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Role of orexin receptors in the ventral tegmental area on acquisition and expression of morphine-induced conditioned place preference in the rats
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Role of orexin receptors in the ventral tegmental area on acquisition and expression of morphine-induced conditioned place preference in the rats Sharareh Farahimanesha,b, Shahram Zarrabianc, Abbas Haghparasta,⁎ a b c
Neuroscience Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Institute for cognitive Science Studies, Tehran, Iran Cognitive and Neuroscience Research Center (CNRC), Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords: Reward Orexin receptors Ventral tegmental area Acquisition Expression Conditioned placed preference
The orexins are hypothalamic neuropeptides and their role in reward processing and drug addiction has been demonstrated. The extent of involvement of each orexin receptor in the acquisition and expression of conditioned place preference (CPP) for morphine is still a matter of controversy. We investigated the functional differences between orexin-1 and -2 receptor blockade in the ventral tegmental area (VTA) on the acquisition and expression of morphine CPP. A total of 86 adult male Wistar rats weighing 250 ± 30 g (age 7–8 weeks) received intra-VTA microinjection of either SB334867 (0.1, 1 and 10 nM), a selective orexin-1 receptor (OX1R) antagonist, or TCS-OX2-29 (1, 5 and 25 nM), a selective orexin-2 receptor (OX2R) antagonist. To measure the acquisition, the animals received each antagonist (SB334867 or TCS-OX2-29) 5 min prior to subcutaneous injection of morphine (5 mg/kg) during the conditioning phase. To measure the CPP expression, the animals received each antagonist on the post-conditioning phase. The CPP conditioning score was recorded by Ethovision software. Data showed that intra-VTA microinjection of OX1-R antagonist significantly attenuated morphine CPP acquisition, during the conditioning phase, and expression, during the post-conditioning phase. Intra-VTA microinjection of OX2-R antagonist also significantly attenuated morphine CPP acquisition and expression in the mentioned phases. Our results showed the orexin role in learning and memory and indicate that orexin receptors (OX1R and OX2R) function in the VTA is essential for both acquisition and expression of morphine reward in rats in the CPP model.
1. Introduction The orexins (hypocretins) were first described in 1998 and comprise orexin A and B with 33 and 28 amino acids in length, respectively. Two distinct receptors respond to orexin stimulation (OX1R and OX2R) (Kukkonen et al., 2002). Both orexin receptors are coupled to Gq, whereas OX2R additionally is coupled to Gi/Go (Coleman et al., 2012). The orexin receptors have equal affinity for orexin A, but orexin B binds relatively specifically to OX2Rs (Sakurai et al., 1998). Orexin-expressing neurons are exclusively in the hypothalamus (the lateral hypothalamus (LH), prefrontal area (PFA) and dorso-medial hypothalamic nuclei (DMH) areas), receive inputs especially from brainstem and reward-related nuclei (Narita et al., 2006). Extensive CNS projections of these neurons were reported in 1998 (Peyron et al., 1998). The axons of orexinergic neurons were seen in the anterior olfactory nucleus, piriform cortex, tenia tecta, CA1-3 regions of hippocampus, septal nucleus, amygdaloid complex, posterior lobe of the pituitary, many parts of the cerebellum, thalamic nuclei, and ventral tegmental area (VTA) (Nambu et al., 1999).
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The OX1Rs are primarily expressed in the VTA, cortical regions, amygdala and bed nucleus of the stria terminalis, prefrontal and infralimbic cortex, hippocampus (CA2), anterior hypothalamus, laterodorsal tegmental nucleus/pedunculopontine nucleus, dorsal raphe, and the locus coeruleus (Lu et al., 2000; Marcus et al., 2001; Trivedi et al., 1998). The OX2R density is enriched in the medial septal nucleus, amygdala, paraventricular nucleus (PVN), CA3 in the hippocampus, nucleus accumbens (NAc), DMH, and VTA. The gathered evidence shows that both receptors have a unique and overlapping expression (Cluderay et al., 2002; Lu et al., 2000; Marcus et al., 2001). The orexin system affects homeostatic functions and orexin neurons functional dichotomy has been described as PFA and DMH neurons are mainly involved in sleep/wakefulness, arousal and stress responses (de Lecea, 2012; Sakurai, 2005) and LH neurons are mainly involved in feeding, reward processing, addiction, memory for stimulus–reward relationships, and synaptic plasticity (Baimel and Borgland, 2012; Harris et al., 2005). A dichotomy in function also has been suggested in which OX1Rs and OX2Rs are associated with reward and arousal,
Corresponding author at: Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, P. O. Box: 19615-1178, Tehran, Iran. E-mail address: [email protected] (A. Haghparast).
http://dx.doi.org/10.1016/j.npep.2017.08.003 Received 10 May 2017; Received in revised form 8 August 2017; Accepted 25 August 2017 0143-4179/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Farahimanesh, S., Neuropeptides (2017), http://dx.doi.org/10.1016/j.npep.2017.08.003
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guides) was used for the injection into the VTA over a 1 min period. The inner cannula was left in place for an additional 30 s to allow diffusion of the solution and to reduce the possibility of reflux. Intra-VTA injections were made 5 min before each experiment.
respectively (Akanmu and Honda, 2005; Aston-Jones et al., 2010; Marcus et al., 2001). The VTA is a key site in reward- and addiction-related behaviors, receives heavy innervation from LH orexin neurons, and has high level of orexin receptors (Fadel and Deutch, 2002; Korotkova et al., 2003; Marcus et al., 2001; Richardson and Aston-Jones, 2012). The LH-VTA pathway role in the expression of morphine CPP and the possible role of orexin in learning the associations between environmental cues and morphine have already been reported (Harris and Aston-Jones, 2007; Richardson and Aston-Jones, 2012; Zhou et al., 2006). The circuitry can be incorporated by the recurrent application of drugs of abuse that can lead to drug dependence as the intensity of reward seeking was reported to correlate with the amount of Fos activation in orexinergic neurons (Harris et al., 2005). Research showed that OX1 signaling plays a major role in withdrawal and morphine seeking (Georgescu et al., 2003; Harris et al., 2005) and the LH projections to the VTA functions both in reward processing and reward based learning and memory (Harris and Aston-Jones, 2006). Considering the above fact, OX1R and OX2R antagonists have been used to reduce the self-administration of heroin (Smith and Aston-Jones, 2012) and ethanol (Shoblock et al., 2011), respectively. Playing roles in stress activation and reward-based learning and memory, the orexin system could be a target for preventing drug relapse (Harris and Aston-Jones, 2006). Focusing further, it becomes clear that OX1Rs and OX2Rs drug seeking functional differences is an area of controversy (Baimel et al., 2015; Li et al., 2011) and recent research opens the ground for further investigation into the role of these two receptors in learning and expression of reward induced by morphine. In order to deepen our understanding of the orexin system roles in the VTA, in this study we inspected the role of OX1 and OX2 receptors in morphine CPP acquisition and expression in rats.
2.3. Drugs The following drugs were used in this study: ketamine and xylazine (Alfasan Chemical Co, Holland), morphine sulfate (referred to as morphine; Temad, Iran). SB-334867, orexin receptor subtype OX1 antagonist and TCS-OX2-29 (also called 4-PT), orexin receptor subtype OX2 antagonist (Tocris Bioscience, UK). Morphine was dissolved in sterile saline (0.9%), SB-334867 and TCS-OX2-29 were dissolved in 12% dimethyl sulfoxide (DMSO; Sigma Aldrich, Germany). The control groups received either saline or 12% DMSO as an antagonist vehicle. The drug doses were selected based on pilot- and our previous studies (Ezzatpanah et al., 2016; Sadeghi et al., 2016). 2.4. Conditioning apparatus and paradigm To measure stimulus–reward associations a three-compartment CPP apparatus was used (30 × 30 × 40 cm) according to an unbiased procedure (i.e. the animals that spent ≥ 70% of the total test time (10 min) in either compartment were considered to have an initial bias and were excluded from the study). The apparatus was made of Plexiglas and divided into two equal-sized cue-different compartments, as previously described (Zarepour et al., 2014). The start box, as the third compartment, connects the two cue-different compartments. A guillotine door separated the two main compartments from the start box. The whole experimental process was performed under controlled light conditions (~ 14 Lux, comprising two 15 W bulbs positioned about 1.5 m above the apparatus) and any aggravating noise was avoided. The procedure consists of a five-day schedule with three distinct phases as follows.
