| Objective: To investigate the roles and working mechanism of prelimbic cortex(PL)in heroin-induced conditioned place preference(CPP)from behavioral,neurochemical and molecular levels.Methods:1.Establishment of heroin-induced CPP model: 20 rats were divided into control group and heroin-induced group(10 rats in each group).Heroin-induced CPP model was established by subcutaneous injection of heroin on rats.The dose was 0.5mg/kg in the first day and increased by 0.5mg/kg daily in 7 days.The rats of the heroin-induced group were placed in the white box of CPP for 45 minutes after subcutaneous injection of heroin in the morning,then after 6 hours,placed in the black box of CPP for 45 minutes after subcutaneous injection of saline.Rats in the control group were subcutaneously injected with the same dose of saline in the morning and afternoon in the white and black box successively.Conditioned place preference software was used to record the behavioral parameters of each item in the rat heroin-induced CPP model,to judge whether the rat heroin-induced CPP model was established successfully,and to analyze the changes of behavioral parameters before and after the establishment of the rat heroin-induced CPP model.2.Lesions of PL: rats were randomly divided into sham operation group and lesion group(10 rats in each group).In the lesion group,0.5ul of IBO was injected into the bilateral PL area.Meanwhile in the sham operation group,0.5ul of normal saline was injected into the bilateral PL area.One week after this operation,the model of heroininduced CPP was established.By analyzing the indicators of CPP before and after heroin injection,it can be speculated whether the damage of PL area on both sides could affect the formation of CPP induced by heroin.The location of PL damage could be observed by the staining of Toluidine blue.3.Determination of NR2 subunit in PL area: rats were randomly divided into two groups: 16 rats in heroin group and 8 rats in control group.In heroin group,16 rats were divided into two states: heroin CPP state and heroin withdrawal state with 8 rats in each state.Then establish the heroin-induced CPP model on the two groups.The brains of the control group and the heroin CPP rats were taken after 7 days of CPP training and perfused by the heart,and then the frozen sections with thickness of 20 um were made.The rats in the state of withdrawal were given normal diet for 7 days without heroin injection.At the end of the withdrawal training,the brain of the withdrawal rats were perfused through the heart,and the frozen sections with thickness of 20 um were made.The expression of NR2 subunit were assessed in PL region by immunofluorescence staining.Results:1.For the heroin-induced group,after 7 days of CPP training,the percentage of activity time and distance in white box increased significantly(P < 0.01),the numbers of shuttles in black box and white box increased(P < 0.05),but the average speed of movement did not change significantly(P > 0.05).2.For the sham operation group,the percentage of activity time in the white box and the percentage of moving distance in the white box increased significantly(P < 0.01),while for the lesion group,the percentage of activity time in the white box,the percentage of activity time in white box,the percentage of distance in white box and the numbers of shuttles in CPP box were not significantly different from those before modeling(P > 0.05).Compared with the lesion group,the numbers of shuttles in the sham operation group increased significantly(P < 0.05).3.For the heroin-induced group with state of withdrawal,there were significant differences in the percentage of activity time in the white box,compared with the state of heroin CPP in the heroin-induced group and before modeling(P < 0.01).4.The results of immunofluorescence staining showed that the expression of NR2 B in PL region in CPP state was significantly higher than that in control group(P < 0.01),and there was no significant change in other NR2 subunits.There was no significant difference in NR2 B expression between heroin withdrawal state and CPP state(P > 0.05),but it was significantly higher than the control group(P < 0.01).Conclusion: The model of heroin-induced CPP in rats can be successfully established by using CPP system.The results showed that the percentage of activity time in the white box,the percentage of moving distance and the numbers of shuttles were increased after 7 days of heroin injection,and the position preference was increased after withdrawal.The excitatory chemical damage in PL region destroyed the formation of CPP induced by heroin,indicating that PL region may be involved in heroin dependence and addiction related learning and memory.NR2 B may be involved in the formation of heroin addiction,while NR2 A and NR2 C are closely related to the expression of drug seeking behavior after heroin withdrawal. |
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