Pharmacology, Biochemistry and Behavior 126 (2014) 109–115
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Microinjection of the mGluR5 antagonist MTEP into the nucleus accumbens attenuates the acquisition but not expression of morphine-induced conditioned place preference in rats Nahid Roohi a, Abdolrahman Sarihi a,⁎, Siamak Shahidi a, Mohammad Zarei a, Abbas Haghparast b,⁎⁎ a b
Neurophysiology Research Center, Hamadan University of Medical Sciences, P.O. Box 65178-38678, Hamadan, Iran Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, P.O. Box 19615-1178, Tehran, Iran
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Article history: Received 26 May 2014 Received in revised form 18 September 2014 Accepted 26 September 2014 Available online 2 October 2014 Keywords: Metabotropic glutamate receptor 5 Nucleus accumbens Acquisition Expression Morphine Conditioned place preference Rat
a b s t r a c t Previous studies suggest that metabotropic glutamate receptor type 5 (mGluR5) plays an important role in modulation of the rewarding properties of morphine. Little is known about the role of mGluR5 in the nucleus accumbens (NAc), as one of the important regions of the reward circuitry. In the present study, we investigated the effects of intra-accumbal injection of mGluR5 antagonist, 3-[(2-methyl-4-thiazolyl) ethynyl] pyridine, MTEP, on the acquisition and expression of morphine induced Conditioned Place Preference (CPP) in the rats. Eighty four adult male Wistar rats (220–260 g) were bilaterally implanted with cannulae into the NAc. Subjects were tested in a CPP paradigm. Different doses of MTEP (0.3, 1 and 3 μg/0.5 μl per side) were delivered bilaterally into NAc during 3 conditioning days (Acquisition) or post-conditioning day (Expression). Results showed that bilateral intra accumbal administration of MTEP (1 and 3 μg) significantly attenuated the acquisition but not expression of morphine-induced CPP in a dose-dependent manner. Our findings indicated that blockade of mGluR5 reduces rewarding properties of morphine. Further studies are needed to know the involved mechanisms. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Opiate addiction is a chronic, relapsing disorder characterized by compulsive drug seeking and a high risk of drug craving which is often precipitated by drug-associated cues, even after long periods of abstinence (De Vries and Shippenberg, 2002). It has been reported that repeated drug exposure elicits changes in central nervous system during the development of addiction (Koob, 2002). The mesolimbic reward system, which extends from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), has been identified as one of the neuronal pathways for reward and drug addiction (Koob, 1992). The NAc, localized within the ventral region of the corpus striatum, is a major target and a central component of the reward system. It receives massive dopaminergic inputs from the VTA and glutamatergic afferents from structures such as the hippocampus, prefrontal cortex, and amygdala (Groenewegen et al., 1996; Vezina and Kim, 1999).
⁎ Correspondence to: A. Sarihi, Neurophysiology Research Center and Department of Physiology, Hamadan University of Medical Sciences, Hamadan, Iran. Tel./fax: +98 811 838 0131. ⁎⁎ Corresponding author. Tel./fax: +98 21 2243 1624. E-mail addresses: [email protected], [email protected] (A. Sarihi), [email protected], [email protected] (A. Haghparast).
http://dx.doi.org/10.1016/j.pbb.2014.09.020 0091-3057/© 2014 Elsevier Inc. All rights reserved.
Glutamate is one of the major neurotransmitters of the central nervous system. It is involved in a wide number of physiological and pathological processes (Kalivas et al., 2009). Glutamate receptors belong to two major groups: viz. ionotropic and metabotropic receptors (Hollmann and Heinemann, 1994). Based on sequence homology, pharmacology, and second messenger activation there are eight metabotropic glutamate receptors (mGluR) which are classified under three groups (Conn and Pin, 1997). Predominant receptor subtypes expressed in NAc include mGluR2/3, mGluR5, and mGluR7 (Pomierny-Chamioto et al., 2014; Tallaksen-Greene et al., 1998), specifically of mGluR5 type. mGluR5 are primarily located on the postsynaptic terminal in medium spiny GABAergic neurons and are rarely found on presynaptic terminals. This could play a role through GABAergic neurons activity modulation in drug addiction (Kenny and Markou, 2004). It has been shown that mGluR5 is located in areas such as the hippocampus, cerebral cortex, NAc, lateral septum, and the dorsal striatum (Kenny and Markou, 2004). Distribution of mGluR5 in the neural circuitry involved in reward consumption and seeking provoked considerable interest as a potential therapeutic target for drug addiction. Chiamulera et al. (2001) showed that mice lacking mGluR5 gene do not self administer cocaine, which shows that mGluR5 has an essential role in reinforcement, and from thereafter mGluR receptors came into the focus of addiction researchers (Chiamulera et al., 2001). In addition, reduction of mGluR5 signaling decreases drug taking and drug-seeking behaviors
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for alcohol (Backstrom et al., 2004), cocaine (Veeneman et al., 2010), methamphetamine (Chesworth et al., 2013; Gass and Olive, 2009), opiates (Brown et al., 2012; Popik and Wrobel, 2002; Veeneman et al., 2010) and nicotine (Paterson et al., 2003). Furthermore, it has been shown that intraperitoneal administration of 2-methyl-6(phenylethynyl) pyridine (MPEP) blocked the development of cocaine and morphine conditioned place preference (CPP) in rats (Herzig and Schmidt, 2004). Since mGluR5 antagonist administration was carried out systemically, the local role of mGluR5 in intended brain structure remains unclear. In this study, we used intra nucleus accumbens microinjection of selective mGluR5 antagonist, 3-[(2-methyl-4-thiazolyl) ethynyl] pyridine (MTEP). On the other hand, CPP paradigm is one of the most widely used experimental protocols used for measuring drug reward in laboratory animals (Bardo and Bevins, 2000). CPP reflects a preference for a context due to the contiguous association between the context and a drugassociated stimulus. Therefore, it is a simple and effective method for assessing the rewarding properties of drugs (Bardo et al., 1995). It has been revealed that systemic administration of MTEP blocks both the acquisition and expression of morphine CPP (Veeneman et al., 2010). In addition, Popik and Wrobel (2002) demonstrated that MPEP injected intraperitoneally also blocked both acquisition and expression phases of CPP for morphine (Popik and Wrobel, 2002). Out of the two, MTEP is a more specific antagonist for mGluR5 rather than MPEP, as MPEP can also block NMDA receptors, which should be considered in the interpretation of mechanisms involved in the results obtained with MPEP (Lea and Faden, 2006). Observations of potentiation in morphine CPP with MPEP suggest that mGluR5 antagonists may partly substitute for opiate reward (Rutten et al., 2011). Recently, Brown et al. (2012) reported that systemic administration of MTEP attenuates operant responding for morphine self-administration and cue-induced morphine-seeking after a period of forced abstinence (Brown et al., 2012). While mGluR5 has been implicated in the opioid's rewarding properties such as a morphine conditioning reward, the role of mGluR5 at NAc has not been elucidated. Therefore, in the present study, we assessed the effects of mGluR5 blockade into the NAc on the acquisition and expression of morphine-induced place preference. 2. Materials and methods 2.1. Animal Eighty four adult male albino Wistar rats (Pasteur Institute, Tehran, Iran) weighing 200–250 g were housed in standard plastic cages in groups of three in a temperature controlled room (temperature 21 ± 3 °C). They were maintained on a 12 h light/dark cycle (lights on at 07:00 a.m.) with food and water ad libitum. The experiments were carried out during the light phase of the cycle. Six to eight animals were used per group and each animal was tested once. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) and were approved by the Research and Ethics Committee of Hamadan University of Medical Sciences. 2.2. Drug The following drugs were used: morphine sulfate (Temad, Iran) dissolved in sterile saline (0.9%); 3-[(2-methyl-4-thiazolyl) ethynyl] pyridine; MTEP; mGluR5 antagonist (Tocris bioscience, UK), dissolved in saline. It is worth mentioning that control animals received saline. 2.3. Stereotaxic surgery Subjects were anesthetized by Xylazine (10 mg/kg) and Ketamine (100 mg/kg) and placed in the stereotaxic apparatus (Stoelting, USA),
with the incisor bar set at approximately 3.3 mm below horizontal zero in order to achieve a flat skull position. After an incision was made to expose the rat's skull, two points were determined and drilled into the skull at the following stereotaxic coordinates 1.4 ± 0.4 mm anterior to bregma, ± 1.5 mm lateral to the sagital suture, and 6.5 mm down from top the skull according to the atlas of rat brain (Paxinos and Watson, 2007).Two guide cannulae (23-Gauge) with 12 mm length were inserted into the holes aiming at NAc. The guide cannulae were anchored with a jeweler's screw and the incision was closed with dental cement. After surgery, dummy inner cannulae that extended 0.5 mm beyond the guide cannulae were inserted into the guide cannulae and left in place until injections were made. All animals were allowed to recover for one week before starting the behavioral testing. 2.4. Intra-accumbal injection Animals were gently restrained by hand and the dummy cannulae were removed from the guide cannulae. Drugs were directly injected into NAc through the guide cannulae by using injector cannulae (30gauge, 1 mm below the tip of the guide cannulae). Polyethylene tubing (PE-20) was used for attaching the injector cannulae to the 1-μl Hamilton syringe. The injection volume was 0.5 μl/side into NAc for all groups. Injections were made bilaterally over a 50 s period, and the injection cannulae were left in the guide cannulae for an additional 60 s in order to facilitate the diffusion of the drugs. The microinjection time for 0.1 μl volume of drugs was 10 s for preventing lesions in these areas (Karimi et al., 2014; Reisi et al., 2014). 2.5. Place conditioning apparatus A three-compartment CPP apparatus was used in these experiments. Place conditioning was conducted using an unbiased protocol, modified slightly from a design previously described (Zarepour et al., 2013). The apparatus was divided into two equal-sized compartments (30 cm × 30 cm × 40 cm) with the third section (30 cm × 15 cm × 40 cm) being the null section which connected the two equal-sized sections. Both compartments had white backgrounds with black stripes in different orientations (vertical vs. horizontal).To provide a tactile difference between the compartments, one of them had a smooth floor, while the other compartment had a net-like floor. In this apparatus, rats showed no consistent preference for either compartment. In the CPP paradigm, the conditioning score and distance traveled were calculated from a video, recorded by a CCD camera with 30 frames per second (fps) resolution (Panasonic Inc., Japan). The camera was placed 2 m above the CPP boxes and locomotion tracking was measured by Maze Router homemade software, a video tracking system for automation of behavioral experiments. In these experiments, on the pre- and post-conditioning phases, each animal was allowed to freely access two compartments for 10 min. The time spent and total distance traveled in each compartment of the CPP apparatus were measured during this period. CPP paradigm took place for 5 continuous days, which consisted of three distinct phases: pre-conditioning, conditioning and post-conditioning. 2.6. CPP protocol The CPP protocol was modified from that described previously by our group (Haghparast et al., 2009; Haghparast et al., 2013; Reisi et al., 2014; Zarepour et al., 2013). Before each session, the rats were habituated to the experimental room for 30 min. On day 1 (preconditioning), they were placed in the central compartment and allowed free access to the entire apparatus. On days 2–4 (conditioning), they received injections of saline or morphine and were immediately confined in one of the two conditioning compartments. An unbiased allocation was used. Rats with a neutral preference (45–55% for either side) were randomly allocated their drug-paired side (unbiased allocation). On test day (post conditioning), the rats were given free access to the CPP apparatus.
