Study On The Effect And Mechanism Of Conotoxin And Its Analogs On Morphine Psychological Addiction In Mice

1 ObjectiveDrug addiction is a both medical and social problem which is more and more serious world-wide. Every country is unfortunately involved in psychoactive drug. Drug addiction is a chronic and rlapsing neurological disease in which compulsive drug-seeking and drug-taking behavior persists despite serious negative consequences. Many learning and memory systems participate in the the formation of addictive behavior. It shows that learning and memory and drug addiction shared many neurobiological changes. Important encephalic regions, functional molecules and signal transduction system associated with learning and memory are involved in the formation of drug addiction and relapse induced by psyco-craving. Therefore, it is very meaningful for exploring the nature of addiction to understand the mechanism characteristic of learning and memory of addiction. Long-term potentiation (LTP) has been generally accepted as the mode of learning and memory, which is also the molecular mechanism of declarative memory. In the induction course of LTP, there must be CaMKII that participates and be activated. CaMKII is the molecule base of memory. Also, CaMKII is the common influence factor of learning and memory and drug addiction. So it can be a kind of index to study the common biological mechanism of drug addiction and learning and memory. CaMKIIα is closely relative to the capability of space learning.To aim at mechanism above, we focus on the international hotspot-conotoxin. 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-,d-,a-,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. There has been studyindicating that calcium channel and glutamate receptor especially NMDA receptor are involved in the formation of drug dependence.In the present study, we investigated the effect of [Glu^3,4,7,10,14]-conantokin G on the expression of CPP induced by morphine in mice, using the computer based vedio tracking CPP system developed by our laboratory. We use [Glu^3,4,7,10,14]-conantokin G as the entry point to testify NMDA receptor are involved in morphine-induced CPP ,and further discuss morphine psychological dependence based on these results. Furthermore, the role of CaMKII which plays in relative encephalic regions of morphine addiction will be approached.2 Materials and methodsAnimals: Male Kunming mice (18-20 g) were supplied by Shanghai Centre of Experimental Animals, Chinese Academy of Sciences. Animals were adapted to the experimental conditions for at least 2 weeks before experiment. Animals, housed four per cage in Individually Ventilated Cages (IVC) with free access to food and water, were maintained at 23±1℃ 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); [Glu^3,4,7,10,14]-conantokin G (Sigma) ; Anti-CaMKII monoclonal antibody (Sigma); Anti-rabbit-IgG(Zhongshan Goldenbridge biotechnology); Monoclonal Anti-β-Actin(Sigma); Anti-mouse-IgG(Zhongshan Goldenbridge biotechnology).Apparatus: The experimental shuttle boxes used in this CPP paradigm were the apparatus consists of four identical plexiglas boxes measuring 30xl5xl5cm, which divided into two chambers (15x15x15 cm) of equal size by a separator, one white with smooth floor and the other black with rough checkerwork floor. The white chamber was design as drug-paired 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 three phases: pre-conditioning phase, conditioning phase and testing 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 separating 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.Drug treatment:To investigate the ability of drugs to induce CPP, saline and 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 [Glu^3,4,7,10,14]-conantokin G on the expression of morphine-induced CPP, [Glu^3,4,7,10,14]-conantokin G or vehicle(saline) were given by intracranial administration 30 min before the test on day 9.Western blotting:For Western blotting analysis, rats were sacrificed immediately after recording. Brains were dissected out and the hippocampus (HP) and prefrontal cortex (PFC) were then separated, and put into liquid nitrogen immediately. The total proteins were extracted by the Cytoplasmic Total Protein Extraction Reagents from the brain tissues. The concentration of proteins of each sample was determined using BCA assay as the standard.Protocols of western blotting1. Preparation of Polyacrylamide resolving gel and stacking gel;2. Adding sample buffers and flash spinning the