| BACKGROUDAt present, the rapid momentum of amphetamine-type stimulants (ATS) abuse is increasing, the number of stimulant abuse has exceeded the number of heroin and cocaine abuse and was global spreading in the21st countries of five continents. A survey of seven European countries is that the ATS has become the second largest category of the most commonly abused addictive substances. In China, amphetamine-type stimulant abuse situation is grim, it can damage to the central nervous system and cardiovascular system seriously, prone to herd riots, personal attacks and other violence, causing social and public health problem, seriously damaging the social climate. Therefore, development of the treatment for its dependence and drug withdrawal symptoms is significance. The history of Chinese medicine in drug treatment has been more than200years, it has accumulated a wealth of experience, the formation of a unique theory and effective way. The treatment of ideology in Chinese medicine drug treatment is strengthening body resistance and eliminate pathogenic factor, highlighting the diagnosis and treatment characteristics. Traditional Chinese Medicine therapy has the unique advantages: low toxicity, low cost, a wide range source of drugs and few of addiction, pay attention to starting from the disease and syndrome, dialectical treatment by traditional Chinese medicine, so the development of new drugs or drug treatment from Chinese medicine is likely to a breakthrough on the problem of withdrawal.A large number of research results show that the central glutamic acid nervous system and drug dependence are closely linked, there are many reports about NMDA receptors and AMPA receptors and drug dependence related, but tyrosine hydroxylase (TH) as a key enzyme in dopamine synthesis also plays an important role in the process of drug dependence, there is few reports about it. Marking TH With green fluorescent protein(GFP) is a new molecular biology techniques, it makes the TH expression changes become more intuitive. GFP is a light-emitting protein expression in living cells without exogenous substrates or cofactors, can issue the green fluorescence by blu-ray excitation. GFP as a fluorescent marker has both sensitive tag detection rate and no radioactive hazards.Conditioned place preference(CPP) is usually used to study on drug-dependence for Evaluation of drug psychological dependence potential. The experimental animals on drug psychological dependence studies is mostly rodents: rats and mice, but With the emergence of increasingly diverse drug, We need to find new available model organism as a complement to traditional model organisms for the field of addiction medicine.Zebrafish as a new model animal, compared with rats or mice and other mammals, their mating behavior is affected by photoperiod control, the amount of spawning, the fertilized egg in vitro fertilization, the reproductive cycle fast, easy feeding; and their early embryo is transparent as a whole, observation in vivo easily, The application prospects of the behavior of the zebrafish are gradually being understood by researchers. OBJECTIVE1. To establish methamphetamine CPP animal models of adult zebrafish, observe the CPP effects in adult zebrafish, assess the dependence of the effect of adult zebrafish from behavior. At the same time, to treat by Chinese medicine active ingredients and to observe the behavioral changes of the methamphetamine-dependent adult zebrafish.2. To establish the model of the adult zebrafish methamphetamine conditioned place preference, Observe the NR2B receptor, the GluR2receptor and the TH receptor expression changes in the methamphetamine conditioned place preference adult zebrafish brain and change of expression after treatment by the as Chinese medicine active ingredients by western blotting.3. To mark a key enzyme—H in the dopamine synthesis with GFP, then cultivate the TH-GFP transgenic zebrafish by transgenic technology, determine its lever of methamphetamine dependence by the fluorescence in zebrafish Larvae head. Preliminary groping for the effective concentration of Uncaria macrophylla leaf aqueous extract about the zebrafish larvae.4. To provide experimental evidence for confirming that the zebrafish is an ideal model to study amphetamine-type stimulants dependence, Further study of methamphetamine-dependent mechanism of the zebrafish and mechanism of Traditional Chinese Medicine treatment.METHODS1. To establish conditioned place preference model in adult zebrafish:According to the literature and pre-experimental results, they shows that the natural preference of adult zebrafish box is brown box, so to definie the transparent box with the kits.20 adult zebrafish which passed the determination of nature place preference were randomly divided into①ormal control group,②odel group of methamphetamine. The fish were moved to the behavior room under maintenance conditions and feeding schedule identical to the fish facility at least2days before each assay. The water level was kept to5cm from the tank bottom to minimize stress. On the third day, the fish was recorded for a15-min trial using Noldus system, The preferred compartment was defined as