2. Materials and methods
2.4.1. Pre-conditioning phase In this phase (day 1), each animal was placed in the start box. The guillotine door was removed and rats were allowed to move freely in all the compartments for 10 min. Time spent in each compartment was recorded using a video camera (Panasonic Inc., Japan) and Ethovision software (Noldus, Version 7). Three animals that showed a preference for one of the compartments on the pre-conditioning phase were excluded from the study (85.9 ± 2.6%).
2.1. Animals A total of 128 adult male albino Wistar rats (Purchased from Pasteur Institute, Iran) were used. The animal's weight and age at the time of surgery was 250 ± 30 g and 7–8 weeks, respectively. The animals randomly were assigned into groups of 5, kept in Plexiglass cages (58 × 38 × 20 cm), and had ad libitum access to food and water. The room temperature and the vivarium were kept at 23 ± 1°C and 12:12 h light/dark cycle (lights on 07:00 h), respectively. The rats were handled about 3 min/day for 2 days before the experiment. Each rat was only used once. All experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) and the experiment protocol was approved by the Research and Ethics Committee of Shahid Beheshti University of Medical Sciences.
2.4.2. Conditioning phase The conditioning phase, also known as the acquisition phase, included days 2 to 4. On the first day (day 2), all the groups received morphine (5 mg/kg) subcutaneously and intra-VTA dose of either SB334867 or TCS-OX2-29 and were confined to the drug paired compartment for 45 min by closing the removable door of the apparatus. Six hours later, all the groups received saline subcutaneously without any intra-VTA treatment. Then, the rats were placed in the non-drug-paired (saline-paired) compartment and their movements were recorded for 45 min. To prevent any time-dependency on drug administration, on the second day of the phase (day 3) the animals received subcutaneous saline in the morning and their active treatment in the afternoon (6 h later). The third day of conditioning phase (day 4) was conducted the same as the first day of this phase.
2.2. Stereotaxic surgery and drug microinjections To stereotactically implant the cannulae, the rats received general anesthesia with an intraperitoneal injection of a mixture of ketamine hydrochloride 10% and xylazine 2% (100 and 10 mg/kg, respectively) and placed in a stereotaxic apparatus (Stoelting, USA). The rats were given the surgical preparation and the area surrounding bregma was cleaned and dried. According to the atlas of the rat brain (Paxinos and Watson, 2007), the VTA coordinates in mm were: AP 4.8 caudal to bregma, ML ± 0.9, and DV 7.3. Two stainless steel guide cannulae (23gauge, 11 mm) were implanted bilaterally and secured to the skull using two stainless steel screws and dental acrylic cement. After one week of recovery, the animals were used for the experiments. A 1-μl Hamilton syringe connected by a polyethylene tube (PE-20) to an internal cannula (30-gauge, terminating 1 mm below the tip of the
2.4.3. Post-conditioning phase In CPP, preference for reward is measured by the amount of time the animal spends in the reward associated chamber minus the time it spends in the non-rewarded chamber, when given free access to both chambers after conditioning. Hence, on day 5 (the test day or the expression phase) in the groups under acquisition investigation, the animals were tested for CPP (under morphine-free condition) with free
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memory expression. The saline/DMSO control group (n = 5) received subcutaneous injection of saline instead of morphine and intra-VTA microinjection of DMSO, as the vehicle of the antagonists. The morphine/DMSO control group (n = 5) received morphine subcutaneously and intra-VTA DMSO (0.3 μl/rat). The animals were put in the drugpaired compartment 5 min after intra-VTA injection of TCS-OX2-29. CPP conditioning scores were calculated during a ten-min period (Supplementary Fig. 1D).
access to all the compartments. To probe the expression of morphine CPP, the animals received either SB-334867 or TCS-OX2-29, at the mentioned doses (Please see the following Section 2.5), 5 min before starting the test. The time spent in each compartment during a ten-min period was recorded. The CPP conditioning score, as a preference index, was calculated as the time spent in the drug-paired compartment minus the time spent in the saline-paired compartment, and the time spent in the start box was not calculated for the CPP conditioning score. 2.5. Experimental design
2.6. Histological verification 2.5.1. Experiment 1: effect of OX1R antagonist microinjection into the VTA on the acquisition of CPP SB-334867 (0.1, 1, and 10 nM/rat; n = 6, 6, 5, respectively), as OX1R antagonist, was microinjected into the VTA 5 min prior to subcutaneous injection of 5 mg/kg morphine during the 3 days of conditioning phase to study the drug effect on the acquisition of morphine reward. The saline/DMSO control group (n = 6) received subcutaneous injection of saline instead of morphine and intra-VTA microinjection of DMSO, as the vehicle of the antagonists. The morphine/DMSO control group (n = 6) received morphine subcutaneously and intra-VTA DMSO (0.3 μl/rat). To investigate the role of SB-334867 in the acquisition phase, the highest dose of SB-334867 (10 nM/rat; n = 5) was microinjected into the VTA 5 min before saline was injected subcutaneously instead of morphine. The animals were put in the drug-paired compartment immediately after subcutaneous injection of morphine. The CPP conditioning scores were calculated during a ten-min period (Supplementary Fig. 1A).
The following procedures were performed on the animals after the completion of the experimental sessions: deep anesthetization with ketamine and xylazine, transcardial perfusion with 0.9% saline and 10% formalin solution, brain removal and coronal sectioning (50 μm). Sections were examined to determine the location of the cannulae aimed for the VTA according to the rat brain atlas (Paxinos & Watson, 2007). In total, five rats were excluded from the statistical analysis due to misplacement (on only one side or both sides) of the guide cannulae. Supplementary Fig. 2 shows the three coronal schematic microinjection sites in the ventral tegmental area.
2.7. Statistical analysis Data are expressed as mean ± SEM. CPP conditioning score represents the difference of time spent in the morphine- and saline-paired compartments. Prism (GraphPad Software, Version 5.0) was used for data processing. Un-paired samples t-test was conducted to compare the CPP conditioning scores in the morphine/DMSO group with the saline/ DMSO group. The comparison between morphine/DMSO group and the experimental groups was performed using one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc analysis. The same tests were also used between all the groups for the comparison of locomotor activity. A P-value < 0.05 was considered to be statistically significant.
2.5.2. Experiment 2: effect of OX1R antagonist microinjection into the VTA on the expression of CPP SB-334867 (0.1, 1, and 10 nM/rat; n = 5, 5, 5, respectively) was microinjected into the VTA 5 min prior to the test (the post-conditioning phase) to study the drug effect on the reward memory expression. The saline/DMSO control group (n = 6) received subcutaneous injection of saline instead of morphine and intra-VTA microinjection of DMSO, as the vehicle of the antagonists. The morphine/DMSO control group (n = 7) received morphine subcutaneously and intra-VTA DMSO (0.3 μl/rat). The animals were put in the drugpaired compartment 5 min after intra-VTA injection of SB-334867. CPP conditioning scores were calculated during a ten-min period (Supplementary Fig. 1B).
