Effect Of Conotoxin On The Expression And Reinstatement Of Conditioned Place Preference Induced By Morphine In Mice

1 ObjectiveOpiate addiction is a phenomenon with complex physiological and social causes and consequences. Opioid drugs such as morphine and heroin can elicit intense euphoric effects, which may cause psychic dependence or craving for drugs, and contribute to the vulnerability to relapse. Past investigations indicated that rewarding effect of abusive drugs is considered as the major reason, which results in addiction especially psychological dependence. Despite a great deal of research, the exact mechanisms underlying the development of dependence to opiates remain unclear. The high rate of relapse to opioid is a major clinical problem in detoxification of opioid and remains the most difficult challenge for treatment of drug abuse.Conotoxin (CTX) is an active peptide isolated from the Conus venom. In terms of the action target , CTX can be divided into several large groups: ω-, δ-, α-, conantokins and so on. ω-CTX MVIIA is a potent and specific blocker of N-type voltage-gated calcium channels (N-VGCCs), and the conantokins exhibit antagonism of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptor. Calcium channel antagonist like, verapamil and nimodipin have been found decrease the signs of opiate withdrawal and development of opiate tolerance. Moreover, CTX MVIIA have beenfound to decrease the total ratings of morphine withdrawal precipitated in rats by naloxone. The results indicated that co-CTX might be of use in the treatment of addicts. Recently more researches focus on the NMDA receptor, which involves in development of addiction through interaction with dopamine receptor. Therefore, conantokins might been speculated to be a potential agent for the treatment of drug addiction.Conditioned place preference (CPP) is commonly used to measure the rewarding or incentive properties of drugs, especially psychological dependence. At the same time, CPP model can be used as an effective tool to investigate the mechanisms underlying drug-induced reinstatement of drug seeking after extinction. In the present study, we investigated the effect of CTX on the expression and reinstatement of CPP induced by morphine in mice, use GST-CTX MVIIA and conantokinG(ConG) as the entry point to testify N-VGCCs and NMDA receptor are involved in morphine-induced CPP ,and further discuss morphine psychological dependence based on these results.2 Materials and methodsAnimals: Male Kunming mice (20-22 g) were supplied by Shanghai Centre of Experimental Animals, Chinese Academy of Sciences. Animals were adapted to the experimental conditions for at least 2 week before experiment. Animals, housed four per cage in Individually Ventilated Cages (IVC) with free access to food and water, were maintained at 23±1°C on a 12/12-h reverse light/dark cycle (lights on at 7:00 pm). The experiments were carried out during the dark phase of the cycle. Chemical reagents: Morphine hydrochloride (First Pharmaceutical Company of ShenYang, China);A fusion protein of CTX MVIIA and GST, which was a purified recombinant product and kindly provided by Professor Jinbiao Zhan's lab at Department of Biochemistry, Zhejiang University School of Medicine;Conantokin-G(AnaSpec)Apparatus: The experimental shuttle boxes used in this CPP paradigm were the apparatus consists of four identical plexiglas boxes measuring 30x15x15cm, whichdivided into two chambers (15x15x15 cm) of equal size by a separator, one white with smooth floor and other black with rough checkerwork floor. The white chamber was design as drug pair side. A video camera was placed 70 cm above the floor and was linked with a compatible computer system. The mice behavior was recorded by the video camera and analyzed by MiceTrack software in another separated room. The experiments were conducted under dim illumination (17-20 Lux) and stable noise (29-31 dB). CPP procedure:The CPP procedure consisted of five phases: pre-conditioning phase, conditioning phase, testing phase, CPP extinction phase and CPP reinstatement phase. Pre-conditioningEach animal was free to explore the two compartments for 3 days before the start of the experiment. On day 3, animals received a single pre-exposure test in which they can access to the entire apparatus for 15 min. The amount of time spent in each chamber was monitored. ConditioningMice received morphine/saline in white/black chamber in the following 8 days, once per day. After morphine was administered in a dose of 5.0 mg/kg (i.p.), the mice were placed into the assigned chamber for 50 min. On alternate days, mice received saline injections before being placed in the other chamber. At that time the guillotine door separated the two compartments was closed. There were a total of four drug sessions and four saline sessions. The control group was treated with a daily saline injection for eight consecutive days in the morphine- and saline-paired chambers. Post-conditioningOn the test day (day 9) session, the guillotine door separating the two compartments was opened again, and then the drug-free mice were placed in the middle with free access to both compartments for the next 15 min. The time spent in each box was measured. Conditioned place preference was defined by an increase in the time spent in the drug-paired compartment during a preference test. Extinction of CPPFollowing the establishment of CPP, all animals received a saline injection for eight consecutive days in the morphine- and saline-paired compartments. On the next day (day 18), animals received nothing (no injection) and the time they spent in the each compartments was recorded for 15 minutes. This measurement was determined as post-extinction time spent in the drug-paired compartment. Reinstatement of CPPOne day after the extinction of CPP (day 19) the control group were treated with saline and morphine group were challenged with morphine (2.5 mg/kg). 