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All conditioning sessions were 30 min in duration and occurred at the same time each day. Place preference was calculated as a preference score (time spent in drug paired zone − time spent in the saline paired zone). Locomotor data were also collected throughout CPP testing in order to assess the development of behavioral sensitization. 2.6.1. Pre-conditioning phase On day 1, each rat was separately placed in the apparatus for 10 min, with free access to all compartments. Animal movements were recorded by Maze Router tracking software and analyzed on the same day. No significant differences between times spent in either of the two compartments were considered as unbiased conditioning. Rats with any compartment preference were omitted from the experiment. In this experimental model, only the animals that did not show a preference in baseline test, to either of the compartments, was used for conditioning phase. These rats were randomly assigned to one of the two groups (odd and even) for place conditioning, which 6–8 animals were used for each subsequent experiment. 2.6.2. Conditioning phase Each group of animals was randomly divided into even or odd. Odd animals received saline/morphine pairing in alternative morning and afternoon design with an interval of 6 h. The vice versa program for even animals was done. This phase consisted of a 3-day schedule of conditioning sessions. A total of six sessions (30 min each) was carried out. During these 3 conditioning days, in 3 sessions, animals were confined to one compartment, under the drug influence. During other three sessions, they were injected with saline while confined to the other compartment. Access to the other compartments was blocked on these days. 2.6.3. Post-conditioning phase On the 5th day, the partition was removed and the rats could access the entire apparatus. The mean time spent for each rat in both compartments during a 10-min period was recorded. In order to calculate the conditioning score, the difference in time spent for the drug- and saline-paired places was considered as the preference criteria. In the acquisition tests, no injection was given on the post-conditioning day. Total distance traveled for each animal was also measured on pre- and post-conditioning days for a 10-min period as the locomotor activity in control and experimental groups. 2.7. Experimental design The effects of subcutaneous (sc) administrations of morphine on induction of place conditioning in animals were determined in previous studies (Reisi et al., 2014; Taslimi et al., 2012; Zarepour et al., 2013). Each morphine dose (0.5, 1, 2.5, 5, 7.5 and 10 mg/kg; sc) was injected on a 3-day schedule of conditioning as described above. Then, animals were tested in a morphine-free state in order to eliminate the influence of morphine induced motor effects on responses (Olmstead and Franklin, 1997). Conditioning score and distance traveled were calculated for each rat (Haghparast et al., 2009; Rezayof et al., 2011). In this experiment, morphine conditioned place preference paradigm was established by using a dose of morphine 5 mg/kg. Based on our previous studies, we have found that the lowest effective dose was 5 mg/kg. To confirm the findings, once again, we included 2.5 mg/kg dosage along with 5 mg/kg dosage regime. Morphine at 5 mg/kg resulted in conditioned place preference while 2.5 mg/kg failed to do so. This reiterates our earlier finding that 5 mg/kg is the lowest effective dose. As stated previously, control animals received saline (1 ml/kg; sc). 2.7.1. Effects of intra-accumbal injection of mGluR5 antagonist, MTEP on the acquisition of morphine-induced conditioned place preference To test the effects of MTEP microinjected into NAc on development of morphine rewarding properties, experiments were performed on
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saline and morphine-treated groups, which received the effective dose of morphine (5 mg/kg; sc) during the acquisition period. MTEP (0.3, 1 and 3 μg/0.5 μl) (Gass and Olive, 2009) was bilaterally microinjected into NAc, 5 min prior to the injection of either morphine or saline during the 3-day conditioning phase. The control group received vehicle (saline) into NAc. Conditioning score and distance traveled were measured during a 10-min period on the test day. 2.7.2. Effects of intra-accumbal injection of mGluR5 antagonist on the expression of morphine-induced conditioned place preference In this set of experiments, for evaluating the effects of single injection of mGluR5 receptor antagonist on the expression of morphine rewarding properties in conditioned place preference paradigm, different doses of MTEP (0.3, 1 and 3 μg/0.5 μl) were bilaterally microinjected into NAc, just 5 min prior to CPP test in saline- and morphine-treated groups which received effective dose of morphine during the acquisition period. Conditioning score and distance traveled were measured during 10 min on the test day. 2.8. Locomotor activity measurement In order to evaluate the effect of drugs on locomotor activity in animals, total distance traveled (cm) during 10-min test period, on the pre- and post-conditioning phases was measured by Maze Router homemade software. 2.9. Histology After completion of behavioral testing, the animals were deeply anesthetized with ketamine and xylazine. Then, they were transcardially perfused with 0.9% saline and 5% formalin solution. Their brains were removed, blocked and cut coronally in 50 μm sections through cannulae placements. The neuroanatomical location of cannulae tips placements was confirmed using rat brain atlas (Paxinos and Watson, 2007). Only the animals with correct cannulae placements in NAc were included in the data analysis (Fig. 1). 2.10. Statistics Data were processed by commercially available software GraphPad Prism® 5.0. In order to compare the conditioning scores and distance traveled obtained from all groups (vehicle and experimental groups), one-way analysis of variance (ANOVA) followed by post hoc analysis (Newman–Keuls multiple comparison test) was used, as appropriate. In the first experiment, Dunnett's multiple comparison test was used. P-values less than 0.05 (P b 0.05) were considered to be statistically significant. 3. Results In this study, using two doses of 2.5 and 5 mg/kg of morphine and by adapting the same protocol of our previous studies (Azizi et al., 2009; Moaddab et al., 2009; Zarepour et al., 2013), it was found that lowest effective dose of morphine was 5 mg/kg. At this dosage, Dunnett's multiple comparison test showed significant differences in conditioning scores among the experimental and vehicle (saline) groups [F(6,45) = 10.49; P b 0.0001;] (Fig. 2). 3.1. Effects of concurrent intra-accumbal administration of MTEP during acquisition period on rewarding properties of morphine To evaluate the role of mGluR5 in NAc during development of morphine-induced CPP, the effect of concurrent administration of intra-accumbal MTEP and systemic morphine during acquisition period were examined. In this set of experiments, we examined the effects of three doses of MTEP (0.3, 1 and 3 μg/0.5 μl saline) microinjected into
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one-way ANOVA followed by Newman–Keuls multiple comparison test [F (5, 41) = 22.11; P b 0.0001] revealed that there were significant differences in conditioning scores among the vehicle (saline bilaterally microinjected into NAc) treated and MTEP treated animals, in both saline and morphine treated animals. The data showed that intra-NAc administration of MTEP in combination with effective systemic morphine during the acquisition period notably attenuated the rewarding properties of morphine in CPP paradigm (Fig. 3A). Additionally, administration of the highest dose of MTEP (3 μg/0.5 μl saline) alone could not affect the conditioning scores in saline-treated animals (Fig. 3A). One-way ANOVA indicated that none of the groups showed significant differences in locomotor activity [F(5,41) = 0.1451, P = 0.8951; Fig. 3B] (Fig. 3). 3.2. Effects of single microinjection of MTEP into NAc on expression of morphine-induced conditioned place preference In this set of experiments, in order to find out the impact of mGluR5 within NAc on expression of morphine-induced CPP, the effects of single intra-NAc injection of 3 doses of MTEP (0.3, 1 and 3 μg/0.5 μl saline) on post-conditioning day were examined. Control animals received saline (0.5 μl/side) into NAc, instead of MTEP. As shown in Fig. 4A, one-way ANOVA revealed that none of the groups showed significant differences in conditioning scores among the vehicle (saline bilaterally microinjected into NAc) treated and MTEP treated animals [F(5,41) = 10.73, P b 0.0001]. On the other hand, one-way ANOVA indicated that none of the groups showed significant differences in locomotor activity [F(5,41) = 0.1533, P = 0.8981; Fig. 4B] (Fig. 4). 4. Discussion
Fig. 1. Schematic illustrations of coronal sections of the rat brain showing the approximate location of NAc injection sites in the experiments. The numbers indicate anterior coordinates relative to bregma. Atlas plates are adapted from Paxinos and Watson (n = 84).