3. Results 3.1. Effect of OX1R antagonist microinjection into the VTA on the acquisition of CPP
2.5.3. Experiment 3: effect of OX2R receptor antagonist microinjection into the VTA on the acquisition of CPP TCS-OX2-29 (1, 5, and 25 nM/rat; n = 5, 6, 6, respectively), as OX2R antagonist, was microinjected into the VTA 5 min prior to subcutaneous injection of 5 mg/kg morphine during the 3 days of conditioning phase to study the drug effect on the acquisition of reward. The saline/DMSO control group (n = 6) received subcutaneous injection of saline instead of morphine and intra-VTA microinjection of DMSO, as the vehicle of the antagonists. The morphine/DMSO control group (n = 6) received morphine subcutaneously and intra-VTA DMSO (0.3 μl/rat). To investigate the role of TCS-OX2-29 in the acquisition phase, the highest dose of TCS-OX2-29 (25 nM/rat; n = 6) was microinjected into the VTA 5 min before saline was injected subcutaneously instead of morphine. The animals were put in the drug-paired compartment immediately after subcutaneous injection of morphine. CPP conditioning scores were calculated during a ten-min period (Supplementary Fig. 1C).
In this set of experiments, we examined the effects of different doses of SB-334867 (0.1, 1, and 10 nM/rat) microinjected into the VTA on morphine (5 mg/kg) reward acquisition in the conditioning phase. The morphine/DMSO control group received intra-VTA DMSO (0.3 μl/rat) instead of SB-334867. As Fig. 1A-left panel shows, morphine application (the morphine/DMSO control group) caused a significant increase in the CPP score in comparison to the saline/DMSO control group [t (10) = 5.996, P < 0.001]. One-way ANOVA test followed by Dunnett's post-hoc test [F(3, 22) = 11.29, P = 0.0002; Fig. 1A-middle panel] showed that the intra-VTA administration of SB-334867 at the doses 1 and 10 nM/rat significantly decreased CPP conditioning scores in the conditioning phase as compared to the morphine/DMSO control group. The data showed that intra-VTA microinjection of SB-334867 prior to the application of morphine could significantly prevent morphine reward acquisition. To investigate a possible effect of SB-334867 on the acquisition of CPP, the saline/SB control group received the intra-VTA highest dose of SB-334867 (10 nM/rat) and saline instead of morphine. The independent t-test showed no significant difference between the two groups [t(9) = 0.3153, P = 7597; Fig. 1A-right panel], indicating that SB-334867, per se, did not have a significant effect on the CPP score in the acquisition phase. One-way ANOVA test showed that none of the treatments in this set of experiment affected the locomotor activity [F(5, 33) = 0.2797, P = 0.9203] (Fig. 1B).
2.5.4. Experiment 4: effect of OX2R antagonist microinjection into the VTA on the expression of CPP TCS-OX2-29 (1, 5, and 25 nM/rat; n = 4, 4, 5, respectively), as OX2R antagonist, was microinjected into the VTA 5 min prior the test (the post-conditioning phase) to study the drug effect on the reward 3
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Fig. 1. A: Left panel) The effect of morphine on the acquisition of morphine CPP in the rats. The rats received subcutaneous saline or morphine (5 mg/kg) and intra-VTA microinjection of DMSO prior to the test. Middle panel) The effect of OX1R antagonist (SB334867) on the acquisition of morphine CPP in the rats. The rats received intra-VTA microinjection of DMSO (0.3 μl/rat) or SB-334867 (0.1, 1, and 10 nM/rat) 5 min before receiving subcutaneous morphine. Right panel) The effect of OX1R antagonist (SB334867) on the CPP score in the acquisition phase. The rats received intra-VTA microinjection of the highest dose of SB-334867 (10 nM/rat) 5 min before receiving subcutaneous saline as the vehicle of morphine. B: The locomotor activity of all the groups in this set of experiment. The data are presented as mean ± SEM of CPP conditioning score for 5–6 animals in each column. ***P < 0.001 compared with the saline/DMSO control group. †† P < 0.01, †††P < 0.001 compared with morphine/DMSO control group.
Fig. 2. A: Left panel) The effect of morphine on the expression of morphine CPP in the rats. The rats received subcutaneous saline or morphine (5 mg/kg) prior to the test. Right panel) The effect of OX1R antagonist (SB-334867) on the expression of morphine CPP in the rats. The rats received intra-VTA microinjection of DMSO (0.3 μl/rat) or SB-334867 (0.1, 1, and 10 nM/rat) 5 min before receiving subcutaneous morphine. B: The locomotor activity of all the groups in this set of experiment. The data are presented as mean ± SEM of CPP conditioning score for 5–7 animals in each column. ***P < 0.001 compared with the saline/DMSO control group. † P < 0.05, ††P < 0.01 compared with morphine/DMSO control group.
3.2. Effect of OX1R antagonist microinjection into the VTA on the expression of CPP
In this set of experiments, we examined the effects of different doses of TCS-OX2-29 (1, 5, and 25 nM/rat) microinjected into the VTA on morphine (5 mg/kg) reward acquisition in the conditioning phase. The morphine/DMSO control group received intra-VTA DMSO (0.3 μl/rat) instead of TCS-OX2-29. As Fig. 3A-left panel indicates, morphine application (the morphine/DMSO control group) caused a significant increase in the CPP conditioning score in comparison to the saline/DMSO control group [t (10) = 7.967, P < 0.001]. One-way ANOVA test followed by Dunnett's post-hoc test F(3,22) = 12,32, P < 0.0001; Fig. 3A-middle panel showed that the intra-VTA administration of TCSOX2-29 at the doses 5 and 25 nM/rat significantly decreased CPP conditioning scores in the conditioning phase as compared to the morphine control group. The data showed that intra-VTA microinjection of TCS-OX2-29 prior to the application of morphine could significantly prevent morphine reward acquisition. To investigate a possible effect of TCS-OX2-29 on the acquisition of CPP, the saline/TCS control group received the intra-VTA highest dose of TCS-OX2-29 (25 nM/rat) and saline instead of morphine. The independent t-test showed no significant difference between the two groups
3.3. Effect of OX2R antagonist microinjection into the VTA on the acquisition of CPP
In this set of experiments, we examined the effects of different doses of SB-334867 (0.1, 1, and 10 nM/rat) microinjected into the VTA on morphine reward memory expression in CPP. The morphine/DMSO control group received intra-VTA DMSO (0.3 μl/rat) instead of SB334867. As Fig. 2A-left panel shows, morphine application (the morphine/DMSO control group) caused a significant increase in the CPP score in comparison to the saline group (the saline/DMSO group) [t (11) = 7.977, P < 0.001]. One-way ANOVA test followed by Dunnett's post-hoc test [F(3, 21) = 5.58, P = 0.0069; Fig. 2A-right panel] showed that the intra-VTA administration of SB-334867 at the doses 1 and 10 nM/rat decreased CPP conditioning scores in the expression phase as compared to the morphine/DMSO control group. The data showed that intra-VTA microinjection of SB-334867 could significantly prevent the expression of morphine CPP. One-way ANOVA test showed that none of the treatments in this set of experiment affected the locomotor activity [F(4, 27) = 0.0829, P = 0.9869] (Fig. 2B). 4
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Fig. 3. A: Left panel) The effect of morphine on the acquisition of morphine CPP in the rats. The rats received subcutaneous saline or morphine (5 mg/kg) prior to the test. Middle panel) The effect of OX2R antagonist (TCS-OX2-29) on the acquisition of morphine CPP in the rats. The rats received intra-VTA microinjection of DMSO (0.3 μl/rat) or TCS-OX2-29 (1, 5, and 25 nM/rat) 5 min before receiving subcutaneous morphine. Right panel) The effect of OX2R antagonist (TCS-OX2-29) on the CPP score in the acquisition phase. The rats received intra-VTA microinjection of the highest dose of TCS-OX2-29 (25 nM/rat) 5 min before receiving subcutaneous saline as the vehicle of morphine. B: The locomotor activity of all the groups in this set of experiment. The data are presented as mean ± SEM of CPP conditioning score for 5–6 animals in each column. ***P < 0.001 compared with the saline/DMSO control group. †† P < 0.01 compared with morphine/DMSO control group.
