Regulatory Role Of D4R In Methamphetamine-induced Addiction In Mice

Background and Objective:There are a large number of methamphetamine（methamphetaine,METH）users in the world,which leads to an endless stream of illegal and criminal activities,which poses a great threat to social public health and safety.It is urgent to study the abusive detoxification methods of METH and how to prevent METH relapse.METH is an addictive central nervous system stimulant that produces a strong central excitatory effect after taking it.Dopamine（dopamine,DA）is the most critical neurotransmitter in the process of drug addiction,and the research on its receptor is also an important target to study the mechanism of drug addiction and to find anti-addictive drugs.Dopamine receptors（dopamine receptor,DR）can be divided into dopamine D1-like receptors and dopamine D2-like receptors.Dopamine D1-like receptors include D1R and D5R,which mainly act on postsynaptic membrane excitatory G protein-coupled receptors.Dopamine D2-like receptors include D2R,D3R and D4R,and mainly act on inhibitory G protein-coupled receptors after activation.Brain dopamine D4receptor（dopamine D4 receptor,D4R）,as a dopamine receptor that does not cause CPP,has no addictive potential and is a feasible treatment for addiction.However,due to its complex function,the regulatory role of D4R in METH-induced addiction in mice and the mechanism of gene expression changes are still unclear.Based on the above background,this study took C57BL/6 mice as the research object,applied the bar place preference（conditioned place preference,CPP）model,first established the mouse addiction model induced by METH,and intervened the METH addicted mice with highly selective D4R agonist（A-412997）and antagonist（A-381393）to explore the precise regulation of D4R in the process of METH addiction in mice.At the same time,the expression of D4R m RNA in prefrontal cortex（prefrontal cortex,PFc）,nucleus accumbens（nucleus accumbens,NAc）,hippocampus（hippocampus,Hip）and ventral tegmental area（ventral tegmental area,VTA）of METH addicted mice was detected by real-time fluorescence quantitative PCR（quantitative real-time polymerase chain reaction,q RT-PCR）to explore the regulatory role of D4R in METH addiction.The purpose of this study is to provide a theoretical basis for further clinical study of highly effective detoxification drugs targeting D4R.Methods:（1）The establishment of C57BL/6 mouse CPP model induced by METH:the CPP model in this experiment includes three stages:pre-test period（1–2 days）,training period（3–10 days）and post-test period（11 days）.The mice were divided into saline group（S group）,METH control group（M group）and D4R ligand intervention group（6 subgroups）.All mice were not injected with drugs during the pretest period（days 1–2）.The mice in group S were intraperitoneally injected with normal saline every day during the training period（3–10 days）,and the mice in group M were intraperitoneally injected with saline（10m L/kg）and METH（1mg/kg）alternately during the training period（3–10 days）,that is,METH（1mg/kg）was injected intraperitoneally on the 3rd,5th,7th,9th and 8th day,and normal saline（10m L/kg）was injected intraperitoneally on the 2nd,4th,6th and 8th day.The mice in group S and group M were intraperitoneally injected with saline（10m L/kg）during the 11th day at the 10 mins before the test,and the mice in group A-412997 and A-381393 were injected intraperitoneally with A-412997 1mg/kg,4mg/kg,and 16mg/kg,A-381393 1mg/kg,4mg/kg,and 16mg/kg respectively at the 10 mins before the test,.The video capture and analysis system collected the residence time,activity distance and shuttle times of mice in each test stage（15 min）in white box and black box.（2）The changes of D4R expression in different brain regions:in this study,four kinds of brain nuclei which were closely related to drug addiction and distributed in D4R were used as subjects.Immediately after the behavior experiment,the mice were killed by cervical dislocation method,and the nuclei of PFc,NAc,Hip and VTA were quickly separated.The changes of D4R m RNA expression in different brain regions were detected by SYBR Green method by quantitative real-time PCR（Quantitative Real-time PCR,q RT-PCR）.The relative expression level of D4R m RNA in different brain regions was calculated by 2^-ΔΔCT method,and the changes of D4R m RNA in different brain regions were analyzed.To explore the regulatory role of D4R in METH-induced addiction in mice.Results:（1）D4R agonist（A-412997,1mg/kg,4mg/kg）could significantly promote the expression of CPP in experimental mice,but the activation of D4R had no effect on the total activity of experimental mice during the test period（15min）.（2）D4R antagonist（A-381393）significantly promoted the expression of CPP in experimental mice,but the antagonist of D4R had no effect on the total activity of experimental mice during the test period（15min）.（3）The expression of D4R m RNA in PFc,NAc,Hip,VTA and other brain regions of METH addicted mice was significantly higher than that in S group,while the expression of D4R m RNA in D4R agonist group and D4R antagonist group was significantly lower than that in M group.Conclusion:（1）METH can induce addiction in mice and lead to compensatory increase of D4R expression in corticostriatal pathway-related brain nuclei of addicted mice,indicating that D4R plays an important role in the process of METH addiction.（2）D4R agonist（4mg/kg）and D4R antagonist could antagonize the effect of METH and decrease the expression of D4R in various brain regions of mice,indicating that D4R in brain regions such as PFc,NAc,Hip and VTA play an important role in the process of METH addiction.The decrease of D4R level in these brain regions may enhance the excitability of the cortical striatal pathway in the brain,resulting in the increased expression of CPP in METH addicted mice.