the compartment in which a fish spends most of its time during the measurement on day3, and the place preference(PP) is calculated as the percentage of time that the fish spends in the preferred compartment. On day4, day6and day8, anesthetized in tricaine for a few seconds. It was immobilized by hand into a Petri dish containing tank water, and then received methamphetamine (40μg/g) by intraperitoneal injection using syringe. After waking up, the fish was confined to the non-preferred compartment for45min. The restriction to this compartment was achieved using a transparent slider. Thus, visual contact with the preferred compartment remained, and the difference between the conditioning and recording conditions was minimized. The experimental tank and conditions were otherwise identical to the ones used for place preference determination, and each fish was tested separately. After45min, the fish was removed from the experimental tank and kept in a1.5L tank on a color-neutral background. On day5and day7, fish was injected intraperitoneally with a saline solution, then restricted for45min into the preferred compartment. Between each injection session, the experimental tank was cleaned with70%ethanol and rinsed with fish facility water. Place preference was then measured again on day9and compared the difference of stay-time in the prefer department.2. The impact of Uncaria alkaloids for Methamphetamine-dependent adult zebrafish:2.1The impact of Uncaria alkaloids for CCP of Methamphetamine-dependent adult zebrafish:50adult zebrafish which passed the determination of nature place preference were randomly divided into①normal control group,②odel group of methamphetamine,③odel group+low dose of group(50μg/g),④odel group+high dose of group(100μg/g),⑤model group+ketamine group (150μg/g) The fish were moved to the behavior room under maintenance conditions and feeding schedule identical to the fish facility at least2days before each assay. The water level was kept to5cm from the tank bottom to minimize stress. On the third day, the fish was recorded for a15-min trial using Noldus system, The preferred compartment was defined as the compartment in which a fish spends most of its time during the measurement on day3, and the place preference(PP) is calculated as the percentage of time that the fish spends in the preferred compartment. On day4, day6and day8, anesthetized in tricaine for a few seconds. It was immobilized by hand into a Petri dish containing tank water, and then received methamphetamine (40μg/g) by intraperitoneal injection using syringe, except control group; control group received equal volume of normal saline. After waking up, the fish was confined to the non-preferred compartment for45min. The restriction to this compartment was achieved using a transparent slider. Thus, visual contact with the preferred compartment remained, and the difference between the conditioning and recording conditions was minimized. The experimental tank and conditions were otherwise identical to the ones used for place preference determination, and each fish was tested separately. After45min, the fish was removed from the experimental tank and kept in a1.5L tank on a color-neutral background. After12hours, except control group, injected equal volume of normal saline in model group; injected low dose of in low dose of rhynchphylla total alkaloids group; injected high dose of rhynchphylla total alkaloids in high dose of rhynchphylla total alkaloids group; injected ketamine in ketamine group. On day5and day7, fish was injected intraperitoneally with a saline solution, then restricted for45min into the preferred compartment. After12hours, the steps are as similar as day4. Between each injection session, the experimental tank was cleaned with70%ethanol and rinsed with fish facility water. Place preference was then measured again on day9and compared the difference of stay-time in the prefer department.2.2The impact of Uncaria alkaloids for expression of NR2B receptor, GluR2receptor and TH receptor in the brain of Methamphetamine-dependent adult zebrafish: to extract total protein in adult zebrafish brain and to computer protein content. According to the protocol of the SCT1111Lab in Hong Kong Baptist University, finished the western blotting.3. To establish methamphetamine-dependent animal model of zebrafish larvae and to groping for the effective concentration of Uncaria macrophylla leaf aqueous extract about the zebrafish Larvae:microinjected the TH—FP plasmid into yoke of one cell stage zebrafish eggs. After5days,12TH—FP transgenic zebrafish larvae which is alive were randomly divided into①model group (60mg/L),②model group (6mg/L),③model group (0.6mg/L),④model group (0.06mg/L). Zebrafish larval of each group into corresponding concentrations of methamphetamine solution in freely moving with fasting. After3days, to observe the fluorescence intensity in of TH—FP transgenic zebrafish larvae brain.After microinjection5days,21transgenic zebrafish larvae were randomly divided into①Uncaria macrophylla leaf aqueous extract group (1g/mL),②Uncaria macrophylla leaf aqueous extract group (0.33g/mL),③Uncaria macrophylla leaf aqueous extract group (0.033g/mL),④Uncaria