Fig. 4. A: Left panel) The effect of morphine on the expression of morphine CPP in the rats. The rats received subcutaneous saline or morphine (5 mg/kg) prior to the test. Right panel) The effect of OX2R antagonist (TCS-OX2-29) on the expression of morphine CPP in the rats. The tats received intra-VTA microinjection of DMSO (0.3 μl/rat) or TCS-OX2-29 (1, 5, and 25 nM/rat) 5 min before receiving subcutaneous morphine. B: The locomotor activity of all the groups in this set of experiment. The data are presented as mean ± SEM of CPP conditioning score for 4–5 animals in each column. ***P < 0.001 compared with the saline/DMSO control group. †† P < 0.01, †††P < 0.001 compared with morphine/DMSO control group.
decreased CPP conditioning scores in the expression phase as compared to the morphine/DMSO control group. The data showed that intra-VTA microinjection of TCS-OX2-29 could significantly prevent the expression of morphine CPP. One-way ANOVA test showed that none of the treatments in this set of experiment affected the locomotor activity [F(4, 22) = 0.2461, P = 0.9083; Fig. 4B].
[t(10) = 0.0695, P = 0.9475; Fig. 3A-right panel], indicating that TCSOX2-29, per se, did not have a significant effect on the CPP score in the acquisition phase. One-way ANOVA test showed that none of the treatments in this set of experiment affected the locomotor activity [F(5, 34) = 0.1609, P = 0.975] (Fig. 3B).
4. Discussion The present study showed that intra-VTA administration of SB334867, an OX1R antagonist, at the effective doses (1 and 10 nM/rat), could attenuate both development and expression of morphine CPP. The administration of TCS-OX2-29, an OX2R antagonist, at the effective doses (5 and 25 nM/rat) also blocked the development and expression of morphine CPP in the rats. In the present study, the subcutaneous injection of morphine together with the intra-VTA injection of antagonist vehicle (DMSO) caused a significant difference in CPP conditioning score. The reinforcing effect of morphine in the VTA was established as early as 1980 (Phillips and LePiane, 1980) and later it was demonstrated that orexin knockout mice are more resistant to acquire morphine dependence (Georgescu et al., 2003). The intra-VTA injection of
3.4. Effect of OX2R antagonist microinjection into the VTA on the expression of CPP In this set of experiments, we examined the effects of different doses of TCS-OX2-29 (1, 5, and 25 nM/rat) microinjected into the VTA on morphine reward memory expression in CPP. The morphine/DMSO control group received intra-VTA DMSO (0.3 μl/rat) instead of TCSOX2-29. As Fig. 4A-left panel indicates, morphine application (the morphine/DMSO control group) caused a significant increase in the CPP score in comparison to the saline/DMSO group [t (8) = 6.247, P < 0.001]. One-way ANOVA test followed by Dunnett's post-hoc test [F(3, 17) = 11.6, P = 0.0004; Fig. 4A-right panel] showed that the intra-VTA administration of TCS-OX2-29 at the doses 5 and 25 nM/rat 5
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Beheshti University of Medical Sciences, Tehran, Iran. The Vice-Chancellor for Research & Technology had no further role in the design of the study; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
SB-334867 inhibited acquisition and expression of CPP for morphine in the rats, a result that is in agreement with the previous investigations in mice (Harris et al., 2005; Narita et al., 2006). We have previously reported that CPP can be induced in a dose dependent manner by the injection of carbachol (a cholinergic agonist), an effect that can be blocked by the application of SB-334867 (Taslimi et al., 2011). Also, it was reported that systemic administration of SB-334867 in mice that were not dependent to morphine can block both acquisition and expression of CPP for morphine (Tabaeizadeh et al., 2013). A possible mechanism for the involvement of hypothalamus orexin neurons ending in the VTA is the activation of the mesolimbic dopaminergic neurons and disinhibition of GABAergic neurons, both of which result in expression of reward related responses (Narita et al., 2006). Based on the observed results, it can be inferred that blockade of OX1Rs in the VTA prevented excitation of the dopaminergic neurons, which in turn led to the acquisition and expression prevention of morphine's rewarding properties. It should be noted that TCS-OX-29 binds to the OX2R with moderate to high affinity, which is a 250-fold selectivity over OX1R (Mould et al., 2014). We found that intra-VTA injection of TCS-OX2-29, as the OX2R antagonists, in the rats resulted in attenuation of acquisition and expression of morphine CPP. A similar result for morphine with the administration of systematic TCS-OX2-29 was reported by Tabaeizadeh et al. in mice (Tabaeizadeh et al., 2013) and for ethanol by Shoblock et al. in rats (Shoblock et al., 2011). It is been demonstrated that application of dual orexin receptor antagonist (almorexant) at doses effective on OX2R slows the firing rate of VTA dopaminergic neurons (Malherbe et al., 2009), suggesting a possible reason for the observed attenuation in morphine CPP acquisition and expression. A literature review indicates an important role for LH orexin neurons (and not those in DMH/PFA areas) in the development of CPP for morphine (Harris et al., 2005; Sharf et al., 2010a; Sharf et al., 2010b), and in restoration of the cue-induced reinstatement (Harris et al., 2005; Mahler et al., 2013). Considering the innervations of the VTA by the orexinergic afferents from LH (Nambu et al., 1999), morphine CPP acquisition was shown to be prevented by the application of both systematic and intraVTA application of SB-334867 (Harris et al., 2005; Narita et al., 2006). The role of orexin in neuronal plasticity of dopaminergic neurons in VTA (Borgland et al., 2006), which depends on the NMDA receptors has been confirmed (Baimel et al., 2015) and explains orexin effects on learning acquisition for morphine CPP (Mahler et al., 2012). Although it was previously reported that for drugs other than ethanol, OX2R is mostly involved in arousal rather than reward (Mahler et al., 2012), we have shown here that OX2R is involved in the reward related behavior for morphine. A literature review indicates that orexin effects on behaviors related to drug abuse not only depends on the applied drug but also depends on the model applied in the investigation (Estabrooke et al., 2001; Georgescu et al., 2003). It also must be considered that the VTA is one of the key players in morphine preference and the role of other areas like the mesocorticolimbic system (Baldo et al., 2003) and the extended amygdala (Fadel and Deutch, 2002) in the evaluation of reward should not be neglected. In conclusion, our results showed the role of orexin in learning and memory and indicate that orexin receptors (OX-1R and OX2-R) function in the VTA is essential for both acquisition and expression of morphine reward in rats in CPP model. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.npep.2017.08.003.
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Acknowledgment The authors would like to thank Zahra Fatahi for her constructive comments. This work was supported by the Neuroscience Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Funding for this study was provided by the grant (no. 47834/ 95/08/05) from Vice-Chancellor for Research & Technology of Shahid 6
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orexinergic systems in the activation of the mesolimbic dopamine pathway and related behaviors induced by morphine. J. Neurosci. 26, 398–405. Paxinos, G., Watson, C., 2007. The rat brain in stereotaxic coordinates, 6th ed. Elsevier, San Diego. Peyron, C., Tighe, D.K., van den Pol, A.N., de Lecea, L., Heller, H.C., Sutcliffe, J.G., Kilduff, T.S., 1998. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J. Neurosci. 18, 9996–10015. Phillips, A.G., LePiane, F.G., 1980. Reinforcing effects of morphine microinjection into the ventral tegmental area. Pharmacol. Biochem. Behav. 12, 965–968. Richardson, K.A., Aston-Jones, G., 2012. Lateral hypothalamic orexin/hypocretin neurons that project to ventral tegmental area are differentially activated with morphine preference. J. Neurosci. 32, 3809–3817. Sadeghi, B., Ezzatpanah, S., Haghparast, A., 2016. Effects of dorsal hippocampal orexin-2 receptor antagonism on the acquisition, expression, and extinction of morphine-induced place preference in rats. Psychopharmacology 233, 2329–2341. Sakurai, T., 2005. Roles of orexin/hypocretin in regulation of sleep/wakefulness and energy homeostasis. Sleep Med. Rev. 9, 231–241. Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R.M., Tanaka, H., Williams, S.C., Richarson, J.A., Kozlowski, G.P., Wilson, S., Arch, J.R., Buckingham, R.E., Haynes, A.C., Carr, S.A., Annan, R.S., McNulty, D.E., Liu, W.S., Terrett, J.A., Elshourbagy, N.A., Bergsma, D.J., Yanagisawa, M., 1998. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92 (1) (page following 696). Sharf, R., Guarnieri, D.J., Taylor, J.R., DiLeone, R.J., 2010a. Orexin mediates morphine place preference, but not morphine-induced hyperactivity or sensitization. Brain Res. 1317, 24–32.