NAc 5 min before subcutaneous injection of an effective dose of morphine (5 mg/kg; sc) during 3-day conditioning phase. Control group animals received saline (0.5 μl/side) into NAc, instead of MTEP. In Fig. 3A,
Fig. 2. Effect of subcutaneous (sc) administration of different doses of morphine on induction of conditioned place preference in rat. Each morphine dose (0.5, 1, 2.5, 5, 7.5 and 10 mg/kg; sc) was injected in the 3-day conditioning phase. Each point shows the mean ± S.E.M. for 6–7 rats. **P b 0.01, ***P b 0.001 different from the saline control group.
In the present study, the effect of MTEP as an mGluR5 antagonist within NAc on development of morphine-induced CPP in rats was investigated. The major outcomes of the present study were: (1) using our CPP protocol we found that, 5 mg/kg of morphine was the lowest effective conditioning dose; (2) administration of MTEP into NAc decreases development of morphine CPP, and (3) administration of MTEP into NAc had no effect on expression of morphine CPP. The present results are consistent with previous findings in which morphine, dosedependently induced reward and conditioned place preference (Azizi et al., 2009; Moaddab et al., 2009; Reisi et al., 2014; Zarepour et al., 2013). To our knowledge, this is the first study which has examined the effect of intra-accumbal mGluR5 blockage on morphine-induced CPP. In agreement with our findings, previous studies have shown that systemic administration of MTEP attenuates morphine-induced CPP (Veeneman et al., 2010). It is noticeable that previous studies had indicated that blockade of mGluR5 attenuated the rewarding properties of other drugs of abuse, including cocaine (Veeneman et al., 2010), methamphetamine (Chesworth et al., 2013; Gass et al., 2008), alcohol (Backstrom et al., 2004) and nicotine (Paterson et al., 2003). Besheer et al. (2010) showed that microinjection of MPEP into NAc reduced ethanol self administration (SA) without alteration of locomotor activity (Besheer et al., 2010). While D'Souza and Markou (2011) have recently shown that low doses of MPEP micro infused into NAc shell had no effect on nicotine SA, higher MPEP doses attenuated the reinforcing effects of nicotine (D'Souza and Markou, 2011). On the other hand, McGeehan and Olive (2003) have shown that blockade of mGluR5 had no effect on morphine CPP (McGeehan and Olive, 2003). Moreover, other studies showed that, mGluR5 knockout mice exhibit normal conditioned place preference to cocaine, in comparison with wild-type mice (Fowler et al., 2011). In this study, we examined the effect of blockade mGluR5 in the entire NAc because of leakage of the drug to the adjacent area is too difficult to divide the effects of drug on such a closed structure (core vs. shell sub regions of NAc). Several studies have suggested that mGluR5 antagonists can impair associative learning (Bikbaev et al., 2008; Gravius et al., 2008; Naie and Manahan-Vaughan, 2004) which is consistent with our results regarding
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Fig. 3. Effect of bilateral administration of different doses of MTEP during acquisition into the Nucleus accumbens on (A) conditioning score and (B) locomotor activity recorded in the post conditioning (test) day in the rats. Animals in all groups except saline and MTEP groups received morphine (5 mg/kg) and MTEP (0, 0.3, 1, 3, μg/0.5 μl saline) 5 min before morphine during acquisition period. Each point shows the mean ± S.E.M. for 6–8 rats. *P b 0.05, **P b 0.01, ***P b 0.001 different from the saline control group. ††P b 0.01 different from the respective vehicle group. ††P b 0.01 and †††P b 0.001.
the positive role for mGluR5 in morphine induced CPP. It is possible that MTEP impairs the formation of an association between the environmental cues in the morphine-paired compartment and the rewarding properties of the drug (Fendt and Schmid, 2002; Gravius et al., 2005), without influencing the rewarding effects of morphine.
Kumaresan et al. (2009) examined the effect of two mGluR5 antagonists MPEP and MTEP on reinstatement of cocaine seeking, and showed that both drugs attenuated reinstatement of drug seeking in a dose-dependent manner (Kumaresan et al., 2009). We showed here that acute blockade of mGluR5 into NAc with MTEP did not affect
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response found by MTEP. Future studies are needed to characterize the specific mechanisms of action of mGluR5 in acquisition and expression of morphine-induced place preference in rats. Acknowledgment The authors would like to thank Dr. Sankar Venkatachalam for his comments and editing our manuscript. This study was supported by the grant from Hamadan University of Medical Science, Hamadan, Iran (39452-90). References
Fig. 4. Effects of administration of different doses of MTEP into the NAc in the post conditioning (test) day on (A) conditioning score and (B) locomotor activity in CPP protocol recorded in the test day. Each point shows the mean ± S.E.M. for 6–8 rats. **P b 0.01, ***P b 0.001 different from the saline control group.
