioning test day displayed significantpreference toward the drug-paired chamber compared to salineconditioned animals (P < 0.001) (Fig. 5.1).

A significant interaction between morphine treatment andOX2R antagonist administration, received on the post-conditioningtest date, on the expression of morphine CPP was indicated bytwo-way ANOVA (factor morphine, F1,12 = 48.255, P < 0.001; factorTCS-OX2-29, F2,21 = 1.725, P > 0.05; factor morphine × TCS-OX2-29,F3,52 = 15.299, P < 0.001) in dependent mice. Further analysis withTukey–Kramer’s multiple comparison tests in the fourth experi-mental group indicated that morphine conditioned animals whichdid not receive TCS-OX2-29 on the post-conditioning test dayshowed a significant preference to the drug-paired chamber com-pared to all TCS-OX2-29 treated groups (P < 0.001 for both 5 and10 mg/kg) and saline treated animals (P < 0.001) (Fig. 5.2).

Considering all SB 334867 treated groups and their controls,animals, which received morphine before conditioning, showedenhanced locomotor activity during conditioning sessions (one-way ANOVA, F1,49 = 95.935, P < 0.001).

Morphine-conditioned animals did not show significant differ-ent lomomotor activity in the post-conditioning test compared toSB 334867 pretreated group (one-way ANOVA F3,24 = 0.511, P > 0.05,post hoc analysis P > 0.05 for 10 mg/kg, 20 mg/kg and 30 mg/kg),however they displayed enhanced locomotion compared to TCS-OX2-29 pretreated group (one-way ANOVA F2,21 = 5.568, P < 0.05,post hoc analysis P < 0.05 for 5 mg/kg and P < 0.05 for 10 mg/kg) andsaline control group (one-way ANOVA, F1,11 = 12.991, P < 0.01).

Considering all TCS-OX2-29 treated groups and their controls,Animals, which received morphine before conditioning, showedenhanced locomotor activity during conditioning sessions (one-way ANOVA, F1,43 = 56.836, P < 0.001).

4. Discussion

Orexin’s contribution to drug-related behaviors appears tobe drug specific and model-specific too [9,16,17,31–36]. Theseresponses may be due to different anatomical targets whichorexin selects or uneven expression of orexin receptor typesthroughout these sites [10–12,16,19,35], as well as interactionwith other neurotransmitters from distinct circuits and pathways

[10,12,15–17,37]. Moreover, it is possible that, chronic administra-tion of drugs would act differently [16,17].

We designed our study to investigate the effect of systemicadministration of selective orexin receptor (both OX1R and OX2R)

46 M. Tabaeizadeh et al. / Behavioural Brain Research 237 (2013) 41– 48

Fig. 4. The interaction of morphine and orexin antagonists on acquisition of CPP in dependent mice. (1) SB 334867 (OX1R antagonist) different doses (10, 20 and 30 mg/kg) orvehicle (saline) were administered 30 min prior to morphine (5 mg/kg) or vehicle (saline) before each session of conditioning. SB 334867 pretreated morphine-conditionedanimals are compared to vehicle pretreated morphine-conditioned animals, whereas SB 334867 pretreated animals not conditioned with morphine are compared to saline-treated group. (2) TCS-OX2-29 (OX2R antagonist) different doses (5 and 10 mg/kg) or vehicles (saline) were administered 30 min prior to morphine (5 mg/kg) or vehicle( nditiow ared t(

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laiaN

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saline) before each session of conditioning. TCS-OX2-29 pretreated morphine-cohereas TCS-OX2-29 pretreated animals not conditioned with morphine are comp

CIP). Significant differences are illustrated as *P < 0.05 and **P < 0.001.

ntagonists on morphine’s CPP acquisition and expression in naïvend morphine-dependent mice while selective OX1R antagonist,B 334867, had been demonstrated to block both acquisition andxpression in naïve mice in previous studies [10,11], the finding,hich also was confirmed in our study. It is reasonable to evaluate

cquisition and expression of CPP separately because it has beenhown previously that orexin may act on any of these stages differ-ntly, for example Aston-Jones et al. reported that administrationf 30 mg/kg SB 334867 prior to the Pavlovian conditioning sessionad no effect on subsequent cue-elicited reinstatement; nev-rtheless, it reduced the expression of conditioned-cue-elicitedrug-seeking when administered just prior to a subsequenteinstatement session [38].