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Neuropeptides journal homepage: www.elsevier.com/locate/npep
Role of orexin receptors in the ventral tegmental area on acquisition and expression of morphine-induced conditioned place preference in the rats Sharareh Farahimanesha,b, Shahram Zarrabianc, Abbas Haghparasta,⁎ a b c
Neuroscience Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Institute for cognitive Science Studies, Tehran, Iran Cognitive and Neuroscience Research Center (CNRC), Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords: Reward Orexin receptors Ventral tegmental area Acquisition Expression Conditioned placed preference
The orexins are hypothalamic neuropeptides and their role in reward processing and drug addiction has been demonstrated. The extent of involvement of each orexin receptor in the acquisition and expression of conditioned place preference (CPP) for morphine is still a matter of controversy. We investigated the functional differences between orexin-1 and -2 receptor blockade in the ventral tegmental area (VTA) on the acquisition and expression of morphine CPP. A total of 86 adult male Wistar rats weighing 250 ± 30 g (age 7–8 weeks) received intra-VTA microinjection of either SB334867 (0.1, 1 and 10 nM), a selective orexin-1 receptor (OX1R) antagonist, or TCS-OX2-29 (1, 5 and 25 nM), a selective orexin-2 receptor (OX2R) antagonist. To measure the acquisition, the animals received each antagonist (SB334867 or TCS-OX2-29) 5 min prior to subcutaneous injection of morphine (5 mg/kg) during the conditioning phase. To measure the CPP expression, the animals received each antagonist on the post-conditioning phase. The CPP conditioning score was recorded by Ethovision software. Data showed that intra-VTA microinjection of OX1-R antagonist significantly attenuated morphine CPP acquisition, during the conditioning phase, and expression, during the post-conditioning phase. Intra-VTA microinjection of OX2-R antagonist also significantly attenuated morphine CPP acquisition and expression in the mentioned phases. Our results showed the orexin role in learning and memory and indicate that orexin receptors (OX1R and OX2R) function in the VTA is essential for both acquisition and expression of morphine reward in rats in the CPP model.
1. Introduction The orexins (hypocretins) were first described in 1998 and comprise orexin A and B with 33 and 28 amino acids in length, respectively. Two distinct receptors respond to orexin stimulation (OX1R and OX2R) (Kukkonen et al., 2002). Both orexin receptors are coupled to Gq, whereas OX2R additionally is coupled to Gi/Go (Coleman et al., 2012). The orexin receptors have equal affinity for orexin A, but orexin B binds relatively specifically to OX2Rs (Sakurai et al., 1998). Orexin-expressing neurons are exclusively in the hypothalamus (the lateral hypothalamus (LH), prefrontal area (PFA) and dorso-medial hypothalamic nuclei (DMH) areas), receive inputs especially from brainstem and reward-related nuclei (Narita et al., 2006). Extensive CNS projections of these neurons were reported in 1998 (Peyron et al., 1998). The axons of orexinergic neurons were seen in the anterior olfactory nucleus, piriform cortex, tenia tecta, CA1-3 regions of hippocampus, septal nucleus, amygdaloid complex, posterior lobe of the pituitary, many parts of the cerebellum, thalamic nuclei, and ventral tegmental area (VTA) (Nambu et al., 1999).
⁎
The OX1Rs are primarily expressed in the VTA, cortical regions, amygdala and bed nucleus of the stria terminalis, prefrontal and infralimbic cortex, hippocampus (CA2), anterior hypothalamus, laterodorsal tegmental nucleus/pedunculopontine nucleus, dorsal raphe, and the locus coeruleus (Lu et al., 2000; Marcus et al., 2001; Trivedi et al., 1998). The OX2R density is enriched in the medial septal nucleus, amygdala, paraventricular nucleus (PVN), CA3 in the hippocampus, nucleus accumbens (NAc), DMH, and VTA. The gathered evidence shows that both receptors have a unique and overlapping expression (Cluderay et al., 2002; Lu et al., 2000; Marcus et al., 2001). The orexin system affects homeostatic functions and orexin neurons functional dichotomy has been described as PFA and DMH neurons are mainly involved in sleep/wakefulness, arousal and stress responses (de Lecea, 2012; Sakurai, 2005) and LH neurons are mainly involved in feeding, reward processing, addiction, memory for stimulus–reward relationships, and synaptic plasticity (Baimel and Borgland, 2012; Harris et al., 2005). A dichotomy in function also has been suggested in which OX1Rs and OX2Rs are associated with reward and arousal,
Corresponding author at: Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, P. O. Box: 19615-1178, Tehran, Iran. E-mail address: [email protected] (A. Haghparast).
http://dx.doi.org/10.1016/j.npep.2017.08.003 Received 10 May 2017; Received in revised form 8 August 2017; Accepted 25 August 2017 0143-4179/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Farahimanesh, S., Neuropeptides (2017), http://dx.doi.org/10.1016/j.npep.2017.08.003
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guides) was used for the injection into the VTA over a 1 min period. The inner cannula was left in place for an additional 30 s to allow diffusion of the solution and to reduce the possibility of reflux. Intra-VTA injections were made 5 min before each experiment.
respectively (Akanmu and Honda, 2005; Aston-Jones et al., 2010; Marcus et al., 2001). The VTA is a key site in reward- and addiction-related behaviors, receives heavy innervation from LH orexin neurons, and has high level of orexin receptors (Fadel and Deutch, 2002; Korotkova et al., 2003; Marcus et al., 2001; Richardson and Aston-Jones, 2012). The LH-VTA pathway role in the expression of morphine CPP and the possible role of orexin in learning the associations between environmental cues and morphine have already been reported (Harris and Aston-Jones, 2007; Richardson and Aston-Jones, 2012; Zhou et al., 2006). The circuitry can be incorporated by the recurrent application of drugs of abuse that can lead to drug dependence as the intensity of reward seeking was reported to correlate with the amount of Fos activation in orexinergic neurons (Harris et al., 2005). Research showed that OX1 signaling plays a major role in withdrawal and morphine seeking (Georgescu et al., 2003; Harris et al., 2005) and the LH projections to the VTA functions both in reward processing and reward based learning and memory (Harris and Aston-Jones, 2006). Considering the above fact, OX1R and OX2R antagonists have been used to reduce the self-administration of heroin (Smith and Aston-Jones, 2012) and ethanol (Shoblock et al., 2011), respectively. Playing roles in stress activation and reward-based learning and memory, the orexin system could be a target for preventing drug relapse (Harris and Aston-Jones, 2006). Focusing further, it becomes clear that OX1Rs and OX2Rs drug seeking functional differences is an area of controversy (Baimel et al., 2015; Li et al., 2011) and recent research opens the ground for further investigation into the role of these two receptors in learning and expression of reward induced by morphine. In order to deepen our understanding of the orexin system roles in the VTA, in this study we inspected the role of OX1 and OX2 receptors in morphine CPP acquisition and expression in rats.