expression of morphine induced CPP in adult rats. Contrarily, Popik and Wrobel (2002) have reported that MPEP can inhibit expression of morphine induced CPP (Popik and Wrobel, 2002). This may be due to the difference in the mode of administration of the mGluR5 antagonist. We injected directly to the NAc, whereas in Popik and Wrobel (2002) used a systemic administration. Secondly, we used MTEP which is more selective than MPEP for mGluR5. MPEP can block both mGluR5 and NMDA receptors. Finally, intraperitoneal injection of MPEP in Popik and Wrobel's (2002) study might have acted through NMDA or mGluR5 in other brain structures like hippocampus. Whether NMDA or mGluR5 in hippocampus or any other brain structure is involved in morphine induced CPP remains to be elucidated. Taken together, the available evidences indicate an important modulatory rather than necessary role for mGluR5 in NAc based morphine reward. It is concluded that mGluR5 within NAc modulates the rewarding effect of morphine during acquisition of conditioned place preference in rats. It can be proposed that the plasticity related to mGluR5 downstream proteins during CPP of morphine could account for the behavioral
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Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Microinjection of the mGluR5 antagonist MTEP into the nucleus accumbens attenuates the acquisition but not expression of morphine-induced conditioned place preference in rats Nahid Roohi a, Abdolrahman Sarihi a,⁎, Siamak Shahidi a, Mohammad Zarei a, Abbas Haghparast b,⁎⁎ a b
Neurophysiology Research Center, Hamadan University of Medical Sciences, P.O. Box 65178-38678, Hamadan, Iran Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, P.O. Box 19615-1178, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 26 May 2014 Received in revised form 18 September 2014 Accepted 26 September 2014 Available online 2 October 2014 Keywords: Metabotropic glutamate receptor 5 Nucleus accumbens Acquisition Expression Morphine Conditioned place preference Rat
a b s t r a c t Previous studies suggest that metabotropic glutamate receptor type 5 (mGluR5) plays an important role in modulation of the rewarding properties of morphine. Little is known about the role of mGluR5 in the nucleus accumbens (NAc), as one of the important regions of the reward circuitry. In the present study, we investigated the effects of intra-accumbal injection of mGluR5 antagonist, 3-[(2-methyl-4-thiazolyl) ethynyl] pyridine, MTEP, on the acquisition and expression of morphine induced Conditioned Place Preference (CPP) in the rats. Eighty four adult male Wistar rats (220–260 g) were bilaterally implanted with cannulae into the NAc. Subjects were tested in a CPP paradigm. Different doses of MTEP (0.3, 1 and 3 μg/0.5 μl per side) were delivered bilaterally into NAc during 3 conditioning days (Acquisition) or post-conditioning day (Expression). Results showed that bilateral intra accumbal administration of MTEP (1 and 3 μg) significantly attenuated the acquisition but not expression of morphine-induced CPP in a dose-dependent manner. Our findings indicated that blockade of mGluR5 reduces rewarding properties of morphine. Further studies are needed to know the involved mechanisms. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Opiate addiction is a chronic, relapsing disorder characterized by compulsive drug seeking and a high risk of drug craving which is often precipitated by drug-associated cues, even after long periods of abstinence (De Vries and Shippenberg, 2002). It has been reported that repeated drug exposure elicits changes in central nervous system during the development of addiction (Koob, 2002). The mesolimbic reward system, which extends from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), has been identified as one of the neuronal pathways for reward and drug addiction (Koob, 1992). The NAc, localized within the ventral region of the corpus striatum, is a major target and a central component of the reward system. It receives massive dopaminergic inputs from the VTA and glutamatergic afferents from structures such as the hippocampus, prefrontal cortex, and amygdala (Groenewegen et al., 1996; Vezina and Kim, 1999).
⁎ Correspondence to: A. Sarihi, Neurophysiology Research Center and Department of Physiology, Hamadan University of Medical Sciences, Hamadan, Iran. Tel./fax: +98 811 838 0131. ⁎⁎ Corresponding author. Tel./fax: +98 21 2243 1624. E-mail addresses: [email protected], [email protected] (A. Sarihi), [email protected], [email protected] (A. Haghparast).
http://dx.doi.org/10.1016/j.pbb.2014.09.020 0091-3057/© 2014 Elsevier Inc. All rights reserved.
Glutamate is one of the major neurotransmitters of the central nervous system. It is involved in a wide number of physiological and pathological processes (Kalivas et al., 2009). Glutamate receptors belong to two major groups: viz. ionotropic and metabotropic receptors (Hollmann and Heinemann, 1994). Based on sequence homology, pharmacology, and second messenger activation there are eight metabotropic glutamate receptors (mGluR) which are classified under three groups (Conn and Pin, 1997). Predominant receptor subtypes expressed in NAc include mGluR2/3, mGluR5, and mGluR7 (Pomierny-Chamioto et al., 2014; Tallaksen-Greene et al., 1998), specifically of mGluR5 type. mGluR5 are primarily located on the postsynaptic terminal in medium spiny GABAergic neurons and are rarely found on presynaptic terminals. This could play a role through GABAergic neurons activity modulation in drug addiction (Kenny and Markou, 2004). It has been shown that mGluR5 is located in areas such as the hippocampus, cerebral cortex, NAc, lateral septum, and the dorsal striatum (Kenny and Markou, 2004). Distribution of mGluR5 in the neural circuitry involved in reward consumption and seeking provoked considerable interest as a potential therapeutic target for drug addiction. Chiamulera et al. (2001) showed that mice lacking mGluR5 gene do not self administer cocaine, which shows that mGluR5 has an essential role in reinforcement, and from thereafter mGluR receptors came into the focus of addiction researchers (Chiamulera et al., 2001). In addition, reduction of mGluR5 signaling decreases drug taking and drug-seeking behaviors
