| 1 ObjectiveOpiate dependence is a chronic and relapsing mental disorder,which mainly appears as forced drug-seeking,unlimited medication,drug withdrawal symptoms after interruption.Because of the exact mechanisms underlying the development of opiate dependence,especially psychic dependence,remain unclear.The high rate of relapse to opioid(95%) is a serious clinical problem.Therefore,if we can resolve the development of psychic dependence and relapse,there will be extremely important social and scientific significance.Many investigations indicated that the mesolimbic dopamine system(MLDS) is the rewarding center of drug addiction,other research also reported that the nucleus accumbens(NAc),hippocampus(Hp),prefrontal cortex(PFC) and other memory-related brain reagions' NR2B subunit activities were increased resulting in morphine addiction memory formation,maintain and relapse.When administrated the selective NR2B antagonist Ifenprodil or administered in the nucleus system,blocking the NR2B subunit in,NAc or Hp CA1 region could inhibit drugs or morphine-induced conditioned place preference relapse,while the acquisition and reproduce of the food required for the maintenance,social and other natural reward memory and spatial memory had no significant changes.This showed that NR2B subunit is expected to be a new target for opioid addiction. The N-methyl-D-aspartate receptor(NMDAR),a member of excitatory glutamate receptor family,is a ligand-and voltage-gated ion channel composed of an obligatory NR1(a-h) subunit and one or more modulatory NR2(A-D) or NR3 subunits and displays high Ca2+ permeability,involving in synaptic signal transduction,neural plasticity,learning and memory and other important physiological processes.It is well known that the protein tyrosine kinase is necessary in the process of synaptic transmission, plasticity,learning and memory,among which the Src family tyrosine kinases(SFKs),a kind of non-receptor-type tyrosine kinases,have a critical role in regulating the synaptic plasticity and memory formation.As PFC and Hp brain areas have extensive relationships with the other addiction-related regions and involved in a variety of reward-related learning and memory.Many studies also show that a variety of proteins abnormal changed in PFC and Hp regions of the mice undergoing chronic morphine treatment,yet the role of SFKs in drug addiction has not clarified.Conantokins,derived from the venom of Conus predatory marine snails,are the natural peptide antagonist targeted to NMDAR.Some studies indicate that Con-G is a potent inhibitor of morphine-induced physical and psychological dependence,but the important modifiedγ-carboxyglutamate residues in the peptide are difficult to get by chemosynthesis or biosynthesis,whereas it is limited in the drug development and clinical application.Recently,we have found that selectively NMDAR antagonist[Glu3, 4,7,10,14]-Conantokin-G(Glu-Con-G),could inhibit the morphine-reduced rewarding effect,easy to get and low cost,which provide new ideas for development of conantokins.Besides,it is suggested that Con-G could decrease excitotoxic calcium responses to NMDAR induced by different agonists and the SFKs can be activated by many intracellular moleculessuch as calcium,so we suppose that Glu-Con-G may indirectly modulate the activation of SFKs by inhibiting NMDAR-mediated excitotoxic calcium influx.In the present study,we further focus on the anti-morphine psychic dependence effects of Con-G and Glu-Con-G by CPP.Meanwhile,we used the western blotting to testify the changes of the levels of Tyr416-phosphorylated SFKs[pSFKs(Tyr416)]in morphine-addicted mice PFC and Hp to discuss the role of SFKs in morphine psychological dependence. 2 Materials and methods2.1 AnimalsMale Kunming mice(18-22g) were adapted to the experimental conditions for at least 2 weeks before experiment.2.2 Chemical reagentsMorphine hydrochloride;Conantokin G(Sigma,product number:C4311,properties assay＞75%);[Glu3,4,7,10,14]-Conantokin G(Sigma,product number:C1733,properties assay＞90%);Phospho-Src Family(Tyr416) antibody(Cell Signaling Technology);Src antibody(Cell Signaling Technology);Anti-Actin antibody(Cell Signaling Technology); HRP-conjugated anti-rabbit IgG.2.3 ApparatusThe computer-based video-tracking CPP system is composed of soundproof bin, shuttle boxes,video system and a computer.The experimental shuttle boxes (45×45×15cm)used in this CPP paradigm were the apparatus consists of four identical Plexiglas boxes measuring 30×15×15cm,which divided into two chambers (15×15×15cm) of equal size by a separator,one white with smooth floor and the other black with rough checker work floor.The white chamber was design as drug-paired side.A video camera was placed in the plafond of the soundproof bin,and was linked with a compatible computer system.The mice behaviors were recorded by the video camera.The