Role of orexinergic receptors in the dentate gyrus of the hippocampus in the acquisition and expression of morphine-induced conditioned place preference in rats

Journal Pre-proof Role of orexinergic receptors in the dentate gyrus of the hippocampus in the acquisition and expression of morphine-induced conditioned place preference in rats Mohammadreza Shirazy, Kimia RayatSanati, Shole Jamali, Abbas Haghparast

PII:

S0166-4328(19)30997-0

DOI:

https://doi.org/10.1016/j.bbr.2019.112349

Reference:

BBR 112349

To appear in:

Behavioural Brain Research

Received Date:

27 June 2019

Revised Date:

6 November 2019

Accepted Date:

7 November 2019

Please cite this article as: Shirazy M, RayatSanati K, Jamali S, Haghparast A, Role of orexinergic receptors in the dentate gyrus of the hippocampus in the acquisition and expression of morphine-induced conditioned place preference in rats, Behavioural Brain Research (2019), doi: https://doi.org/10.1016/j.bbr.2019.112349

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Role of orexinergic receptors in the dentate gyrus of the hippocampus in the acquisition and expression of morphine-induced conditioned place preference in rats

Mohammadreza Shirazy a, Kimia RayatSanati a,+, Shole Jamali b, Abbas

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Haghparast b,*

Student Research Committee, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Neuroscience Research Center, School of Medicine, Shahid Beheshti University of

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b

Contributed equally as co-first author

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Medical Sciences, Tehran, Iran

* Correspondence should be sent to: Abbas Haghparast, PhD

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Neuroscience Research Center, School of Medicine Shahid Beheshti University of Medical Sciences P.O. Box: 19615-1178, Tehran, Iran Tel & Fax: +98-21-2243-1624

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Email: [email protected]; [email protected] (A. Haghparast)

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Highlights

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The blockade of OX1 receptor in the DG decreased both acquisition and expression of morphine-induced CP.

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High dose of intra-DG OX2R antagonist decreased the acquisition of morphine-

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induced CPP in the rat.

Intra-DG OX2R antagonist dose-dependently reduced the expression of morphineinduced CPP.

The contribution of OX2 receptor within the DG in the expression phase was more

Abstract

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Orexinergic projections derived from the lateral hypothalamus (LH) play a crucial role in the acquisition and expression of morphine-conditioned place preference (CPP). It has been demonstrated in previous that orexinergic receptors are expressed in the dentate gyrus (DG) region of the hippocampus, which receives projections of LH

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orexinergic neurons. This study examined the effects of intra-DG orexin-1 (OX1) and orexin-2 (OX2) receptor antagonists on the acquisition and expression of CPP induced by morphine. Two separate cannulas were inserted bilaterally into the DG, and a CPP paradigm was performed. The CPP scores and locomotor activities were recorded using Ethovision software. The results showed that intra-DG microinjection of SB334867 as a selective OX1R antagonist (0.5, 2.5, 12.5 nM/0.5 µl DMSO) or TCSOX229 as a

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selective OX2R antagonist (0.5, 2.5, 12.5 nM/0.5 µl DMSO) before a morphine subcutaneous injection (5 mg/kg) during a three-day conditioning phase dosedependently represses the acquisition of morphine-induced CPP in rats. Furthermore, these antagonists reduced the CPP scores in the expression phase. Consequently, it was established that orexinergic receptors in the DG are involved in the acquisition and

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expression of morphine-induced CPP.

Keywords: Reward; Orexinergic receptors; Dentate gyrus; Morphine; Conditioned

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place preference; Rat

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1. Introduction

Pleasure has been nominated “evolution’s boldest trick” (Berridge et al., 2015), with reward described as that which is given in return for good behavior. Reward is associated with learning, considering that its performance is a positive stimulator and that it can also evoke approach and consummatory behavior (Haghparast et al., 2017).

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Repeated exposure to reward cues such as drugs can cause drug addiction (Berridge et al., 2015). Over the last few decades, numerous neuroimaging studies have indicated

that various reward cues activate an overlapping brain system known as the “common

currency” reward network due to interactions between several brain structures (Berridge

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et al., 2015), which include the ventral tegmental area (VTA), hippocampus (HIP),

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nucleus Accumbens (NAc), hypothalamus, and prefrontal cortex (PFC) (Harris and Aston-Jones 2006; Koob and Volkow 2010). It has been established that there is a

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strong connection between learning, memory, and reward processing (Borgland et al., 2006; Hyman et al., 2006; Kelley, 2004; Xu et al., 2001). Furthermore, it has been

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determined that the hippocampus plays a crucial role not only in learning and memory but also in the acquisition and expression of reward-related learning pertaining to drug abuse and addiction (Farr et al., 2000; Nestler, 2001; Yang et al., 2013). Further, the hippocampus is described as the region that associates the rewarding effects of opioids

