Male Wistar rats (200–250g) were obtained from animal breeding colony of Hamadan University of Medical Sciences (Hamadan, Iran). They were maintained on 12/12h light/dark cycle (light on at 7 AM) and had access to freely available food and water in their home cages (temperature 22°C ± 2°C). 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 institutional ethics committee at Hamadan University of Medical Sciences.
I the current study in order to conduct the experiments the following drugs were used as following: Morphine sulfate (Temad, Iran) was dissolved in normal saline (0.9% NaCl) (S)-3,4-Dicarboxyphenylglycine (S-3,4-DCPG) (Tocris, UK), a selective mGluR8 allosteric agonist, was also dissolved in normal saline (0.9% NaCl). It is worth mentioning that control and vehicle groups received saline.
Stereotaxic Surgery and Drug Administration
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 holed into the skull at stereotaxic coordinates of 1.4 ± 0.4 mm anterior to bregma, ±1.5 mm lateral to the sagital suture, and 6.5 mm down from top of 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 the NAc. The guiding 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 guiding cannulae were inserted into the guiding cannulae and left in place until injections were made. All rats were allowed to recover for one week before starting the behavioral testing.
The rats were gently restrained by hand and the dummy cannulae were removed from the guiding cannulae. Drugs were directly injected into the NAc through the guiding cannulae using injector cannulae (30- gauge, 1 mm below the tip of the guiding cannula). Polyethylene tubing (PE-20) was used for attaching the injector cannula to the 1-μl Hamilton syringe. Doses of selective mGluR8 allosteric agonist, S-3,4-DCPG, (0.03, 0.3, and 3 μg/0.5 μL saline per side) were administered into the NAc. The injection volume into the NAc was 0.5 μl/side for all groups. Injections were made bilaterally over a 50s period and the injection cannulae were left in the guiding cannulae for an additional 60s in order to facilitate the diffusion of the drugs.
Place Conditioning Apparatus and Protocol
A three-compartment CPP apparatus was used in the experiments. 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. The CPP protocol has been previously described (45). An unbiased allocation was used. Rats with a neutral preference (45–55% for either side) were randomly allocated their drug-paired side (unbiased allocation). In the CPP paradigm, the conditioning score (CS) and distance traveled were calculated based on a video recorded by a CCD camera with 30 frames per second (30 fps) resolution. The camera was placed 2m above the CPP boxes and the locomotion tracking was measured by Maze Router homemade software, a video tracking system for automation of behavioral experiments. CPP paradigm took place for 5 continuous days, which consisted of three distinct phases: pre-conditioning, conditioning and post-conditioning (5, 6).
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. Rats with any compartment preference were omitted from the experiment. 3 rats were excluded from this study due to compartment preference. Then rats were randomly assigned to one of the two groups (odd and even) for place conditioning (45).
The morphine conditioning phase, also known as the acquisition phase, were conducted on days 2,3 and 4. Each group of animals was randomly divided into even or odd. Odd animals received subcutaneous (SC) injection of saline and morphine (5mg/kg) 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. Place preference was calculated as a preference score (time spent in drug paired zone − time spent in the saline paired zone) (5, 6). During this phase, saline group animals received saline in both compartments during alternative morning and afternoon design with an interval of 6 h. Locomotor data were also collected throughout CPP testing in order to assess the development of behavioral sensitization.
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 the 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.
The effect of intra-accumbal administration of mGluR8 allosteric agonist (S-3,4-DCPG) on the acquisition of morphine-induced CPP
To investigate the effects of mGluR8 agonist on the acquisition of morphine-induced CPP, bilaterally intra-accumbal injection of S-3,4-DCPG (3, 0.3 and 0.03μg/0.5μL) (46) was done 5min prior to each morphine injection (5 mg/kg; SC) during the three days of conditioning phase. During this phase, a vehicle-control group received saline (0.5μL) instead of S-3,4-DCPG into the NAc, prior to SC injection of morphine. Moreover, to rule out the possibility that S-3,4-DCPG administration alone had rewarding or aversive effects on the CPP, a separate group of rats received the highest doses (3μg/0.5μL) of S-3,4-DCPG prior to saline injection (1mL/kg; SC) instead of morphine during the conditioning days. Saline group received saline SC injection instead of morphine during the conditioning phase.
The effects of intra-accumbal S-3,4-DCPG injection on the expression of morphine-induced CPP
In order to examine the effects of the highest dose of S-3,4-DCPG (3μg/ 0.5μL) on the expression of morphine-induced CPP, the rats were bilaterally given S-3,4-DCPG into the NAc 5min prior to CPP test. In addition, a control vehicle group received saline (0.5μL) through the NAc instead of S-3,4-DCPG before CPP test on post-conditioning phase. The saline group received saline instead of morphine during the conditioning phase.
Locomotor Activity Measurement
The locomotor activity of each rat was recorded using the locomotion tracking apparatus by a video tracking system (Router maze software). In these experiments, the total distance traveled (in centimeters) by each rat was measured in pre- and post-tests for all groups.
After behavioral testing, all the rats implanted with injection cannulae were deeply anesthetized with Ketamine and Xylazine. They were then transcardially perfused with 0.9% saline and then a 10% formalin solution. The brains were removed, blocked, and cut coronally in 50μm sections through to the cannulae. All the rats with cannula placement 1 mm distant from the intended injection site were removed from the data. (Fig. 1). It should be noted that some points are completely or partially overlapped.
Data were processed by commercially available software GraphPad Prism® 8.0.2 In order to compare the conditioning scores (CS) and the traveled distance obtained from morphine CPP animals, one-way analysis of variance (ANOVA) followed by post hoc analysis (Newman–Keuls multiple comparison test) was used. Multiple student's t-test was used to compare pre-conditioning with saline or highest dose of S-3,4-DCPG (3 μg/0.5 μL). P-values less than 0.05 (P < 0.05) were considered to be statistically significant.