We found that in contrary to naïve mice, the suppress-ng effect of OX1R antagonist, SB 334867, on acquisition andxpression of morphine CPP, was not seen in morphine depend-nt mice, however, OX2R antagonist, TCS-OX2-29, appeared tonhibit acquisition and expression of morphine CPP not only inaïve mice, but also in morphine-dependent mice. Previously,nother study demonstrated that selective blockade of OX2Rs byNJ-10397049 attenuated the acquisition, expression and reinstate-

ent of ethanol CPP while SB-408124, selective OX1R antagonist,id not have any of these effects [39].

It is possible that the effect of orexin on reward seeking andearning the association between reward and cue is mainly medi-

ted through OX2R in dependent mice whereas OX1R has beenmplicated to have the essential role in naïve mice. In fact, OX1Rnd OX2R have been reported to be expressed in both VTA andA but orexin’s actions in the NA have been attributed to OX2R

ig. 5. The interaction of morphine and orexin antagonists on expression of CPP in depenr vehicle (saline) were administered 30 min prior to post-conditioning test. SB 334867 tonditioned animals, whereas SB 334867 treated animals not conditioned with morphineoses (5 and 10 mg/kg) or vehicles (saline) were administered 30 min prior to post-condehicle treated morphine-conditioned animals; whereas TCS-OX2-29 treated animals nhown as mean ± SEM of change in preference (CIP). Significant differences are illustrated

ned animals are compared to vehicle pretreated morphine-conditioned animals,o saline-treated group. All data are shown as mean ± SEM of change in preference

binding because of the very low levels of OX1R in this area [6,37];therefore a suggestion might be that direct action of Orexin at theNA via projections from LH is the dominant circuit in turn of indi-rect pathway modulated by VTA. Previous studies indicated thatchronic cocaine administration increased OX2R levels in NA in along-lasting manner and naloxane-precipitated morphine with-drawal is accompanied by an increase in c-Fos expression in theNA shell [16–19]. In addition, although we did not find the sameevidence about morphine, it has been reported that chronic admin-istration of cocaine up-regulates OX2R – but not OX1R – levels inNA. The up-regulation has not occurred in frontal cortex, VTA andthe dorsal striatum. This effect in the NA persisted even monthsafter the cessation of the cocaine treatment [16]. A limitation ofour study is that we could not take this hypothesis as a directconclusion of the results and it may require to be examined bylocal injection of both orexin receptors specific antagonists, intodifferent sites of the circuit including NA. It should also be con-sidered that the role of OX2R is not necessarily limited to NA; onestudy demonstrated that dual receptor antagonist, almorexant, inconcentrations, which are exclusively efficient on OX2R can slowthe firing rate of VTA dopaminergic neurons effective in reward-related behaviors, and suggested this effect, which is more potentthan the influence of OX1R antagonists, as an evidence of greaterrole of OX2R in response to orexin A in this area. The same effectwas also observed by the administration of EMPA, a selective OX2R

antagonist [40].

CPP in morphine dependent mice may include a stress compo-nent which increases Fos activation in DMH and PFA rather than LHand VTA [15] and is therefore less sensitive to antagonistic effect

dent mice. (1) SB 334867 (OX1R antagonist) different doses (10, 20 and 30 mg/kg)reated morphine-conditioned animals are compared to vehicle-treated morphine-

are compared to saline-treated group. (2) TCS-OX2-29 (OX2R antagonist) differentitioning test. TCS-OX2-29 treated morphine-conditioned animals are compared toot conditioned with morphine are compared to saline-treated group. All data are

as *P < 0.05 and **P < 0.001. cm.

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f SB 334867 in comparison to naïve mice, however, we treatedhe dependent mice with a daily dose of morphine on the days ofonditioning and expression to remove the confounding effect oforphine withdrawal and it could neutralize that stress compo-

ent of CPP in the dependent mice too. Animals, which appearedhysical withdrawal signs, were also excluded from the exper-

ments. This daily maintenance dose of morphine was injectedate in the evening after conditioning or test sessions to avoid thenterference with morphine conditioning. This timeline was alsoelected to prevent the generating of state-dependent memory onhe test day. We also observed the physical activity of animalsuring conditioning and test sessions. Morphine-treated animalsisplayed enhanced locomotion after receiving morphine duringonditioning sessions. SB 334867 or TCS-OX2-29 did not suppresshis effect of morphine, which might be a clue that suppress-ng effect of receptor antagonists is limited to attention requiredor conditioning or other stages of learning process; otherwisehysical aspects of drug sensitization may be mediated by otherechanisms. Conditioned animal also revealed enhanced activity

