The Effects And Mechanisms Of Glucagon Like Peptide-1 Receptor Agonist In Cocaine-induced Conditioned Place Preference Behaviors In Mice

Part 1:The effects of glucagon like peptide-1 receptor agonist on the cocaine-induced conditioned place preference behaviorsObjective Drug addiction causes serious catastrophic outcomes and lack of effective pharmacotherapies to date.Recently,Glp-1 agonists were reported to improve cocaine-associated rewarding behavior.In the first part of this study,cocaine-induced CPP and CPP reinstatement paradigms were established by intraperitoneal injection of cocaine in mice.And then the effects of exendin-4 on the acquisition,expression as well as extinction and reinstatement of cocaine-induced CPP were behaviorally observed.Methods（1）After 2-day acclimation of CPP apparatus,C57BL/6J mice are required to experience baseline preference test.（2）After the establishment of cocaine-induced CPP and CPP reinstatement paradigms,the effects of intraperitoneal injection of exendin-4 on the acquisition,expression,extinction and reinstatement of cocaine-induced CPP were observed.Results（1）The scores of natural preference showed that the vast majority of mice displayed a significant preference for the black box（P<0.001）.（2）Compared to natural preference,CPP scores were significantly higher in the Coc+Sal group（P<0.001）,indicating that cocaine induced a CPP paradigm.Compared with the Coc+Sal group,the scores of exendin-4（10.0μg/kg,1.0μg/kg,and 0.1μg/kg）groups were decreased（P<0.01）,suggesting that exendin-4 pretreatment inhibited the CPP acquisition in mice.（3）Identical to the above results,after the completion of cocaine-induced CPP;in comparison with natural preference,the Coc+Sal group also scored significantly higher（P<0.01）;compared to the Coc+Sal group,the scores of exendin-4（10.0μg/kg,1.0μg/kg,and 0.1μg/kg）groups were significantly lower（P<0.01）,revealing that single intraperitoneal injection of exendin-4 inhibited the expression of CPP in mice.（4）The first CPP extinction test showed that relative to natural preference,the scores of the Coc+Sal group were still significantly higher（P<0.01）;compared to the Coc+Sal group,exendin-4（10.0μg/kg,1.0μg/kg,and 0.1μg/kg）groups had a lower score（P<0.05）,indicating that the CPP has not achieved the criteria of CPP extinction and exendin-4 promoted the earlier CPP extinction;the second CPP extinction test exhibited no significant difference in exendin-4 10.0μg/kg,1.0μg/kg,and 0.1μg/kg)scores compared with the Coc+Sal group（P>0.05）;compared with natural preference,there was no significant difference in the scores of each group（P>0.05）,indicating that Coc+Sal group and exendin-4（10.0μg/kg 1.0μg/kg 0.1μg/kg）group has reached the standard of CPP extinction.In addition,we also observed the effects of intraperitoneal injection of exendin-4 on CPP reinstatement during CPP extinction,and then found that compared with natural preference,the scores of blank control group were significantly increased again（P<0.001）;compared with the Coc+Sal group,CPP scores of exendin-4（10.0μg/kg,1.0μg/kg,and 0.1μg/kg）groups were significantly decreased(P_{（10.0μg/kg）}<0.01,P_{（1.0μg/kg）}<0.01 and P_{（0.1μg/kg）}<0.05)on CPP reinstatement test day.（5）Unlike the CPP reinstatement procedure described above,exendin-4 was not injected into the mice until the day of CPP reinstatement test.Compared with natural preference,the scores in each group were significantly increased after cocaine treatment（20mg/kg）（P<0.01）compared with Coc+Sal group,exendin-4（10.0μg/kg,1.0μg/kg,and 0.1μg/kg）groups had no significant reduction（P>0.05）,suggesting that a single intraperitoneal injection of exendin-4 on the CPP reinstatement day could not attenuate cocaine-induced CPP reinstatement.Conclusion（1）Intraperitoneal injection of 20mg/kg and 10mg/kg cocaine successfully induced a conditioned place preference and behavioral reinstatement paradigm in mice.