samples;3. Samples with equal amounts of protein and the prestained marker were loaded into the wells;4. Run the gel with constant voltage, 110 V for 1.5 h;5. Proteins were transferred to NC membrane, 110 V for 2 h;6. Membranes were incubated in Tris-buffered saline with 0.5% Tween 20 containing 5% non-fat milk for 2 hours at room temperature;7. Incubated with monoclonal rabbit anti-CaMKII (1:3000) antibodies at 4℃ overnight;8. Wash the membrane 25 min in TBST buffer;9. Incubated with the secondary antibody, goat anti-rabbit IgG conjugated with horseradish peroxidase at a 1:3000 dilution in blocking buffer for 2 h;10. The membranes were then washed 25 min in TBST buffer;11. The blots were developed by enhanced chemiluminescence method;12. The membranes were then washed 20 min in TBST buffer;13. Incubated with monoclonal rabbit anti-Actin (1:5000) antibodies for 2 h;14. Wash the membrane 25 min in TBST buffer;15. Incubated with the secondary antibody, goat anti-mouse IgG conjugated with horseradish peroxidase at a 1:3000 dilution in blocking buffer for 2 h;16. The membranes were then washed 25 min in TBST buffer;17. The blots were developed by enhanced chemiluminescence method;18. Visualized by exposure to GS-800 Calibrated Densitometer (Bio-Rad).Data analysis and statisticsPlace Preference: the time spent in the drug-paired side; Locomotor Activity: the distance that mice moved in chamber or different area; Exploring Activity: shuttle number; the total distance that mice moved in the center area of both chambers; the total time spent in the center area of both chambers; the distance that mice moved in the center area of white chamber; the time spent in the cener area of white chamber. All the data was expressed as the mean±S.E.M. A value of P< 0.05 was considered significant. The densitometry of the bands was calculated by the Biorad quantity one 4.4.0 densitomonter, and datas were analyzed with paired sample t-test. Statistical differences at P<0.05 were considered significant.3 ResultsEffect of CPP expression induced by morphine: Pre-conditioning test shows that mice spent more time in black chamber than in white chamber(504. 0 ± 25. 1,396. 6±25. l,P<0. 05). Following morphine conditioning, mice spent a greater amount of time in the morphine-paired chamber than in the saline-paired chamber (573.2 ± 53.0, 441.1 ± 39.3, P<0.05) . The data were analyzed and revealed a significant preference effect on drug-paired side.Effect of [Glu^3,4,7,10,14]-conantokin G on the expression of morphine induced CPP: After conditioning with morphine for 8 days, on day 9, pretreatment with [Glu^3,4,7,10,14]-conantokin G (30, 60 and 120 pmol) 30 min before the test dose dependently suppressed the expression of morphine induced CPP. Independent from the treatment, mice of the different groups did not show any significant difference in the distance they moved and exploring activity, and they were not related with place preference.Comparison of the effect of [Glu^3,4,7,10,14]-conantokin G,Con G and GST-CTX MVIIA on the CPP expression induced by morphine: Under the same experiment condition and the same dose, the time spent in the drug-paired side islonger with [Glu^3,4,7,10,14]-conantokin G treated mice than Con G treated mice; but there is no significant difference between them. However, GST-CTX MVIIA with a much higher dose has no significantly difference, compared with morphine control.Changes in CaMKIIα protein levels in PFC and hippocampus of mice after CPP expression attenuated by GST-CTX MVIIA: After attenuated CPP expression by GST-CTX MVIIA, as to the saline group, CaMKIIα protein expression was increased in morphine and 0.01 mg/kg GST-CTX MVIIA treated group in the hippocampus and PFC(P<0.05). As to the morphine group, CaMKIIα protein expression was decreased in 0.03 , 0.1 mg/kg GST-CTX MVIIA treated and saline group in the hippocampus and PFC(P<0.05).4 Conclusions4.1 Using the computer based video tracking conditioned place preference (CPP) system, morphine induced mice CPP acquisition model was established. CPP model may be used as an effective tool to investigate the common mechanism of drug addiction and learning and memory.4.2 [Glu^3,4,7,10,14]-conantokin G attenuated the expression of CPP in a dose-dependent manner, which lay the foundation for screening the active component of conotoxin forward.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 The effect of GST-CTX MVIIA on CaMKIIα protein level in addictive mice supplys more evidence for explaining the mechanism of drug dependence.