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for alcohol (Backstrom et al., 2004), cocaine (Veeneman et al., 2010), methamphetamine (Chesworth et al., 2013; Gass and Olive, 2009), opiates (Brown et al., 2012; Popik and Wrobel, 2002; Veeneman et al., 2010) and nicotine (Paterson et al., 2003). Furthermore, it has been shown that intraperitoneal administration of 2-methyl-6(phenylethynyl) pyridine (MPEP) blocked the development of cocaine and morphine conditioned place preference (CPP) in rats (Herzig and Schmidt, 2004). Since mGluR5 antagonist administration was carried out systemically, the local role of mGluR5 in intended brain structure remains unclear. In this study, we used intra nucleus accumbens microinjection of selective mGluR5 antagonist, 3-[(2-methyl-4-thiazolyl) ethynyl] pyridine (MTEP). On the other hand, CPP paradigm is one of the most widely used experimental protocols used for measuring drug reward in laboratory animals (Bardo and Bevins, 2000). CPP reflects a preference for a context due to the contiguous association between the context and a drugassociated stimulus. Therefore, it is a simple and effective method for assessing the rewarding properties of drugs (Bardo et al., 1995). It has been revealed that systemic administration of MTEP blocks both the acquisition and expression of morphine CPP (Veeneman et al., 2010). In addition, Popik and Wrobel (2002) demonstrated that MPEP injected intraperitoneally also blocked both acquisition and expression phases of CPP for morphine (Popik and Wrobel, 2002). Out of the two, MTEP is a more specific antagonist for mGluR5 rather than MPEP, as MPEP can also block NMDA receptors, which should be considered in the interpretation of mechanisms involved in the results obtained with MPEP (Lea and Faden, 2006). Observations of potentiation in morphine CPP with MPEP suggest that mGluR5 antagonists may partly substitute for opiate reward (Rutten et al., 2011). Recently, Brown et al. (2012) reported that systemic administration of MTEP attenuates operant responding for morphine self-administration and cue-induced morphine-seeking after a period of forced abstinence (Brown et al., 2012). While mGluR5 has been implicated in the opioid's rewarding properties such as a morphine conditioning reward, the role of mGluR5 at NAc has not been elucidated. Therefore, in the present study, we assessed the effects of mGluR5 blockade into the NAc on the acquisition and expression of morphine-induced place preference. 2. Materials and methods 2.1. Animal Eighty four adult male albino Wistar rats (Pasteur Institute, Tehran, Iran) weighing 200–250 g were housed in standard plastic cages in groups of three in a temperature controlled room (temperature 21 ± 3 °C). They were maintained on a 12 h light/dark cycle (lights on at 07:00 a.m.) with food and water ad libitum. The experiments were carried out during the light phase of the cycle. Six to eight animals were used per group and each animal was tested once. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) and were approved by the Research and Ethics Committee of Hamadan University of Medical Sciences. 2.2. Drug The following drugs were used: morphine sulfate (Temad, Iran) dissolved in sterile saline (0.9%); 3-[(2-methyl-4-thiazolyl) ethynyl] pyridine; MTEP; mGluR5 antagonist (Tocris bioscience, UK), dissolved in saline. It is worth mentioning that control animals received saline. 2.3. Stereotaxic surgery Subjects were anesthetized by Xylazine (10 mg/kg) and Ketamine (100 mg/kg) and placed in the stereotaxic apparatus (Stoelting, USA),
with the incisor bar set at approximately 3.3 mm below horizontal zero in order to achieve a flat skull position. After an incision was made to expose the rat's skull, two points were determined and drilled into the skull at the following stereotaxic coordinates 1.4 ± 0.4 mm anterior to bregma, ± 1.5 mm lateral to the sagital suture, and 6.5 mm down from top the skull according to the atlas of rat brain (Paxinos and Watson, 2007).Two guide cannulae (23-Gauge) with 12 mm length were inserted into the holes aiming at NAc. The guide cannulae were anchored with a jeweler's screw and the incision was closed with dental cement. After surgery, dummy inner cannulae that extended 0.5 mm beyond the guide cannulae were inserted into the guide cannulae and left in place until injections were made. All animals were allowed to recover for one week before starting the behavioral testing. 2.4. Intra-accumbal injection Animals were gently restrained by hand and the dummy cannulae were removed from the guide cannulae. Drugs were directly injected into NAc through the guide cannulae by using injector cannulae (30gauge, 1 mm below the tip of the guide cannulae). Polyethylene tubing (PE-20) was used for attaching the injector cannulae to the 1-μl Hamilton syringe. The injection volume was 0.5 μl/side into NAc for all groups. Injections were made bilaterally over a 50 s period, and the injection cannulae were left in the guide cannulae for an additional 60 s in order to facilitate the diffusion of the drugs. The microinjection time for 0.1 μl volume of drugs was 10 s for preventing lesions in these areas (Karimi et al., 2014; Reisi et al., 2014). 2.5. Place conditioning apparatus A three-compartment CPP apparatus was used in these experiments. Place conditioning was conducted using an unbiased protocol, modified slightly from a design previously described (Zarepour et al., 2013). The apparatus was divided into two equal-sized compartments (30 cm × 30 cm × 40 cm) with the third section (30 cm × 15 cm × 40 cm) being the null section which connected the two equal-sized sections. Both compartments had white backgrounds with black stripes in different orientations (vertical vs. horizontal).To provide a tactile difference between the compartments, one of them had a smooth floor, while the other compartment had a net-like floor. In this apparatus, rats showed no consistent preference for either compartment. In the CPP paradigm, the conditioning score and distance traveled were calculated from a video, recorded by a CCD camera with 30 frames per second (fps) resolution (Panasonic Inc., Japan). The camera was placed 2 m above the CPP boxes and locomotion tracking was measured by Maze Router homemade software, a video tracking system for automation of behavioral experiments. In these experiments, on the pre- and post-conditioning phases, each animal was allowed to freely access two compartments for 10 min. The time spent and total distance traveled in each compartment of the CPP apparatus were measured during this period. CPP paradigm took place for 5 continuous days, which consisted of three distinct phases: pre-conditioning, conditioning and post-conditioning. 2.6. CPP protocol The CPP protocol was modified from that described previously by our group (Haghparast et al., 2009; Haghparast et al., 2013; Reisi et al., 2014; Zarepour et al., 2013). Before each session, the rats were habituated to the experimental room for 30 min. On day 1 (preconditioning), they were placed in the central compartment and allowed free access to the entire apparatus. On days 2–4 (conditioning), they received injections of saline or morphine and were immediately confined in one of the two conditioning compartments. An unbiased allocation was used. Rats with a neutral preference (45–55% for either side) were randomly allocated their drug-paired side (unbiased allocation). On test day (post conditioning), the rats were given free access to the CPP apparatus.