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with the contextual indicators present during exposure to the drug (Black et al., 2004; Corrigall and Linseman, 1988; Ferbinteanu and McDonald, 2001). Two critical hypothalamic neuropeptides: orexin-A and orexin-B (also known as

hypocretin-1 and hypocretin-2) were noticed for the first time in 1998 (De Lecea et al., 1998; Sakurai et al., 1998). Two distinct G-protein-coupled receptors were reported to

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mediate the actions of the orexin-1 receptor (OX1R) and the orexin-2 receptor (OX2R). Both OX1 and OX2 are Gq-coupled receptors, while OX2 is coupled to Gi/Go. It is significant that both receptors show equal affinity for orexin-A, while orexin-B binds predominantly to the OX2 receptor (De Lecea et al., 1998; Sakurai et al., 1998). Orexinergic neurons emanate exclusively from the LH, prefrontal area (PFA), and dorsomedial hypothalamic nuclei (DMH) areas) and project extensively in the central

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nervous system. The axons of orexinergic neurons are found in different regions of the brain, including the locus coeruleus, VTA, nucleus accumbens, HIP, and the midline thalamic nuclei (Mondal et al., 1999; Sadeghi et al., 2013; Yang et al., 2013). This

extensive orexinergic system is known to modulate plenty of complex physiological

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functions such as sleep, feeding, pain, stress, arousal, and memory-related processes.

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There is significant evidence indicating that the orexinergic system is involved in reward processing and addiction (Harris and Aston-Jones, 2006; Safari et al., 2009;

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Taslimi et al., 2012). Studies confirm that the hippocampal regions are heterogeneous, considering their memory-related activities. Different regions of the HIP, such as CA1,

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CA2, CA3, and DG, have exhibited expression of both OX1 and OX2 receptors (Lu et al., 2000; Marcus et al., 2001). Dentate granule cells are assumed to be the gatekeepers of the HIP (Hamilton et al., 2010). Evidence shows that the DG is a hippocampal region that is significantly involved in memory and learning processes (Kim et al., 1995;

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Logue et al., 1997; Hernández-Ra¬baza et al., 2007). Furthermore, the DG region itself has terminals of both orexin receptors that originate from LH-projected orexinergic neurons (Wayner et al., 2004). Conditioned place preference (CPP) paradigm is an instance of contextdependent reward and is widely used in reward-based studies on animal models

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(Hernandez-Rabaza et al., 2008; Liu et al., 2010). Additionally, CPP is the proper paradigm for studying the effects of morphine as a rewarding drug and is widely used to evaluate reward-related learning in rats (Corrigall & Linseman, 1988). The hippocampus plays a crucial part in reward-related learning functions, such as the CPP paradigm (Haghparast et al., 2013; Rashidy-Pour et al., 2014; Riahi et al., 2013). Furthermore, it is involved in the development of CPP in response to opioids, including

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morphine and heroin (Liu et al., 2010; Vetulani, 2001). Recent studies report that blockage of OX1R and OX2R within the DG region reduces the acquisition and

expression of LH stimulation-induced place preference in rats (Parsania et al., 2016). Thus, to extend our knowledge about the role of orexin receptors within the dentate

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gyrus, this study surveyed the effects of intra-DG microinjection of SB334867 (a

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selective OX1R antagonist) and TCSOX229 (a selective OX2R antagonist) on the

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2. Material and Methods

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acquisition and expression of CPP induced by morphine in rats.

2.1. Animals

Adult male Wistar rats (Pasteur Institute, Tehran, Iran), weighting 220–280g were used in these experiments. The animals were randomly housed in groups of three

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per cage in a controlled temperature and relative humidity condition (temperature 21±2°C; Humidity 55%–60%) with access to chow and tap water, and with a 12/12 h light/dark cycle. All experiments were executed according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised

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1996) and were approved by the Research and Ethics Committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.PHNS.REC.1397.023), Tehran, Iran.

2.2. Stereotaxic surgery Animals were intraperitoneally anesthetized with injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) to implant bilateral guide cannulas targeting the DG

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region of the hippocampus. The scalp of the rat was gathered and the area surrounding the bregma was cleaned. Cannulas were bilaterally placed into 1mm above the

predetermined site of the injection according to the atlas of the rat brain (Paxinos and Watson, 2007). Stereotaxic coordinates for the DG were AP = 3.7 mm caudal to

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bregma, Lat = ±1.7 mm lateral to midline and DV= -2.7 mm ventral from top of the

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skull (cannulas 23-gauge, 8mm).

The guide cannulas were secured in place by using two stainless steel screws

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anchored to the skull and dental acrylic cement. After the cement was entirely dried and hardened, two stainless steel stylets were used to fasten the guide cannulas during a 7-

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day recovery period.