macrophylla leaf aqueous extract group (0.0033g/mL),⑤Uncaria macrophylla leaf aqueous extract group (0.00033g/mL),⑥model group (0.6mg/L),⑦control group. Zebrafish larval of each group into0.6mg/L methamphetamine solution in freely moving with fasting, except①group. After3days, Zebrafish larval of each group into corresponding concentrations of Uncaria macrophylla leaf aqueous extract, Zebrafish larval of⑥group into water in freely moving with fasting, except①group. Next day, to observe the fluorescence intensity in of TH—FP transgenic zebrafish larvae brain.RESULTS1. The experimental results shown that compared with control group and model group by two independent sample t-test (t=-11.791, P=0.000), the difference of stay-time is significant. According the map of zebrafish in the prefer department, after modeling, the track of model group zebrafish is reduced significantly than before; and the change of control group is not significant.2. The experimental results shown that compared with control group, the difference of model group stay-time is significant (P=0.000), compared with model group, the difference of high dose of rhynchphylla total alkaloids group(P=0.000)stay-time and ketamine group (P=0.000) are significant, but the difference of low dose of rhynchphylla total alkaloids group stay-time is not significant (P=0.949)The western blotting results shown that compared with control group, the difference of the TH receptor expression in model group adult zebrafish brain is significant (P=0.001), the difference of high dose of rhynchphylla total alkaloids group (P=0.777) the TH receptor expression and ketamine group (P=0.546) the TH receptor expression are not significant; compared with model group, the difference of high dose of rhynchphylla total alkaloids group (P=0.001) the TH receptor expression and ketamine group (P=0.007) the TH receptor expression are significant, the difference of low dose of rhynchphylla total alkaloids group the TH receptor expression is not significant (P=0.113); compared with control group, the difference of the NR2B receptor expression in model group (P=0.001) and low dose of rhynchphylla total alkaloids group (P=0.01) the NR2B receptor expression are significant, the difference of high dose of rhynchphylla total alkaloids group (P=0.083) the NR2B receptor expression and ketamine group (P=0.105) the NR2B receptor expression are not significant; compared with control group, the difference of the GluR2receptor expression in model group (P=0.04) is significant, the difference of high dose of rhynchphylla total alkaloids group (P=0.334) the GluR2receptor expression and ketamine group(P=0.130)the GluR2receptor expression are not significant, compared with model group, the difference of the GluR2receptor expression in high dose of rhynchphylla total alkaloids group (P=0.003)is significant, the difference of the GluR2receptor expression in low dose of rhynchphylla total alkaloids group (P=0.698) is not significant.3. The experimental results shown that compared with normal zebrafish larvae, the fluorescence of transgenic zebrafish larvae is brighter. Compared with control group, the fluorescence of model group (60mg/L) and model group (6mg/L) and model group (0.6mg/L) are brighter. After zebrafish larval of each group into corresponding concentrations of Uncaria macrophylla leaf aqueous extract, Uncaria macrophylla leaf aqueous extract group (1g/mL), Uncaria macrophylla leaf aqueous extract group (0.33g/mL),Uncaria macrophylla leaf aqueous extract group (0.033g/mL) and Uncaria macrophylla leaf aqueous extract group (0.0033g/mL) are dead, but Uncaria macrophylla leaf aqueous extract group (0.00033g/mL) is alive.CONCLUSION1. Methamphetamine can make adult zebrafish have obvious place preference in the medicine cabinet, prolonging the stay-time in the drug-paired department. It confirmed that adult zebrafish can be used as a model organism for the study of drug dependence. High doses of rhynchphylla total alkaloids (100μg/g) and ketamine (150μg/g) could significantly inhibit methamphetamine dependent adult zebrafish place preference. Low dose of rhynchphylla total alkaloids (50μg/g) on methamphetamine dependence in adult zebrafish place preference shows no obvious inhibition effect.2. Methamphetamine-dependent formation can make the expression of normal adult zebrafish brain TH, NR2B, GluR2was significantly higher. High doses of rhynchphylla total alkaloids (100μg/g) and ketamine (150μg/g) could significantly inhibit the change, make the expression of TH, NR2B, GluR2in methamphetamine-dependence adult zebrafish brain is down regulated, while the lower dose of rhynchphylla total alkaloids(50μg/g) had no obvious effect on the change.3. We can use the TH—GFP transgenic zebrafish, under the microscope by green fluorescence intensity, to observe the zebrafish methamphetamine-dependence, and the effects of the drug treatment.0.6mg/L TH—GFP methamphetamine solution can make transgenic zebrafish head have obvious fluorescence enhancement, and the concentration of0.00033g/mL Uncaria macrophylla leaf water extract can to reduce this fluorescence change. |
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