2.3. Drugs The following drugs were used in this study: ketamine and xylazine (Alfasan Chemical Co, Holland), morphine sulfate (referred to as morphine; Temad, Iran). SB-334867, orexin receptor subtype OX1 antagonist and TCS-OX2-29 (also called 4-PT), orexin receptor subtype OX2 antagonist (Tocris Bioscience, UK). Morphine was dissolved in sterile saline (0.9%), SB-334867 and TCS-OX2-29 were dissolved in 12% dimethyl sulfoxide (DMSO; Sigma Aldrich, Germany). The control groups received either saline or 12% DMSO as an antagonist vehicle. The drug doses were selected based on pilot- and our previous studies (Ezzatpanah et al., 2016; Sadeghi et al., 2016). 2.4. Conditioning apparatus and paradigm To measure stimulus–reward associations a three-compartment CPP apparatus was used (30 × 30 × 40 cm) according to an unbiased procedure (i.e. the animals that spent ≥ 70% of the total test time (10 min) in either compartment were considered to have an initial bias and were excluded from the study). The apparatus was made of Plexiglas and divided into two equal-sized cue-different compartments, as previously described (Zarepour et al., 2014). The start box, as the third compartment, connects the two cue-different compartments. A guillotine door separated the two main compartments from the start box. The whole experimental process was performed under controlled light conditions (~ 14 Lux, comprising two 15 W bulbs positioned about 1.5 m above the apparatus) and any aggravating noise was avoided. The procedure consists of a five-day schedule with three distinct phases as follows.
2. Materials and methods
2.4.1. Pre-conditioning phase In this phase (day 1), each animal was placed in the start box. The guillotine door was removed and rats were allowed to move freely in all the compartments for 10 min. Time spent in each compartment was recorded using a video camera (Panasonic Inc., Japan) and Ethovision software (Noldus, Version 7). Three animals that showed a preference for one of the compartments on the pre-conditioning phase were excluded from the study (85.9 ± 2.6%).
2.1. Animals A total of 128 adult male albino Wistar rats (Purchased from Pasteur Institute, Iran) were used. The animal's weight and age at the time of surgery was 250 ± 30 g and 7–8 weeks, respectively. The animals randomly were assigned into groups of 5, kept in Plexiglass cages (58 × 38 × 20 cm), and had ad libitum access to food and water. The room temperature and the vivarium were kept at 23 ± 1°C and 12:12 h light/dark cycle (lights on 07:00 h), respectively. The rats were handled about 3 min/day for 2 days before the experiment. Each rat was only used once. All experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) and the experiment protocol was approved by the Research and Ethics Committee of Shahid Beheshti University of Medical Sciences.
2.4.2. Conditioning phase The conditioning phase, also known as the acquisition phase, included days 2 to 4. On the first day (day 2), all the groups received morphine (5 mg/kg) subcutaneously and intra-VTA dose of either SB334867 or TCS-OX2-29 and were confined to the drug paired compartment for 45 min by closing the removable door of the apparatus. Six hours later, all the groups received saline subcutaneously without any intra-VTA treatment. Then, the rats were placed in the non-drug-paired (saline-paired) compartment and their movements were recorded for 45 min. To prevent any time-dependency on drug administration, on the second day of the phase (day 3) the animals received subcutaneous saline in the morning and their active treatment in the afternoon (6 h later). The third day of conditioning phase (day 4) was conducted the same as the first day of this phase.
2.2. Stereotaxic surgery and drug microinjections To stereotactically implant the cannulae, the rats received general anesthesia with an intraperitoneal injection of a mixture of ketamine hydrochloride 10% and xylazine 2% (100 and 10 mg/kg, respectively) and placed in a stereotaxic apparatus (Stoelting, USA). The rats were given the surgical preparation and the area surrounding bregma was cleaned and dried. According to the atlas of the rat brain (Paxinos and Watson, 2007), the VTA coordinates in mm were: AP 4.8 caudal to bregma, ML ± 0.9, and DV 7.3. Two stainless steel guide cannulae (23gauge, 11 mm) were implanted bilaterally and secured to the skull using two stainless steel screws and dental acrylic cement. After one week of recovery, the animals were used for the experiments. A 1-μl Hamilton syringe connected by a polyethylene tube (PE-20) to an internal cannula (30-gauge, terminating 1 mm below the tip of the
2.4.3. Post-conditioning phase In CPP, preference for reward is measured by the amount of time the animal spends in the reward associated chamber minus the time it spends in the non-rewarded chamber, when given free access to both chambers after conditioning. Hence, on day 5 (the test day or the expression phase) in the groups under acquisition investigation, the animals were tested for CPP (under morphine-free condition) with free
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memory expression. The saline/DMSO control group (n = 5) received subcutaneous injection of saline instead of morphine and intra-VTA microinjection of DMSO, as the vehicle of the antagonists. The morphine/DMSO control group (n = 5) received morphine subcutaneously and intra-VTA DMSO (0.3 μl/rat). The animals were put in the drugpaired compartment 5 min after intra-VTA injection of TCS-OX2-29. CPP conditioning scores were calculated during a ten-min period (Supplementary Fig. 1D).
access to all the compartments. To probe the expression of morphine CPP, the animals received either SB-334867 or TCS-OX2-29, at the mentioned doses (Please see the following Section 2.5), 5 min before starting the test. The time spent in each compartment during a ten-min period was recorded. The CPP conditioning score, as a preference index, was calculated as the time spent in the drug-paired compartment minus the time spent in the saline-paired compartment, and the time spent in the start box was not calculated for the CPP conditioning score. 2.5. Experimental design
2.6. Histological verification 2.5.1. Experiment 1: effect of OX1R antagonist microinjection into the VTA on the acquisition of CPP SB-334867 (0.1, 1, and 10 nM/rat; n = 6, 6, 5, respectively), as OX1R antagonist, was microinjected into the VTA 5 min prior to subcutaneous injection of 5 mg/kg morphine during the 3 days of conditioning phase to study the drug effect on the acquisition of morphine reward. The saline/DMSO control group (n = 6) received subcutaneous injection of saline instead of morphine and intra-VTA microinjection of DMSO, as the vehicle of the antagonists. The morphine/DMSO control group (n = 6) received morphine subcutaneously and intra-VTA DMSO (0.3 μl/rat). To investigate the role of SB-334867 in the acquisition phase, the highest dose of SB-334867 (10 nM/rat; n = 5) was microinjected into the VTA 5 min before saline was injected subcutaneously instead of morphine. The animals were put in the drug-paired compartment immediately after subcutaneous injection of morphine. The CPP conditioning scores were calculated during a ten-min period (Supplementary Fig. 1A).
The following procedures were performed on the animals after the completion of the experimental sessions: deep anesthetization with ketamine and xylazine, transcardial perfusion with 0.9% saline and 10% formalin solution, brain removal and coronal sectioning (50 μm). Sections were examined to determine the location of the cannulae aimed for the VTA according to the rat brain atlas (Paxinos & Watson, 2007). In total, five rats were excluded from the statistical analysis due to misplacement (on only one side or both sides) of the guide cannulae. Supplementary Fig. 2 shows the three coronal schematic microinjection sites in the ventral tegmental area.
2.7. Statistical analysis Data are expressed as mean ± SEM. CPP conditioning score represents the difference of time spent in the morphine- and saline-paired compartments. Prism (GraphPad Software, Version 5.0) was used for data processing. Un-paired samples t-test was conducted to compare the CPP conditioning scores in the morphine/DMSO group with the saline/ DMSO group. The comparison between morphine/DMSO group and the experimental groups was performed using one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc analysis. The same tests were also used between all the groups for the comparison of locomotor activity. A P-value < 0.05 was considered to be statistically significant.