30 minutes after the injection animals were placed in the test cage and the time spent in each compartments was recorded for 15 minutes. Drug treatmentTo investigate the ability of drugs to induce CPP, saline, morphine were injected before the animals were placed in the drug-paired side. The tests were carried out 24h after the last conditioning session without any preceding injection. In order to test the effects of CTX on the expression of morphine-induced CPP, CTX or vehicle were given by intracranial administration 30 min before the test on day 9. To test the effects of the CTX on the reinstatement of morphine -induced CPP, CTX or vehicle were injected by intracranial administration accompany with morphine (2.5 mg/kg, i.p.) 30 min before the test after extinction of CPP on day 19. Data analysis and statisticsThe time spent in the drug-paired side in the CPP test, the time spent in different area, the distance that mice moved in chamber or different area, shuttle number is expressed as the mean±S.E.M. The time spent in the drug-paired side and in different area were assessed parametric statistically with one-way analysis of variance (ANOVA) followed by LSD test for multiple post hoc comparisons;The distance that mice moved in chamber or different area, shuttle number were assessed nonparametric statistically with Kruskal-Wallis multi-rank sum test, Nemenyi rank sum test;All data were assessed statistically with Pearson n Spearman bivariate correlations test, multiple linear regression. A value of p< 0.05 was considered significant.3 ResultsEffect of morphine induced CPP: Following morphine conditioning, mice spent a greater amount of time in the morphine-paired chamber than in the saline-paired chamber (570.9+10.3, 409.9.1 + 12.4, P<0.0\) .The data were analyzed and revealed a significant effect of chamber.Effect of extinction training: After 8 days saline paired extinction training, the mean time spent in the morphine paired side revealed no differences between two groups (451.6±34.2,434.8±30.1, P>0.05), mice did not show conditioned preference for the previous drug-paired chamber.Reinstatement of CPP by priming dosage of morphine: After received a priming injection of morphine (2.5 mg/kg, i.p.), there was a significant effect between two groups of the time spent in the morphine-paired chamber(585.4±20.1,360.1±39.6, PO.01), mice showed a significant preference for the morphine-paired chamber.Effect of CTX on the expression of morphine induced CPP: After conditioning with morphine for 8 days, on day 9, pretreatment with GST- CTX MVIIA (0.01, 0.03 and 0.1 mg/kg) and ConG(0.001, 0.003 and 0.01 mg/kg) 30 min before the test dose dependently suppressed the expression of morphine induced CPP (F(5,42)=4.510, PO.05;F(5,42)=2.645, P<0.05). Independent from the treatment, the mices of the different groups did not show any significant difference in the distance they moved(F(5,42)=0.376, P>0.05;F(5,42)=0.973, P>0.05) and exploring activity, and they were not related with time spent in drug-paired side.Effect of CTX on the reinstatement of morphine-induced CPP: After extinction training with saline for 8 days, on day 19, pretreatment with GST- CTX MVIIA (0.01, 0.03 and 0.1 mg/kg) and ConG(0.001, 0.003 and 0.01 mg/kg) accompany with morphine (2.5 mg/kg) 30 min before the test dose dependently suppressed the reinstatement of morphine induced CPP(F(5,42) = 5.723, p < 0.05;F(5,38) =3.593, p < 0.05). As for the distance that mice moved and exploring activity in reinstatement tests on day 19, there was no significant difference between these groups , ,and they were not related with CPP induced by morphine.4 Conclusions4.1 Using the computer based video tracking conditioned place preference (CPP) system, morphine induced mice CPP acquisition, extinction and reinstatement model were established. CPP model may be used as an effective tool to investigate the common mechanism of drug addiction and learning memory.4.2 GST- CTX MVIIA and ConG attenuated the expression as well as the reinstatement of CPP in a dose-dependent manner. These findings suggest that these conotoxin are potential agent for the treatment of drug dependence.4.3 The mices of the different groups did not show significant difference in locomotor activity and exploring activity, and they were not related with CPP induced by morphine.4.4 NMDA receptor and N-VGCCs might be involve in the expression and the reinstatement of CPP induced by morphine, These findings indicate NMDA-type glutamate receptors and N-VGCCs might be mechanism of drug addiction.