2.3. Drugs

SB334867 and TCS OX2 29 (Tocris Bioscience, Bristol, UK) (0.5, 2.5, or 12.5

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nM/0.5 μl DMSO) (Parsania et al., 2016) were used as OX1R and OX2R antagonist, respectively. These drugs were dissolved in 12% dimethyl sulfoxide (DMSO Sigma Aldrich, Germany) and injected into the DG. Control groups received DMSO 12% as vehicles. Also, morphine sulfate (Temad, Iran) was dissolved in physiological saline

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(0.9% NaCl) and was subcutaneously (s.c.) injected at 5 mg/kg concentration (Parsania et al., 2016) in the acquisition phase.

2.4. Microinjection procedure Microinjections were done by lowering a stainless-steel injector cannula (30gauge needle) with a length of 1mm longer than the guide cannula into the DG. Injector

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cannulas were attached to the 1-µl Hamilton syringe by Polyethylene tubing (PE-20) and the connection was firmly sealed. Vehicles or drug solutions were slowly injected, bilaterally, in a volume of 0.5 μl/cannula, over a period of 60 s into the nuclei. Injector was left in cannula for an additional 60 s to simplify the diffusion of the drugs and

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prevented the drug backflow.

2.5. Conditioning apparatus and paradigm

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The conditioned place preference (CPP) procedure was used to evaluate the motivation properties, such as rewarding or aversive effects of drugs in animals

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(Haghparast et al., 2011). We used the CPP apparatus in these experiments, which is included a three-compartment (30 × 30 × 40 cm) box. The apparatus was made of Plexiglas and divided into two equal-sized compartments, which were isolated by a guillotine door, leading into a third part known as the null part (30 × 15 × 40 cm). To

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make the compartments different from each other, one of the sections had a smooth floor and the other had a rough floor. Both compartments had white backgrounds with black stripes in dissimilar orientations (vertical vs. horizontal) on their wall. This paradigm, occurred in five consecutive days, which included three distinct phases, including pre-conditioning, conditioning, and post-conditioning (Fig. 1).

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2.5.1. Pre-conditioning phase For habituation, the rats were carried to the test room from the housing room at least 30 min before experiment. On the first day, we placed each rat into the null compartment and the animal was moved freely to entire apparatus for 10 min. The movement and the time spent in each section were recorded by a 3CCD camera (Panasonic Inc., Japan) which placed two meters above the CPP box and were analyzed

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using Ethovision software, a video tracking system for automation of behavioral experiments (Noldus Information Technology, the Netherlands).

As with the experimental design used in this investigation, the animals did not

show any priority for either of the sections, however, individual rats tended to allocate

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more time in one part compared to other part, any animal which spent ≥80% of the total

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test time in each part was excluded.

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2.5.2. Conditioning phase

This phase was started 24 h after the pre-conditioning day and included six 45-

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min sessions in a three-day schedule. These sessions were performed twice each day (from day 2 to day 4) within six-hour timeout. On the first day of conditioning, animals were treated with morphine (5mg/kg, s.c.) in the morning and immediately confined to the morphine-paired compartment for 45 min; at least 6 h later, animals received saline

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(s.c.) and put in the saline-paired compartment for 45 min. The next day, we changed the injection time of morphine and saline. On the third day, the injection time of morphine and saline was similar to the first day. In order to investigate the effect of OXR1 and OXR2 antagonists on the acquisition phase, animals were microinjected with these antagonists before each injection of morphine during conditioning phase.

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2.5.3. Post-conditioning phase On the post-test day (day 5), we removed the sliding door in the apparatus and the animal could access to the entire arena for 10 min. The time spent and distance traveled were recorded by a 3CCD camera and analyzed using the Ethovision software. The time spent in the morphine-paired chamber minus the time spent in the salinepaired chamber was calculated and defined as the conditioning score. Although in

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acquisition experiments, neither morphine nor saline injections were given on the day 5, for comparing the role of orexin receptors in expression of morphine-induced CPP, on the post-conditioning day, animals were microinjected by OX1R or OX2R antagonist into the DG prior to the CPP test. In this part of experiment, CPP score and total

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distance moved each rat were calculated on the test day, during a 10-min period (Fig. 1)

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(Taslimi et al., 2011).

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2.6. Experimental design

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2.6.1. Effects of OX1 and OX2 receptor antagonists microinjection into the DG region on the acquisition of morphine-induced CPP Six treatment groups (n=6 in each group) were administered with different doses of SB334867 or TCS OX2 29 (0.5, 2.5, and 12.5 nM/0.5 μl DMSO; once per day before

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morphine injection), as the OX1 or OX2 receptor antagonist into the DG region 5 min prior to subcutaneous injection of morphine (5 mg/kg) during the 3 days of conditioning phase. The animals were injected by saline (s.c.) without received antagonist, as salinecontrol group. In vehicle control group (n=6), animals received morphine (5 mg/kg, s.c.) and DMSO 12% instead of SB334867 or TCS OX2 29 into the DG during conditioning

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days. Two other control groups (n=6 in each group) received only the maximum dose of the antagonists (12.5 nM/0.5 μl DMSO) into the DG with s.c. injection of saline instead of morphine to determine whether SB334867 or TCS OX2 29 has a preference or aversive effect on the development of CPP. The animals were instantly put in the drugpaired compartment after the s.c. injection of morphine. On day 5 which is the post-test day, the animals were exposed to the CPP test without any treatment. During the testing

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phase, CPP scores and distance traveled were recorded for 10 min.