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All conditioning sessions were 30 min in duration and occurred at the same time each day. Place preference was calculated as a preference score (time spent in drug paired zone − time spent in the saline paired zone). Locomotor data were also collected throughout CPP testing in order to assess the development of behavioral sensitization. 2.6.1. Pre-conditioning phase On day 1, each rat was separately placed in the apparatus for 10 min, with free access to all compartments. Animal movements were recorded by Maze Router tracking software and analyzed on the same day. No significant differences between times spent in either of the two compartments were considered as unbiased conditioning. Rats with any compartment preference were omitted from the experiment. In this experimental model, only the animals that did not show a preference in baseline test, to either of the compartments, was used for conditioning phase. These rats were randomly assigned to one of the two groups (odd and even) for place conditioning, which 6–8 animals were used for each subsequent experiment. 2.6.2. Conditioning phase Each group of animals was randomly divided into even or odd. Odd animals received saline/morphine pairing in alternative morning and afternoon design with an interval of 6 h. The vice versa program for even animals was done. This phase consisted of a 3-day schedule of conditioning sessions. A total of six sessions (30 min each) was carried out. During these 3 conditioning days, in 3 sessions, animals were confined to one compartment, under the drug influence. During other three sessions, they were injected with saline while confined to the other compartment. Access to the other compartments was blocked on these days. 2.6.3. Post-conditioning phase On the 5th day, the partition was removed and the rats could access the entire apparatus. The mean time spent for each rat in both compartments during a 10-min period was recorded. In order to calculate the conditioning score, the difference in time spent for the drug- and saline-paired places was considered as the preference criteria. In the acquisition tests, no injection was given on the post-conditioning day. Total distance traveled for each animal was also measured on pre- and post-conditioning days for a 10-min period as the locomotor activity in control and experimental groups. 2.7. Experimental design The effects of subcutaneous (sc) administrations of morphine on induction of place conditioning in animals were determined in previous studies (Reisi et al., 2014; Taslimi et al., 2012; Zarepour et al., 2013). Each morphine dose (0.5, 1, 2.5, 5, 7.5 and 10 mg/kg; sc) was injected on a 3-day schedule of conditioning as described above. Then, animals were tested in a morphine-free state in order to eliminate the influence of morphine induced motor effects on responses (Olmstead and Franklin, 1997). Conditioning score and distance traveled were calculated for each rat (Haghparast et al., 2009; Rezayof et al., 2011). In this experiment, morphine conditioned place preference paradigm was established by using a dose of morphine 5 mg/kg. Based on our previous studies, we have found that the lowest effective dose was 5 mg/kg. To confirm the findings, once again, we included 2.5 mg/kg dosage along with 5 mg/kg dosage regime. Morphine at 5 mg/kg resulted in conditioned place preference while 2.5 mg/kg failed to do so. This reiterates our earlier finding that 5 mg/kg is the lowest effective dose. As stated previously, control animals received saline (1 ml/kg; sc). 2.7.1. Effects of intra-accumbal injection of mGluR5 antagonist, MTEP on the acquisition of morphine-induced conditioned place preference To test the effects of MTEP microinjected into NAc on development of morphine rewarding properties, experiments were performed on
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saline and morphine-treated groups, which received the effective dose of morphine (5 mg/kg; sc) during the acquisition period. MTEP (0.3, 1 and 3 μg/0.5 μl) (Gass and Olive, 2009) was bilaterally microinjected into NAc, 5 min prior to the injection of either morphine or saline during the 3-day conditioning phase. The control group received vehicle (saline) into NAc. Conditioning score and distance traveled were measured during a 10-min period on the test day. 2.7.2. Effects of intra-accumbal injection of mGluR5 antagonist on the expression of morphine-induced conditioned place preference In this set of experiments, for evaluating the effects of single injection of mGluR5 receptor antagonist on the expression of morphine rewarding properties in conditioned place preference paradigm, different doses of MTEP (0.3, 1 and 3 μg/0.5 μl) were bilaterally microinjected into NAc, just 5 min prior to CPP test in saline- and morphine-treated groups which received effective dose of morphine during the acquisition period. Conditioning score and distance traveled were measured during 10 min on the test day. 2.8. Locomotor activity measurement In order to evaluate the effect of drugs on locomotor activity in animals, total distance traveled (cm) during 10-min test period, on the pre- and post-conditioning phases was measured by Maze Router homemade software. 2.9. Histology After completion of behavioral testing, the animals were deeply anesthetized with ketamine and xylazine. Then, they were transcardially perfused with 0.9% saline and 5% formalin solution. Their brains were removed, blocked and cut coronally in 50 μm sections through cannulae placements. The neuroanatomical location of cannulae tips placements was confirmed using rat brain atlas (Paxinos and Watson, 2007). Only the animals with correct cannulae placements in NAc were included in the data analysis (Fig. 1). 2.10. Statistics Data were processed by commercially available software GraphPad Prism® 5.0. In order to compare the conditioning scores and distance traveled obtained from all groups (vehicle and experimental groups), one-way analysis of variance (ANOVA) followed by post hoc analysis (Newman–Keuls multiple comparison test) was used, as appropriate. In the first experiment, Dunnett's multiple comparison test was used. P-values less than 0.05 (P b 0.05) were considered to be statistically significant. 3. Results In this study, using two doses of 2.5 and 5 mg/kg of morphine and by adapting the same protocol of our previous studies (Azizi et al., 2009; Moaddab et al., 2009; Zarepour et al., 2013), it was found that lowest effective dose of morphine was 5 mg/kg. At this dosage, Dunnett's multiple comparison test showed significant differences in conditioning scores among the experimental and vehicle (saline) groups [F(6,45) = 10.49; P b 0.0001;] (Fig. 2). 3.1. Effects of concurrent intra-accumbal administration of MTEP during acquisition period on rewarding properties of morphine To evaluate the role of mGluR5 in NAc during development of morphine-induced CPP, the effect of concurrent administration of intra-accumbal MTEP and systemic morphine during acquisition period were examined. In this set of experiments, we examined the effects of three doses of MTEP (0.3, 1 and 3 μg/0.5 μl saline) microinjected into
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one-way ANOVA followed by Newman–Keuls multiple comparison test [F (5, 41) = 22.11; P b 0.0001] revealed that there were significant differences in conditioning scores among the vehicle (saline bilaterally microinjected into NAc) treated and MTEP treated animals, in both saline and morphine treated animals. The data showed that intra-NAc administration of MTEP in combination with effective systemic morphine during the acquisition period notably attenuated the rewarding properties of morphine in CPP paradigm (Fig. 3A). Additionally, administration of the highest dose of MTEP (3 μg/0.5 μl saline) alone could not affect the conditioning scores in saline-treated animals (Fig. 3A). One-way ANOVA indicated that none of the groups showed significant differences in locomotor activity [F(5,41) = 0.1451, P = 0.8951; Fig. 3B] (Fig. 3). 3.2. Effects of single microinjection of MTEP into NAc on expression of morphine-induced conditioned place preference In this set of experiments, in order to find out the impact of mGluR5 within NAc on expression of morphine-induced CPP, the effects of single intra-NAc injection of 3 doses of MTEP (0.3, 1 and 3 μg/0.5 μl saline) on post-conditioning day were examined. Control animals received saline (0.5 μl/side) into NAc, instead of MTEP. As shown in Fig. 4A, one-way ANOVA revealed that none of the groups showed significant differences in conditioning scores among the vehicle (saline bilaterally microinjected into NAc) treated and MTEP treated animals [F(5,41) = 10.73, P b 0.0001]. On the other hand, one-way ANOVA indicated that none of the groups showed significant differences in locomotor activity [F(5,41) = 0.1533, P = 0.8981; Fig. 4B] (Fig. 4). 4. Discussion
Fig. 1. Schematic illustrations of coronal sections of the rat brain showing the approximate location of NAc injection sites in the experiments. The numbers indicate anterior coordinates relative to bregma. Atlas plates are adapted from Paxinos and Watson (n = 84).