2.5.2. Experiment 2: effect of OX1R antagonist microinjection into the VTA on the expression of CPP SB-334867 (0.1, 1, and 10 nM/rat; n = 5, 5, 5, respectively) was microinjected into the VTA 5 min prior to the test (the post-conditioning phase) to study the drug effect on the reward memory expression. The saline/DMSO control group (n = 6) received subcutaneous injection of saline instead of morphine and intra-VTA microinjection of DMSO, as the vehicle of the antagonists. The morphine/DMSO control group (n = 7) received morphine subcutaneously and intra-VTA DMSO (0.3 μl/rat). The animals were put in the drugpaired compartment 5 min after intra-VTA injection of SB-334867. CPP conditioning scores were calculated during a ten-min period (Supplementary Fig. 1B).
3. Results 3.1. Effect of OX1R antagonist microinjection into the VTA on the acquisition of CPP
2.5.3. Experiment 3: effect of OX2R receptor antagonist microinjection into the VTA on the acquisition of CPP TCS-OX2-29 (1, 5, and 25 nM/rat; n = 5, 6, 6, respectively), as OX2R antagonist, was microinjected into the VTA 5 min prior to subcutaneous injection of 5 mg/kg morphine during the 3 days of conditioning phase to study the drug effect on the acquisition of reward. The saline/DMSO control group (n = 6) received subcutaneous injection of saline instead of morphine and intra-VTA microinjection of DMSO, as the vehicle of the antagonists. The morphine/DMSO control group (n = 6) received morphine subcutaneously and intra-VTA DMSO (0.3 μl/rat). To investigate the role of TCS-OX2-29 in the acquisition phase, the highest dose of TCS-OX2-29 (25 nM/rat; n = 6) was microinjected into the VTA 5 min before saline was injected subcutaneously instead of morphine. The animals were put in the drug-paired compartment immediately after subcutaneous injection of morphine. CPP conditioning scores were calculated during a ten-min period (Supplementary Fig. 1C).
In this set of experiments, we examined the effects of different doses of SB-334867 (0.1, 1, and 10 nM/rat) microinjected into the VTA on morphine (5 mg/kg) reward acquisition in the conditioning phase. The morphine/DMSO control group received intra-VTA DMSO (0.3 μl/rat) instead of SB-334867. As Fig. 1A-left panel shows, morphine application (the morphine/DMSO control group) caused a significant increase in the CPP score in comparison to the saline/DMSO control group [t (10) = 5.996, P < 0.001]. One-way ANOVA test followed by Dunnett's post-hoc test [F(3, 22) = 11.29, P = 0.0002; Fig. 1A-middle panel] showed that the intra-VTA administration of SB-334867 at the doses 1 and 10 nM/rat significantly decreased CPP conditioning scores in the conditioning phase as compared to the morphine/DMSO control group. The data showed that intra-VTA microinjection of SB-334867 prior to the application of morphine could significantly prevent morphine reward acquisition. To investigate a possible effect of SB-334867 on the acquisition of CPP, the saline/SB control group received the intra-VTA highest dose of SB-334867 (10 nM/rat) and saline instead of morphine. The independent t-test showed no significant difference between the two groups [t(9) = 0.3153, P = 7597; Fig. 1A-right panel], indicating that SB-334867, per se, did not have a significant effect on the CPP score in the acquisition phase. One-way ANOVA test showed that none of the treatments in this set of experiment affected the locomotor activity [F(5, 33) = 0.2797, P = 0.9203] (Fig. 1B).
2.5.4. Experiment 4: effect of OX2R antagonist microinjection into the VTA on the expression of CPP TCS-OX2-29 (1, 5, and 25 nM/rat; n = 4, 4, 5, respectively), as OX2R antagonist, was microinjected into the VTA 5 min prior the test (the post-conditioning phase) to study the drug effect on the reward 3
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Fig. 1. A: Left panel) The effect of morphine on the acquisition of morphine CPP in the rats. The rats received subcutaneous saline or morphine (5 mg/kg) and intra-VTA microinjection of DMSO prior to the test. Middle panel) The effect of OX1R antagonist (SB334867) on the acquisition of morphine CPP in the rats. The rats received intra-VTA microinjection of DMSO (0.3 μl/rat) or SB-334867 (0.1, 1, and 10 nM/rat) 5 min before receiving subcutaneous morphine. Right panel) The effect of OX1R antagonist (SB334867) on the CPP score in the acquisition phase. The rats received intra-VTA microinjection of the highest dose of SB-334867 (10 nM/rat) 5 min before receiving subcutaneous saline as the vehicle of morphine. B: The locomotor activity of all the groups in this set of experiment. The data are presented as mean ± SEM of CPP conditioning score for 5–6 animals in each column. ***P < 0.001 compared with the saline/DMSO control group. †† P < 0.01, †††P < 0.001 compared with morphine/DMSO control group.
Fig. 2. A: Left panel) The effect of morphine on the expression of morphine CPP in the rats. The rats received subcutaneous saline or morphine (5 mg/kg) prior to the test. Right panel) The effect of OX1R antagonist (SB-334867) on the expression of morphine CPP in the rats. The rats received intra-VTA microinjection of DMSO (0.3 μl/rat) or SB-334867 (0.1, 1, and 10 nM/rat) 5 min before receiving subcutaneous morphine. B: The locomotor activity of all the groups in this set of experiment. The data are presented as mean ± SEM of CPP conditioning score for 5–7 animals in each column. ***P < 0.001 compared with the saline/DMSO control group. † P < 0.05, ††P < 0.01 compared with morphine/DMSO control group.
3.2. Effect of OX1R antagonist microinjection into the VTA on the expression of CPP
In this set of experiments, we examined the effects of different doses of TCS-OX2-29 (1, 5, and 25 nM/rat) microinjected into the VTA on morphine (5 mg/kg) reward acquisition in the conditioning phase. The morphine/DMSO control group received intra-VTA DMSO (0.3 μl/rat) instead of TCS-OX2-29. As Fig. 3A-left panel indicates, morphine application (the morphine/DMSO control group) caused a significant increase in the CPP conditioning score in comparison to the saline/DMSO control group [t (10) = 7.967, P < 0.001]. One-way ANOVA test followed by Dunnett's post-hoc test F(3,22) = 12,32, P < 0.0001; Fig. 3A-middle panel showed that the intra-VTA administration of TCSOX2-29 at the doses 5 and 25 nM/rat significantly decreased CPP conditioning scores in the conditioning phase as compared to the morphine control group. The data showed that intra-VTA microinjection of TCS-OX2-29 prior to the application of morphine could significantly prevent morphine reward acquisition. To investigate a possible effect of TCS-OX2-29 on the acquisition of CPP, the saline/TCS control group received the intra-VTA highest dose of TCS-OX2-29 (25 nM/rat) and saline instead of morphine. The independent t-test showed no significant difference between the two groups
3.3. Effect of OX2R antagonist microinjection into the VTA on the acquisition of CPP
In this set of experiments, we examined the effects of different doses of SB-334867 (0.1, 1, and 10 nM/rat) microinjected into the VTA on morphine reward memory expression in CPP. The morphine/DMSO control group received intra-VTA DMSO (0.3 μl/rat) instead of SB334867. As Fig. 2A-left panel shows, morphine application (the morphine/DMSO control group) caused a significant increase in the CPP score in comparison to the saline group (the saline/DMSO group) [t (11) = 7.977, P < 0.001]. One-way ANOVA test followed by Dunnett's post-hoc test [F(3, 21) = 5.58, P = 0.0069; Fig. 2A-right panel] showed that the intra-VTA administration of SB-334867 at the doses 1 and 10 nM/rat decreased CPP conditioning scores in the expression phase as compared to the morphine/DMSO control group. The data showed that intra-VTA microinjection of SB-334867 could significantly prevent the expression of morphine CPP. One-way ANOVA test showed that none of the treatments in this set of experiment affected the locomotor activity [F(4, 27) = 0.0829, P = 0.9869] (Fig. 2B). 4
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Fig. 3. A: Left panel) The effect of morphine on the acquisition of morphine CPP in the rats. The rats received subcutaneous saline or morphine (5 mg/kg) prior to the test. Middle panel) The effect of OX2R antagonist (TCS-OX2-29) on the acquisition of morphine CPP in the rats. The rats received intra-VTA microinjection of DMSO (0.3 μl/rat) or TCS-OX2-29 (1, 5, and 25 nM/rat) 5 min before receiving subcutaneous morphine. Right panel) The effect of OX2R antagonist (TCS-OX2-29) on the CPP score in the acquisition phase. The rats received intra-VTA microinjection of the highest dose of TCS-OX2-29 (25 nM/rat) 5 min before receiving subcutaneous saline as the vehicle of morphine. B: The locomotor activity of all the groups in this set of experiment. The data are presented as mean ± SEM of CPP conditioning score for 5–6 animals in each column. ***P < 0.001 compared with the saline/DMSO control group. †† P < 0.01 compared with morphine/DMSO control group.