2.6.2. Effects of OX1 and OX2 receptor antagonists microinjection into the DG region on the expression of morphine-induced CPP

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In order to investigate the effects of intra-DG administration of OX1 and OX2

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receptor antagonists, on the expression of morphine-induced place preference, six treatment groups of animals (n=6 for each group) received different doses of SB334867

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or TCS OX2 29 (0.5, 2.5, or 12.5 nM/0.5 μl DMSO; once per day before morphine injection) ), as OX1R and OX1R antagonists into the DG region on the post-

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conditioning test day and after 5 min were put into the CPP apparatus and tested for the CPP score and distance traveled for 10-min period. One group received the subcutaneous injection of morphine (5 mg/kg) during the conditioning days without any injection into the DG which considered as saline-control group. Vehicle-control group

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(n=6), received DMSO 12% (0.5 μl) instead of SB334867 or TCS OX2 29 on the posttest day. Anatomical control group administrated by morphine (s.c.) and the highest dose of antagonists (SB334867 or TCS OX2 29; 12.5 nM/0.5 μl DMSO). Two other control groups (n=6 in each group) received only the maximum doses of the antagonists (12.5 nM/0.5 μl DMSO) into the DG with subcutaneous injection of saline instead of

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morphine to determine whether SB334867 or TCS OX2 29 have preference or aversive effect on the expression of morphine induced-CPP.

2.7. Histology Rats were deeply anesthetized with ketamine and xylazine after completion of experimental tests. Then, they were perfused with 0.9% saline and 4%

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paraformaldehyde solution. The brains were removed and fixed in a 4% formalin solution for 3 days and cut coronally in 50 µm sections using vibrating microtome

(Campden Instruments, Germany). Sections were tested to show the location of the

microinjection sites based on the atlas of rat brain (Paxinos and Watson, 2007) in the

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DG region of hippocampus (Fig. 2).

2.8. Statistics

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Data were expressed as mean ± SEM and normality of distribution was tested using Kolmogorov-Smirnov. The data were processed by the commercially available

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software GraphPad Prism (version 5.0). In order to compare the CPP scores and the distance traveled of the control and experimental groups, one-way ANOVA followed by Newman-Keuls multiple comparison tests was used, as either was required. Results

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were statistically considered significant when P < 0.05.

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3. Results

3.1. Effects of intra-DG administration of OX1R antagonist during acquisition phase of morphine-induced CPP One-way ANOVA followed by Newman-Keuls multiple comparison test [F (6, 41) = 59.38; P < 0.0001; Fig. 3A] showed that there were remarkable differences in

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CPP scores between the saline-control group and groups treated by morphine. As it can be derived from Fig. 3A even though animals received SB334867 during 3-

days conditioning phase, morphine CPP was appointed, represented as increased CPP

scores. The one-way ANOVA followed by Newman-Keuls multiple comparison test [F

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(3, 22) = 41.54; P < 0.0001; η2 = 0.87; Fig. 3A] also presented that administration of the

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antagonist (0.5, 2.5 and 12.5 nM/0.5 μl DMSO) dose-dependently reduced morphineinduced CPP during the acquisition phase. Two higher doses of SB334867 (2.5 and 12.5

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nM; P < 0.01 and P < 0.001, respectively) significantly weakened the CPP induced by morphine, unlike the adjacent areas which this antagonist was administered at maximum

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concentration (12.5 nM). Therefore, the acquired results are most likely specific to the OX1R antagonist infusion into the DG during the acquisition phase. In addition, one-way ANOVA followed by Newman-Keuls test [F (6, 41) = 0.242; P = 0.9593; Fig. 3B] demonstrated that administration of different doses of

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SB334867 (0.5, 2.5 and 12.5 nM/0.5 μl DMSO) during the conditioning phase did not change distance traveled during the 10-min test period in comparison with that of the saline-control group. Therefore, the changes in CPP scores following administration of SB334867 during the acquisition phase could not be due to any changes associated with locomotor activity in subjects.