experiments were conducted under dim illumination(15-20 Lux) and stable noise(＜30 dB).2.4 CPP procedureThe CPP procedure consisted of five phases:pre-conditioning phase,conditioning phase,testing phase,extinction phase and reinstatement phase.Pre-conditioningEach animal was free to explore the two compartments for 3 days(d-2,d-1,d0) before the start of the experiment.Every day,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.According to the natural preference circumstances,the mice were randomly divided into 12 groups:the saline control groups(NS,NS-NS),the morphine control groups(Mor,M-NS),the saline plus Glu-Con-G and Con-G groups(NS-G120,NS-C120) and the morphine plus different doses of Glu-Con-G and Con-G groups(M-G30,M-G60,M-G120,M-C30,M-C60, M-C120).ConditioningThe white chamber was chosen as the morphine-paird chamber for the following conditioning according to the natural preference circumstances of mice.Mice received morphine/saline in white/black chamber in the following 8 days,once per day,After morphine was intraperitoneally administered(i.p.) in a dose of 5.0mg/kg,the mice were placed into the white 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.Testing phaseThe CPP test was carried out 24h after the last conditioning session without any preceding injection.On d9,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 phaseFollowing the establishment of CPP,all animals received a saline injection for eight consecutive days in the morphine- and saline-paired compartments.On day18, animals received nothing 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 phaseOne day19,the control group was treated with saline and morphine group was challenged with morphine(2.5mg/kg).30 minutes after the injection animals were placed in the test compartment and the time spent in each compartments was recorded for 15 minutes.2.5 Conotoxin treatmentIn order to test the effects of Con-G and Glu-Con-G on the expression and reinstatement of morphine-induced CPP,Con-G(30,60,120pmol) and Glu-Con G(30, 60,120pmol) or vehicle(saline) were respectively given by intracerebroventicular administration(i.c.v.) 30 min before the test on day 9 and day 19.2.6 Western blotting analysisBrain regions(PFC,Hp) were immediately isolated from all mice for total proteins concentration detection.Protocols of western blotting:1.Preparation of Polyacrylamide resolving gel and stacking gel;2.An aliquot of 30μg was mixed with an equal volume of SDS-PAGE loading buffer,and heated at 100℃for 5 min;3.Samples with equal amounts of protein and the prestained marker were loaded into the wells;4.Run the gel with constant voltage,80V for 3h;5.Proteins were transferred to PVDF membrane,200mA for 2h;6.Membranes were incubated in 5%Bovine Serum Albumin(BSA)-TBST at room temperature for 2h;7.Incubated with the Phospho-Src Family(Tyr416) polyclonal antibodies in 5%BSA-TBST(1:2000) at 4℃overnight;8.Wash membrane for 30 min with TBST buffer;9.Incubated membrane with the HRP-conjugated anti-rabbit IgG in 5%BSA-TBST(1:5000) in blocking buffer with gentle agitation at room temperature for 1 h;10.Wash membrane for 40 min with TBST buffer;11.The blot was developed with ECL according to the manual and was exposed to X-film;12.Wash membrane for 5min with purify water then incubated the membrane in western stripping buffer for 5min and then washed two 5min in purify water;13.Membranes were incubated in 5%BSA-TBST at room temperature for 2h;14.Incubated with the Src and Actin primary antibodies in 5%BSA-TBST(1:4000) at 4℃overnight;15.Wash membrane for 30 min with TBST buffer; 16.Incubated membrane with the HRP-conjugated anti-rabbit IgG in 5%BSA-TBST(1:5000) in blocking buffer with gentle agitation at room temperature for 1h;17.Wash membrane for 40 min with TBST buffer;18.The blot was developed with ECL according to the manual and was exposed to X-film;19.Visualized by exposure to GS-800 Calibrated Densitometer(Bio-Rad).2.7 Data analysis and statisticsPlace Preference:the time spent in white chamber(TW);Locomotor Activity:the total distance that mice moved in different chambers(TD)and shuttle time(ST); Exploratory Behavior:the total time spent in the center area of both chambers(TTC); the ratio of time spent in the center area of white chamber(TCW) to TW;the ratio of total distance that mice moved in the center area(TDC) to TD;the ratio of distance in the center area of white chamber(DCW) to distance in white chamber(DW).The densitometry of the bands was calculated by the Bio-rad Quantity One? software.The relative levels of protein were obtained by taking the ratio of the band intensity of pSFKs(Tyr416) to total SFKs,SFKs to Actin and expressed as percent of saline control group.Statistical analysis was performed on the average protein level of 5 animals per group.All the data were expressed as the Mean±S.E.M.and analyzed with SPSS software.P＜0.05 was