（2）Different-doses of exendin-4 played an important role in inhibiting the acquisition and expression of CPP in cocaine-experienced mice.（3）Repeated intraperitoneal injection of different doses of exendin-4 promoted the extinction of CPP and inhibited the reinstatement of CPP.These results altogether suggest that intraperitoneal injection of exendin-4 plays an essential role in regulating cocaine-induced maladaptive behaviors in mice.Part 2:Possible mechanisms by which GLP-1 receptor agonists improve cocaine-induced conditioned place preference behaviors in miceObjective The cocaine-induced CPP and CPP reinstatement paradigms were established in the same way as the first part of the study.In addition,these experiments were designed to investigate the potential mechanism underlying the intraperitoneal injection of exendin-4 on cocaine-induced behavior by using protein analysis technique in mice.Methods（1）After the establishment of cocaine-induced CPP paradigm in mice,we observed the effects of exendin-4 on the acquisition,expression and reinstatement of cocaine-induced CPP.（2）Expression of TLR4,My D88,and NF-κβp65 in the hippocampus of mice was observed by immunohistochemistry and immunofluorescence labeling.（3）Western blot were used to detect the expression of TLR4,My D88,NF-κβp65,TNF-α,and IL-1βprotein in mice.Results（1）CPP scores:compared with Sal+Sal group,scores of Coc+Sal group were significantly increased（P<0.001）,while scores of Sal+Ex4 group were not significantly different（P>0.05）,suggesting that cocaine successfully induced a CPP paradigm.Compared to the Coc+Sal group,the score of the Coc+Ex4 group was significantly decreased（P<0.01）,suggesting that Ex4 inhibited CPP acquisition.In the early CPP regression tests,in comparison with the Sal+Sal group,Ex4 also promoted the earlier CPP extinction（P<0.05）.In the CPP reinstatement tests,compared with the Sal+Sal group,scores of Coc+Sal group were significantly increased（P<0.001）,while scores of the Sal+Ex4 group were not different（P>0.05）,suggesting that cocaine instead of saline successfully induced CPP reinstatement paradigm.Compared with the Coc+Sal group,the score of the Coc+Ex4 group was significantly decreased（P<0.01）,suggesting that Ex4 inhibited the reinstatement of CPP.（2）The concentration of TLR4,My D88 and NF-κβp65 in the hippocampus of mice was found to be different by immunohistochemistry.（3）In line with these results above,the expression level of the three cytokines mentioned above in the hippocampus of mice was significantly different by immunofluorescence labeling（P<0.01）.Compared with Sal+Sal group,the fluorescence density values of the three cytokines mentioned above in the hippocampus of the Coc+Sal group were significantly increased（P<0.001）.Compared with the Coc+Sal group,the fluorescence density values of the three cytokines mentioned above in the hippocampus of Coc+Ex4 group were significantly decreased（P<0.01）.（4）Consistent with the above experimental results,the expression of TLR4,NF-κβp65,My D88,TNF-αand IL-1βin the hippocampus of mice was also significantly different by Western blot（P<0.01）.Compared with the Sal+Sal group,the expression of the five cytokines mentioned above in the hippocampus of the Coc+Sal group was significantly increased（P<0.01）.Compared with the Coc+Sal group,the expression of the five cytokines mentioned above in the hippocampus of Coc+Ex4 group was significantly decreased（P<0.05）.Conclusion（1）Cocaine did not only successfully induce CPP and CPP reinstatement paradigms in mice,but also activated the TLR4-meidated signaling pathway.（2）Exendin-4not only had a pivotal role in regulating the cocaine-induced CPP acquisition as well as extinction and reinstatement,but also inhibited the TLR4-mediated signaling pathway.Therefore,these experimental results show that TLR4-mediated inflammation pathway is related to cocaine-induced maladaptive behavior in mice.Exendin-4 can ameliorate the maladaptive behavior,which may be associated with the down-regulation of TLR4-mediated inflammatory pathway.