uring test session associated with their preference toward therug-paired chamber. Failure of antagonist to inhibit physical sen-itization is consistent with Sharf et al. study, which demonstratedhat SB 334867 blocked morphine CPP and somatic withdrawalymptoms but did not suppress morphine-induced hyperactivity12]. Their finding like our data is in contrast to previous reports,hich suggest a role for orexin in morphine locomotor enhance-ent [11], Sharf et al. proposed that orexin may contribute to

piate-induced neural plasticity, which becomes evident followingessation of drug-treatment, such as during withdrawal or drug-eeking. They hypothesized that orexin function may not directlyffect morphine-induced locomotor responses, but once morphines no longer present, morphine withdrawal and place preferenceepend on orexin action [12]. It is also interesting that OX2Rntagonist did not interfere with enhancement of locomotor activ-ty because it may be expected to decrease operant behavior bynducing sleep. Shoblock et al. that used another OX2R antagonist,NJ-10397049, and found the inhibiting effect of OX2R antagonistn alcohol-induced CPP also observed that OX2R antagonist didot decrease lever presses by animals for saccharin, or did not

mpair their motor performance on the rotarad. They suggestedhat doses of OX2R antagonists that are effective at promotingleep do not prevent animals from completing challenging whenotivated to do so [39]. The same explanation may be true aboutorphine induced locomotor enhancement although it is not a

eward-seeking action once after injection of morphine.Another possibility, which cannot be derived directly from this

tudy but can be hypothesized to justify the finding, is that neuro-lasticity and transient or consistent changes in neurocircuitriesnd neurotransmitters, which underlie the pathophysiology ofddiction, enhance animals’ susceptibility to rewarding effects ofrugs and learning process. Learning to become addicted critically

nvolves dopamine cells in VTA that release dopamine into the pre-rontal cortex (PFC), amygdalia, and NA. Animal models supporthat during repeated administration of drug, which make the drug-eeking behaviors well learned, glutamatergic projections from PFCo the NA emerges [41–46]. Concisely, chronic administration ofrugs may not only change the main sites for learning drug-seekingehavior, but also expose these sites and the primary sites likeTA to react more easily to lower levels of neurotransmitters likerexins. For example, there is a tonic release of orexin that maylay role in baseline impulse activity of dopamine neurons, whichre involved in brain mechanisms of reward, reinforcement, and

motion [23]. This tonic release, which gets rid of the influence ofB 334867 in the doses applied in our study [23], may suffice tocquire CPP in morphine-dependent animals even in the presencef OX1R antagonist, however TCS-OX2-29 as an OX2R antagonist

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may inhibit this orexin release. This is consistent with the findingof Malherbe et al., which demonstrated the more potent effect ofOX2R antagonists on slowing the firing rate of dopaminergic neu-rons [40]. The current doses which we used in our study (10, 20and 30 mg/kg of SB 334867) all failed to block the development orexpression of CPP in dependent mice. Further studies associatedwith local injection of different doses of both antagonists and mea-suring the level of drugs and orexin in the particular site can beuseful. Lacking molecular survey was a limitation of our study.

Altogether, our results show that systemic administration ofOX1R antagonist attenuates acquisition and expression of mor-phine CPP only in naïve animals but OX2R antagonists has apreventive effect not only in naïve mice, but also in morphine-dependent mice. It would be important because any manipulationof the orexin (hypocretin) system intending to treat or controlthe addiction, is more applicable in substance-dependent subjectsand this finding, if confirmed in other experiments, proposes clin-ical and practical benefits of OX2R antagonists and encourages usto evaluate their effects on addicted individuals. This is specifi-cally reasonable because sleep-promoting effects of these drugs canpotentiate their activity against rewarding effects of opioids, mak-ing them more favorable medications to treat addicted persons.

5. Conclusions

In this study, we have shown that systemic administration ofan OX1R antagonist, SB 334867, suppressed CPP acquisition andexpression in naïve mice; however, it failed to block CPP acquisi-tion and expression in morphine dependent animals. In contrast,selective OX2R antagonist, TCS-OX2-29 inhibited CPP acquisitionand expression not only in naïve mice but also in morphine depend-ent mice. It is suggested that the effect of orexin on reward seekingand learning the association between reward and cue, might bemainly mediated through OX2R in dependent mice; whereas bothreceptors could be implicated to manifest the essential role in naïvemice.

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