NAc 5 min before subcutaneous injection of an effective dose of morphine (5 mg/kg; sc) during 3-day conditioning phase. Control group animals received saline (0.5 μl/side) into NAc, instead of MTEP. In Fig. 3A,
Fig. 2. Effect of subcutaneous (sc) administration of different doses of morphine on induction of conditioned place preference in rat. Each morphine dose (0.5, 1, 2.5, 5, 7.5 and 10 mg/kg; sc) was injected in the 3-day conditioning phase. Each point shows the mean ± S.E.M. for 6–7 rats. **P b 0.01, ***P b 0.001 different from the saline control group.
In the present study, the effect of MTEP as an mGluR5 antagonist within NAc on development of morphine-induced CPP in rats was investigated. The major outcomes of the present study were: (1) using our CPP protocol we found that, 5 mg/kg of morphine was the lowest effective conditioning dose; (2) administration of MTEP into NAc decreases development of morphine CPP, and (3) administration of MTEP into NAc had no effect on expression of morphine CPP. The present results are consistent with previous findings in which morphine, dosedependently induced reward and conditioned place preference (Azizi et al., 2009; Moaddab et al., 2009; Reisi et al., 2014; Zarepour et al., 2013). To our knowledge, this is the first study which has examined the effect of intra-accumbal mGluR5 blockage on morphine-induced CPP. In agreement with our findings, previous studies have shown that systemic administration of MTEP attenuates morphine-induced CPP (Veeneman et al., 2010). It is noticeable that previous studies had indicated that blockade of mGluR5 attenuated the rewarding properties of other drugs of abuse, including cocaine (Veeneman et al., 2010), methamphetamine (Chesworth et al., 2013; Gass et al., 2008), alcohol (Backstrom et al., 2004) and nicotine (Paterson et al., 2003). Besheer et al. (2010) showed that microinjection of MPEP into NAc reduced ethanol self administration (SA) without alteration of locomotor activity (Besheer et al., 2010). While D'Souza and Markou (2011) have recently shown that low doses of MPEP micro infused into NAc shell had no effect on nicotine SA, higher MPEP doses attenuated the reinforcing effects of nicotine (D'Souza and Markou, 2011). On the other hand, McGeehan and Olive (2003) have shown that blockade of mGluR5 had no effect on morphine CPP (McGeehan and Olive, 2003). Moreover, other studies showed that, mGluR5 knockout mice exhibit normal conditioned place preference to cocaine, in comparison with wild-type mice (Fowler et al., 2011). In this study, we examined the effect of blockade mGluR5 in the entire NAc because of leakage of the drug to the adjacent area is too difficult to divide the effects of drug on such a closed structure (core vs. shell sub regions of NAc). Several studies have suggested that mGluR5 antagonists can impair associative learning (Bikbaev et al., 2008; Gravius et al., 2008; Naie and Manahan-Vaughan, 2004) which is consistent with our results regarding
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Fig. 3. Effect of bilateral administration of different doses of MTEP during acquisition into the Nucleus accumbens on (A) conditioning score and (B) locomotor activity recorded in the post conditioning (test) day in the rats. Animals in all groups except saline and MTEP groups received morphine (5 mg/kg) and MTEP (0, 0.3, 1, 3, μg/0.5 μl saline) 5 min before morphine during acquisition period. Each point shows the mean ± S.E.M. for 6–8 rats. *P b 0.05, **P b 0.01, ***P b 0.001 different from the saline control group. ††P b 0.01 different from the respective vehicle group. ††P b 0.01 and †††P b 0.001.
the positive role for mGluR5 in morphine induced CPP. It is possible that MTEP impairs the formation of an association between the environmental cues in the morphine-paired compartment and the rewarding properties of the drug (Fendt and Schmid, 2002; Gravius et al., 2005), without influencing the rewarding effects of morphine.
Kumaresan et al. (2009) examined the effect of two mGluR5 antagonists MPEP and MTEP on reinstatement of cocaine seeking, and showed that both drugs attenuated reinstatement of drug seeking in a dose-dependent manner (Kumaresan et al., 2009). We showed here that acute blockade of mGluR5 into NAc with MTEP did not affect
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response found by MTEP. Future studies are needed to characterize the specific mechanisms of action of mGluR5 in acquisition and expression of morphine-induced place preference in rats. Acknowledgment The authors would like to thank Dr. Sankar Venkatachalam for his comments and editing our manuscript. This study was supported by the grant from Hamadan University of Medical Science, Hamadan, Iran (39452-90). References
Fig. 4. Effects of administration of different doses of MTEP into the NAc in the post conditioning (test) day on (A) conditioning score and (B) locomotor activity in CPP protocol recorded in the test day. Each point shows the mean ± S.E.M. for 6–8 rats. **P b 0.01, ***P b 0.001 different from the saline control group.
expression of morphine induced CPP in adult rats. Contrarily, Popik and Wrobel (2002) have reported that MPEP can inhibit expression of morphine induced CPP (Popik and Wrobel, 2002). This may be due to the difference in the mode of administration of the mGluR5 antagonist. We injected directly to the NAc, whereas in Popik and Wrobel (2002) used a systemic administration. Secondly, we used MTEP which is more selective than MPEP for mGluR5. MPEP can block both mGluR5 and NMDA receptors. Finally, intraperitoneal injection of MPEP in Popik and Wrobel's (2002) study might have acted through NMDA or mGluR5 in other brain structures like hippocampus. Whether NMDA or mGluR5 in hippocampus or any other brain structure is involved in morphine induced CPP remains to be elucidated. Taken together, the available evidences indicate an important modulatory rather than necessary role for mGluR5 in NAc based morphine reward. It is concluded that mGluR5 within NAc modulates the rewarding effect of morphine during acquisition of conditioned place preference in rats. It can be proposed that the plasticity related to mGluR5 downstream proteins during CPP of morphine could account for the behavioral
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