Fig. 4. A: Left panel) The effect of morphine on the expression of morphine CPP in the rats. The rats received subcutaneous saline or morphine (5 mg/kg) prior to the test. Right panel) The effect of OX2R antagonist (TCS-OX2-29) on the expression of morphine CPP in the rats. The tats received intra-VTA microinjection of DMSO (0.3 μl/rat) or TCS-OX2-29 (1, 5, and 25 nM/rat) 5 min before receiving subcutaneous morphine. B: The locomotor activity of all the groups in this set of experiment. The data are presented as mean ± SEM of CPP conditioning score for 4–5 animals in each column. ***P < 0.001 compared with the saline/DMSO control group. †† P < 0.01, †††P < 0.001 compared with morphine/DMSO control group.
decreased CPP conditioning scores in the expression phase as compared to the morphine/DMSO control group. The data showed that intra-VTA microinjection of TCS-OX2-29 could significantly prevent the expression of morphine CPP. One-way ANOVA test showed that none of the treatments in this set of experiment affected the locomotor activity [F(4, 22) = 0.2461, P = 0.9083; Fig. 4B].
[t(10) = 0.0695, P = 0.9475; Fig. 3A-right panel], indicating that TCSOX2-29, per se, did not have a significant effect on the CPP score in the acquisition phase. One-way ANOVA test showed that none of the treatments in this set of experiment affected the locomotor activity [F(5, 34) = 0.1609, P = 0.975] (Fig. 3B).
4. Discussion The present study showed that intra-VTA administration of SB334867, an OX1R antagonist, at the effective doses (1 and 10 nM/rat), could attenuate both development and expression of morphine CPP. The administration of TCS-OX2-29, an OX2R antagonist, at the effective doses (5 and 25 nM/rat) also blocked the development and expression of morphine CPP in the rats. In the present study, the subcutaneous injection of morphine together with the intra-VTA injection of antagonist vehicle (DMSO) caused a significant difference in CPP conditioning score. The reinforcing effect of morphine in the VTA was established as early as 1980 (Phillips and LePiane, 1980) and later it was demonstrated that orexin knockout mice are more resistant to acquire morphine dependence (Georgescu et al., 2003). The intra-VTA injection of
3.4. Effect of OX2R antagonist microinjection into the VTA on the expression of CPP In this set of experiments, we examined the effects of different doses of TCS-OX2-29 (1, 5, and 25 nM/rat) microinjected into the VTA on morphine reward memory expression in CPP. The morphine/DMSO control group received intra-VTA DMSO (0.3 μl/rat) instead of TCSOX2-29. As Fig. 4A-left panel indicates, morphine application (the morphine/DMSO control group) caused a significant increase in the CPP score in comparison to the saline/DMSO group [t (8) = 6.247, P < 0.001]. One-way ANOVA test followed by Dunnett's post-hoc test [F(3, 17) = 11.6, P = 0.0004; Fig. 4A-right panel] showed that the intra-VTA administration of TCS-OX2-29 at the doses 5 and 25 nM/rat 5
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Beheshti University of Medical Sciences, Tehran, Iran. The Vice-Chancellor for Research & Technology had no further role in the design of the study; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
SB-334867 inhibited acquisition and expression of CPP for morphine in the rats, a result that is in agreement with the previous investigations in mice (Harris et al., 2005; Narita et al., 2006). We have previously reported that CPP can be induced in a dose dependent manner by the injection of carbachol (a cholinergic agonist), an effect that can be blocked by the application of SB-334867 (Taslimi et al., 2011). Also, it was reported that systemic administration of SB-334867 in mice that were not dependent to morphine can block both acquisition and expression of CPP for morphine (Tabaeizadeh et al., 2013). A possible mechanism for the involvement of hypothalamus orexin neurons ending in the VTA is the activation of the mesolimbic dopaminergic neurons and disinhibition of GABAergic neurons, both of which result in expression of reward related responses (Narita et al., 2006). Based on the observed results, it can be inferred that blockade of OX1Rs in the VTA prevented excitation of the dopaminergic neurons, which in turn led to the acquisition and expression prevention of morphine's rewarding properties. It should be noted that TCS-OX-29 binds to the OX2R with moderate to high affinity, which is a 250-fold selectivity over OX1R (Mould et al., 2014). We found that intra-VTA injection of TCS-OX2-29, as the OX2R antagonists, in the rats resulted in attenuation of acquisition and expression of morphine CPP. A similar result for morphine with the administration of systematic TCS-OX2-29 was reported by Tabaeizadeh et al. in mice (Tabaeizadeh et al., 2013) and for ethanol by Shoblock et al. in rats (Shoblock et al., 2011). It is been demonstrated that application of dual orexin receptor antagonist (almorexant) at doses effective on OX2R slows the firing rate of VTA dopaminergic neurons (Malherbe et al., 2009), suggesting a possible reason for the observed attenuation in morphine CPP acquisition and expression. A literature review indicates an important role for LH orexin neurons (and not those in DMH/PFA areas) in the development of CPP for morphine (Harris et al., 2005; Sharf et al., 2010a; Sharf et al., 2010b), and in restoration of the cue-induced reinstatement (Harris et al., 2005; Mahler et al., 2013). Considering the innervations of the VTA by the orexinergic afferents from LH (Nambu et al., 1999), morphine CPP acquisition was shown to be prevented by the application of both systematic and intraVTA application of SB-334867 (Harris et al., 2005; Narita et al., 2006). The role of orexin in neuronal plasticity of dopaminergic neurons in VTA (Borgland et al., 2006), which depends on the NMDA receptors has been confirmed (Baimel et al., 2015) and explains orexin effects on learning acquisition for morphine CPP (Mahler et al., 2012). Although it was previously reported that for drugs other than ethanol, OX2R is mostly involved in arousal rather than reward (Mahler et al., 2012), we have shown here that OX2R is involved in the reward related behavior for morphine. A literature review indicates that orexin effects on behaviors related to drug abuse not only depends on the applied drug but also depends on the model applied in the investigation (Estabrooke et al., 2001; Georgescu et al., 2003). It also must be considered that the VTA is one of the key players in morphine preference and the role of other areas like the mesocorticolimbic system (Baldo et al., 2003) and the extended amygdala (Fadel and Deutch, 2002) in the evaluation of reward should not be neglected. In conclusion, our results showed the role of orexin in learning and memory and indicate that orexin receptors (OX-1R and OX2-R) function in the VTA is essential for both acquisition and expression of morphine reward in rats in CPP model. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.npep.2017.08.003.
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Acknowledgment The authors would like to thank Zahra Fatahi for her constructive comments. This work was supported by the Neuroscience Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Funding for this study was provided by the grant (no. 47834/ 95/08/05) from Vice-Chancellor for Research & Technology of Shahid 6
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