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3.2. Effects of intra-DG administration of OX1R antagonist during expression phase of morphine-induced CPP One-way ANOVA followed by Newman-Keuls multiple comparison test [F (6, 40) = 23.24; P < 0.0001; Fig. 4A] showed a remarkable difference in CPP score between the saline-control group and groups treated by morphine. Fig. 4A demonstrated that although animals received the antagonist on day 5 (post-conditioning test),

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morphine CPP was induced, represented as increased CPP scores. However, one-way ANOVA followed by Newman-Keuls multiple test [F (3, 23) = 13.31; η2 = 0.66; and P < 0.0001; Fig. 4A] also revealed that administration of

SB334867 (0.5, 2.5 and 12.5 nM/0.5 μl DMSO) dose-dependently reduced morphine

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CPP during the expression phase, but it was less pronounced compared to the

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acquisition phase. Here also, higher doses of SB334867 (2.5 and 12.5 nM) significantly blocked OX1 receptors within the DG and weakened the induction of place preference

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unlike the adjacent areas which were administered SB334867 at maximum concentration (12.5 nM). Accordingly, it can be derived that the results are specific to

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the infusion of SB334867 into the DG during the expression phase. In addition, one-way ANOVA followed by Newman-Keuls comparison test [F (6, 40) = 0.1531; P = 0.9871; Fig. 4B] demonstrated that administration of SB334867 (0.5, 2.5 and 12.5 nM) during the expression phase did not change distance traveled

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during the 10-min test period compared to that of the saline-control group. Therefore, administration of SB334867 during the expression phase impressed the CPP scores which this effect was not because of the changes in locomotor activity in the rats.

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3.3. Effects of OX1R antagonist on the induction of morphine CPP during the acquisition compared to the expression phase Changes of CPP scores of DMSO-control group compared to those of the saline group were considered 100 %. Figure 5 shows that logarithmic dose response curves have been presented to calculate the 50% effective dose (ED50) of intra-DG SB334867 microinjection during acquisition phase compared to expression phase. The ED50 of

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SB334867 during acquisition and expression phase are equal to 3.27 and 3.92, respectively. It can be inferred that the OX1R antagonist reduced morphine-induced CPP during acquisition period in lower dose than expression period. It revealed that

OX1R antagonist microinjection into the DG on reduction of morphine-induced CPP

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during acquisition phase was more than its effect in expression phase.

3.4. Effects of intra-DG administration of OX2R antagonist during acquisition

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phase of morphine-induced CPP

One-way ANOVA followed by Newman-Keuls multiple comparison test [F (6,

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41) = 33.37; P < 0.0001; Fig. 6A] displayed that there were remarkable differences in CPP scores between the saline-control group and groups treated by morphine. As it is shown in Fig. 6A, morphine CPP was set up, embodied as increased CPP scores; although animals received TCS OX2 29 during 3-day conditioning phase.

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Furthermore, one-way ANOVA followed by Newman-Keuls multiple

comparison test [F (3, 23) = 5.019; P = 0.0094; η2 = 0.43; Fig. 6A] also indicated that administration of the antagonist (0.5, 2.5, and 12.5 nM/0.5 μl DMSO) diminished morphine place preference during the acquisition phase. However, the highest concentration of TCS OX2 29 (12.5 nM) considerably blocked OX2R in the DG area

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and weakened the induction of place preference (P < 0.01), contrasting to the neighboring regions which were operated the antagonist at maximum concentration (12.5 nM). Thus, it can be a proof that the attained results are exact to the OX2R antagonist infusion into the DG during the acquisition phase. Also, one-way ANOVA followed by Newman-Keuls multiple comparison test [F (6, 41) = 0.1595; P = 0.989; Fig. 6B] showed that administration of TCS OX2 29

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(0.5, 2.5, and 12.5 nM) during the conditioning phase did not change distance traveled during the 10-min test period in comparison with that of the saline-control group (Fig. 6B). Hence, it indicates that the effect of TCS OX2 29 administration during the

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acquisition phase is not related to locomotor activity in rats.

phase of morphine-induced CPP

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3.5. Effects of intra-DG administration of OX2R antagonist during expression

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One-way ANOVA followed by Newman-Keuls multiple comparison test [F (6, 39) = 47.66; P < 0.0001; Fig. 7A] confirmed substantial differences in CPP scores

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between the saline-control group and morphine-treated groups on the test day. Figure 7A displays that morphine CPP was induced, characterized as increased CPP scores; although animals received intra-DG DMSO as a vehicle instead of the OX2R antagonist on day 5.

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On the other hand, one-way ANOVA followed by Newman-Keuls multiple

comparison test [F (3, 21) = 32.63; P < 0.0001; η2 = 0.84; Fig. 7A] also showed that administration of TCS OX2 29 (0.5, 2.5, and 12.5 nM/0.5 μl DMSO) dose-dependently reduced CPP scores during the expression phase compared to those in vehicle-control group, but it was more definite in comparison with the acquisition phase. Likewise, two

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high concentrations of TCS OX2 29 (2.5 and 12.5 nM) remarkably blocked OX2Rs into the DG area and reduced the CPP induction contrasting to the neighboring regions which were administered TCS OX2 29 at maximum concentration (12.5 nM). Thus, it can suggest that the obtained results are precise to the infusion of TCS OX2 29 into the DG region during the expression phase. Furthermore, one-way ANOVA followed by Newman-Keuls multiple

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comparison test [F (6, 39) = 0.1361; P = 0.9905; Fig. 7B] demonstrated that administration of TCS OX2 29 (0.5, 2.5, and 12.5 nM) during the expression phase did not change distance traveled during the 10-min test period in comparison to the saline-

control group. Thus, the changes in CPP scores resulting administration of TCS OX2 29

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during the expression phase could not be due to any changes related to locomotor

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activity in subjects.