considered statistically significant.3 ResultsTest of preference in preconditioning phasePre-conditioning test shows that all groups mice spent more time in black chamber than in white chamber(529.8±6.1 and 369.2±6.1,respectively,P＜0.05),that suggests that mice prefer the black chamber;Meanwhile,mice of the different groups did not show any significant difference(P＞0.05) in the time spent in black chamber.Effect of CPP expression induced by morphineFollowing morphine conditioning,mice spent a greater amount of time in the morphine-paired chamber than in the saline-paired chamber(513.5±7.6 and 378.0±17.3, respectively,P＜0.05),and the mice in morphine group(Mor) spent more time in the white chamber compared with that of mice in the saline group(NS)(513.5±7.6 and 403.2±13.8,respectively,P＜0.05). Effect of CPP extinction induced by morphineAfter 8 days saline paired extinction training,the mean time spent in the morphine paired side revealed no differences between two groups(P＞0.05),mice did not show conditioned preference for the previous drug-paired chamber.Effect of Con-G and Glu-Con-G on the expression and reinstatement of morphine induced CPP,locomotor activity and exploratory behaviorOn d9,compared with the saline control group(NS-NS),the mice in morphine control group(M-NS) spent more time in the white chamber(535.6±21.4 and 435.0±30.7,respectively,P＜0.05),and testing dose of Con-G and Glu-Con-G(120pmol) neither prolonged nor reduced time mice spent in white chamber(P＞0.05).After conditioning with morphine for 8 days,pretreatment with Glu-Con-G and Con-G(30,60, 120pmol) 30 min before the test displayed a dose-effect dependent decrease the expression of morphine induced CPP(Glu-Con-G:30 pmol:386.2±50.2,P=0.000; 60pmol:381.1±32.3,P=0.000;120pmol:362.3±25.4,P=0.000;Con-G:30 pmol: 430.0±15.7,P=0.006;60pmol:417.2±40.4,P=0.003;120pmol:379.5±12.0,P=0.000).On d19,compared with the saline control group(NS-NS),the mice in morphine control group(M-NS) spent more time in the white chamber(534.6±29.2 and 307.3±26.7,respectively,P＜0.05),and testing dose of Con-G and Glu-Con-G(120pmol) neither prolonged nor reduced time mice spent in white chamber(P＞0.05).After conditioning with morphine for 8 days,pretreatment with Glu-Con-G and Con-G(30,60, 120pmol) 30 min before the test displayed a dose-effect dependent decrease the expression of morphine induced CPP(Glu-Con-G:30pmol:331.2±34.0,P=0.000; 60pmol:287.8±21.4,P=0.000;120pmol:282.9±54.3,P=0.000;Con-G:30pmol: 352.7±32.2,P=0.000;60pmol:354.3±30.8,P=0.001;120pmol:339.7±40.0,P=0.000).Independent from the treatment,mice of the different groups did not show any significant difference in locomotor activity and exploratory behavior,and correlation analysis indicated they were not related with place preference.Protein levels of pSFKs(Tyr416) and total SFKs in PFC and Hp of mice on CPP expression and reinstatement phasesDuring CPP expression phase,the pSFKs(Tyr416) protein levels in PFC and Hp brain areas of mice were decreased in 60 and 120pmol Glu-Con-G,but there were unchanged by 30pmol Glu-Con-G treated;the pSFKs(Tyr416) protein levels in PFC brain areas of mice were decreased in 30,60 and 120pmol Con-G,but only 120pmol Con-G could inhibit the increasement of pSFKs(Tyr416) protein levels in PFC,there were unchanged by 30,60pmol dose of Con-G treated and the SFKs protein levels were no difference(P＞0.05).During CPP reinstatement phase,the pSFKs(Tyr416) protein levels in Hp brain area of mice were decreased in 60 and 120 pmol Glu-Con-G and Con-G;the pSFKs (Tyr416) protein levels in PFC brain areas of mice were decreased in 30,60 and 120 pmol Con-G,but only 120 pmol Glu-Con-G could inhibit the increasement of the pSFKs(Tyr416) protein levels in PFC,meanwhile the SFKs protein levels were no difference(P＞0.05).4 Conclusions1.Glu-Con-G and Con-G(30,60,120pmol) by single intracerebroventricular administration attenuated the expression and reinstatement phases of morphine-induced CPP in a dose-effect dependent manner.2.Glu-Con-G and Con-G(30,60,120pmol) by single intracerebroventricular administration had no invisible behavioral disorder under our experiment conditions.3.The levels of Tyr416-phosphorylated SFKs were increased in PFC and Hp brain areas of morphine addictive mice,meanwhile the protein levels of SFKs were no difference.4.Glu-Con-G and Con-G(30,60,120pmol) by single intracerebroventricular administration attenuated the levels of Tyr416-phosphorylated SFKs in PFC and Hp brain areas of morphine addictive mice.5.The levels of Tyr416-phosphorylated SFKs were increased in PFC and Hp brain areas when normal mice were single intracerebroventricular administered 120pmol Glu-Con-G,but only single intracerebroventricular administered 120pmol Con-G could increased the levels of Tyr416-phosphorylated SFKs in Hp during CPP reinstatement phase and the protein levels of SFKs were no difference. |