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3.6. Effects of OX2R antagonist on the induction of morphine CPP during the acquisition compared to the expression phase

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In Fig. 8, a logarithmic dose response curves have been presented to calculate the 50% effective dose (ED50) of intra-DG TCS OX2 29 administration during acquisition and expression phase of morphine-induced CPP. The ED50 of TCS OX2 29 during acquisition and expression phases was 29.9 and 3.08, respectively. The data

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showed that the microinjection of OX2R antagonist into the DG considerably reduced CPP scores during expression phase compared to acquisition phase. It revealed that TCS OX2 29, as OX2R antagonist microinjection into the DG had an imperative effect in expression phase. It can be suggested that OX2Rs in this area are prominently involved in the mediation of expression phase of morphine-induced CPP in the rats.

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4. Discussion

The main discoveries of this study are as follows: (i) bilateral microinjection of SB334867 (a selective OX1R antagonist) into the DG area reduces the acquisition (contextual learning) of CPP induced by morphine; (ii) expression phase-related CPP scores are attenuated by the bilateral microinjection of SB334867 into the DG area; (iii)

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bilateral microinjection of TCS OX2 29 (a selective OX2R antagonist) into the DG area decreases the development of morphine-induced CPP; (iv) blockage of OX2Rs in the DG reduces the expression of CPP induced by morphine; (v) blockage of intra-DG OX1Rs is more pronounced in the acquisition phase of morphine-induced place

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preference than in the expression phase; and (vi) the effectiveness of blockage of the

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OX2 receptors is more significant in the expression phase of morphine-induced place preference in comparison with the acquisition phase. There were no significant change

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in the CPP score due to microinjecting orexin receptor antagonists in other regions near the DG during the conditioning phase or on the post-test day, which indicates that the

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data captured is primarily related to antagonist infusion in the DG area. Cell damage does not appear to be a significant issue in this protocol because it has been used in many prior studies. Furthermore, intra-DG administration of the drug did not significantly change locomotor activities compared to the vehicle control groups.

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This study shows that intra-DG microinjection of OX1R and OX2R antagonists

reduce the acquisition and expression of drug-seeking behaviors. These discoveries demonstrate that orexin transition in the hippocampal DG area through recruitment of OX1R and OX2R are crucial in both acquisition and expression of morphine-induced CPP. This result confirms previous discoveries from our laboratory, which show the

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critical role of orexin receptors within the DG in the acquisition and expression of CPP induced by LH stimulation (Parsania et al., 2016). Increased stimulation of orexin neurons in the LH has been found in the conditioning phase of morphine place preference (Harris et al., 2007; Zarepour et al., 2013). The administration of SB334867 reduces the acquisition and expression of morphine place preference (Harris et al., 2007; Riahi et al., 2013; Sharf et al., 2010). Furthermore, bilateral intra-DG administration of

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SB334687 on conditioning days suppresses the acquisition of morphine CPP (Narita et al., 2006). Previous studies have demonstrated that OX1 receptor activity in the dorsal HIP is involved in the development of morphine CPP, and the antagonism of OX1

receptors by SB334867 introduced into the HIP during the conditioning phase causes

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intervention in the morphine reward (Riahi et al., 2013). Furthermore, blockage of OX1

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receptors in the hippocampal CA1 region could reduce the acquisition of place preference induced by LH stimulation (Rashidy- Pour et al., 2014). Although numerous

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studies have been conducted to clarify the effect of OX1 receptors on reward processing, not many studies have explored the role of OX2 receptors. Shoblock et al.

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reported that blockage of OX2 receptors could interrupt alcohol-associated rewards (Shoblock et al., 2011). Furthermore, Tabaeizadeh et al. demonstrated that OX2R antagonism can attenuate the acquisition and expression of morphine CPP in mice (Tabaeizadeh et al., 2013). We reported in earlier studies that the microinjection of TCS

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OX2 29 (a selective OX2 antagonist) in the CA1 region can diminish the acquisition of LH stimulation-induced CPP (Rashidy-Pour et al., 2014). These findings are in agreement with the results of this study, demonstrating that

the expression and acquisition of morphine CPP requires orexin transmission via OX1Rs and OX2Rs in the DG area. Our results emphasized the critical roles of intra-

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DG OX1Rs and OX2Rs in mediating the expression and acquisition of morphineseeking behavior. Conversely the results of this study recommend that OX1Rs appear to have a significantly greater effect on the acquisition of morphine-induced CPP compared to the expression phase. New concepts show that the stimulation of OX2R may be significantly less effective than stimulation of OX1Rs in mediating a plurality of neurochemical signals (Narita et al., 2006); therefore, continued microinjection of an

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OX1R antagonist (SB334867) during the acquisition phase (three injections compared to one injection during the expression day) could decrease CPP scores more in the

acquisition phase than in the expression phase. However, the effectiveness of the OX2R antagonist in decreasing CPP scores was more pronounced in the expression phase than

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in the acquisition phase. Furthermore, OX1R and OX2R have a differential distribution

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within the brain, and that has an impact on their functions (Cluderay et al., 2002; Hervieu et al., 2001; Marcus et al., 2001; Trivedi et al., 1998). It is supposed that the

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differential effects of OX1R and OX2R antagonists on the expression and acquisition of drug-seeking behavior are related to the arousal-related functions of OX2Rs (Mahler et

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al., 2012). Several lines of evidence indicate that arousal is largely related to the role of OX2R and reward is most closely related to the role of OX1R (Mahler et al., 2012). Consistent with the effect of OX1Rs in rewarding processes, in this experiment, the OX1R antagonist appears to be more effective on the rewarding property of morphine

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within the DG than the OX2R antagonist. In addition, earlier studies demonstrated that intra-DG orexin receptors participate in the rewarding effect of drugs (Parsania et al., 2016). Cumulative proof has implicated the essential behavioral role of the orexin system in morphine CPP (Baimel and Borgland, 2012). The LH orexinergic neurons project to reward-associated brain sites, including the DG (Parsania et al., 2016).

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Furthermore, the intra-DG administration of SB334867 as an OX1R antagonist and TCS OX2 29 as an OX2R antagonist blocked the acquisition and expression of place preference by LH stimulation (Parsania et al., 2016). These results are in agreement with other recent studies that have emphasized the importance of orexinergic neurons in morphine conditioning and consequently, suggest the involvement of orexins in drugseeking and other motivational behaviors (Harris et al., 2005; Scammell and Saper,

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2005). These facts suggest the critical role of LH orexin neurons in reward function and motivation.

In conclusion, this study demonstrates that the blockage of OX1Rs and OX2Rs

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in the DG reduces the acquisition and expression of morphine-induced CPP, and the

role of OX1Rs in the acquisition phase is more than the expression phase. Furthermore,

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the OX2Rs play a more effective role in the expression phase than acquisition phase.

this phenomenon.

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Acknowledgment

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However, there is need for more research to describe the precise mechanism involved in

This project was supported by the Student Research Committee, Shahid

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Beheshti University of Medical Sciences (Grant no. 1396/56470), Tehran, Iran. Furthermore, this project was supported by the Vice-Chancellor for Research & Technology of Shahid Beheshti University of Medical Sciences (Grant no. 1589661851/97/10/25). Also, the authors would like to thank the Neuroscience Research Center (97-230-A), School of Medicine, Shahid Beheshti University of Medical Sciences for valuable cooperation.

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Figure Legends

Fig.1. The experimental protocols (schematic design) for the induction of morphineinduced CPP including the pre-test, acquisition, post-test (expression).

Fig. 2. Three coronal schematic administration regions in the dentate gyrus for (A) SB334867 [○ Vehicle (12%DMSO); ● treatment (SB334867);  Anatomical Control],

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(B) TCS OX2 29 [□Vehicle (12%DMSO); ■ treatment (TCS OX2 29);  Anatomical Control] microinjection.

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Fig. 3. Effect of bilateral microinjection of different doses of SB334867, as an OX1

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receptor antagonist, in the DG region of hippocampus on (A) CPP scores and (B) locomotor activity in the acquisition phase of CPP induced by morphine. The animals

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received different doses of SB334867 (0.5, 2.5 and 12.5 nM/0.5 µl DMSO) into the DG, 5 min prior to administration of morphine (5 mg/kg, s.c.) during conditioning days.

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Saline-control group received saline instead of morphine without microinjection to DG. DMSO-control group treated by morphine (5 mg/kg) and received DMSO into DG area instead of antagonist in acquisition phase. Anatomical control group received maximum dose of SB334867 in the adjacent regions before morphine injection into the DG. Results

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show that administration of 2.5 and 12.5 nM SB334867 in animals during acquisition phase decreased the CPP score compared to the DMSO-control group. Locomotor activity did not change in any of the treatment or control groups. All data are expressed as mean ± SEM. for 4-6 rats. *P < 0.05, ***P < 0.001 compared to saline-control group. ††P < 0.01, †††P < 0.001 compared to DMSO-control group.

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Fig. 4. Effect of bilateral microinjection of different doses of SB334867, as an OX1 receptor antagonist, in the DG region of hippocampus on (A) CPP scores and (B) locomotor activity on expression phase of CPP induced by morphine. The animals received different doses of SB334867 (0.5, 2.5 and 12.5 nM/0.5 µl DMSO) into the DG, 5 min prior to administration of morphine (5 mg/kg, subcutaneous) on the post conditioning (test) day. Saline-control group injected by saline during acquisition phase

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instead of morphine. DMSO-control group treated by morphine during acquisition phase (5 mg/kg) and received DMSO into DG area instead of antagonist in expression phase (test day). Anatomical control group received maximum dose of SB334867 in the

adjacent regions before morphine injection (s.c.) on the test day. Results show that

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administration of 2.5 and 12.5 nM SB334867/0.5 μl DMSO 12% per side decreased the

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CPP score in expression phase compared to the DMSO-control group. Locomotor activity did not change in any of the treatment or control groups. All data are expressed as mean

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± SEM for 4–6 rats. **P < 0.01, ***P < 0.001 are different from the saline-control group.

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†P < 0.05, †††P < 0.001 are different from the DMSO-control group.

Fig. 5. A log dose response curve for SB334867, an OX1 receptor antagonist, during acquisition and expression phases, within the DG in animals which received subcutaneous morphine during acquisition and expression phases of CPP. In this figure we set the CPP

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scores of the DMSO-control (animals that received 5 mg/kg morphine subcutaneously and 0.5 µl DMSO into the DG) to 100%, and represent of the remaining scores (animals that received different doses of SB334867 in the DG) as % changes in their responses to generate an effective dose 50% (ED50) of SB334867 in acquisition phase (3.27) and in expression phase (3.92).

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Fig. 6. The effect of bilateral administration of TCS OX2 29 (0.5, 2.5 and 12.5 nM/0.5 µl DMSO) into the DG on (A) CPP scores (s) and (B) locomotor activity during the acquisition phase of CPP induced by morphine. Animals received TCS OX2 29, 5 min before the subcutaneous injection of morphine (5 mg/kg) during 3 days of conditioning. Saline-control and DMSO-control groups just received saline and DMSO 12 % (0.5 μl per side) into the DG, respectively while saline-control group treated by saline instead of

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morphine (s.c.) during acquisition days. Anatomical control group received the highest dose of TCS OX2 29 (12.5 nM/0.5 µl DMSO) into some neighboring sites of the DG.

Also, a group just received a 12.5 nM solution of TCS OX2 29 into the DG, 5 min before

the subcutaneous injection of saline. Results show that administration of highest dose of

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TCS OX2 29 (12.5 nM) decreased the CPP score compared to DMSO control group.

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Locomotor activity did not change in any of the treatment or control groups. Each bar is represented the mean ± SEM for six rats (seven rats in DMSO group). **P < 0.01, ***P

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< 0.001 compared to saline-control group. ††P < 0.01 compared to DMSO-control group.

Fig. 7. The effect of bilateral microinjection of TCS OX2 29 (0.5 μl of 0.5, 2.5, and 12.5 nM solutions per side) into the DG on (A) CPP scores (s) and (b) locomotor activity with or without morphine during the expression phase. Following the experimental procedure

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of morphine place conditioning, animals received TCS OX2 29 on the fifth day. After subcutaneous injection of morphine during 3 days of conditioning, DMSO- control group received DMSO 12 % (0.5 μl per side) into the DG on test day (expression phase). After 3 days of conditioning with morphine, anatomical control group received a 12.5 nM solution of TCS OX2 29 into some neighboring sites of the DG, on day 5. Also, a group

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just received a 12.5 nM solution of TCS OX2 29 following saline injection (s.c.) in expression phase. These data show that two higher dose of TCS OX2 29 (2.5 and 12.5 nM) decreased CPP score compared to DMSO-control group. Locomotor activity did not change in any of the treatment or control groups. Each bar is represented by the mean ± SEM for six rats (seven rats in DMSO group). ***P < 0.001 compared to saline-control

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group. ††P < 0.01, †††P < 0.001 compared to DMSO-control group.

Fig. 8. A log dose-response curve for intra-DG administration of TCS OX2 29 during either the acquisition or expression experiments. The changes of CPP scores of DMSO

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group compared to those of the saline-control group were set as 100 %. The remaining

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scores (animals that received TCS OX2 29 during the acquisition or expression phases) were represented as changes in their responses compared to DMSO group. The effect of

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TCS OX2 29 on reduction of morphine CPP during the acquisition was more pronounced than that of the expression (P < 0.05). This figure illustrates the effective dose 50%

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(ED50) of TCS OX2 29 (0.5, 2.5 and 12.5 nM/0.5 µl DMSO) during acquisition phase

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(29.9 nM) and expression phase (3.08 nM).

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