| Objective: Heroin-induced conditioned positional preference(CPP)model of rat was established,and behavioral,morphological and molecular biological experimental methods were adopted to detect the expression of neurotrophic factors and glial cell response levels in the prelimbic area(PL)and inferior limbic cortex(IL)of the medial prefrontal cortex of rats,thus to explore their role in the process of heroin accdition and related mechanisms.Methods:1.Establishment of heroin-induced CPP model: The experimental rats were randomly divided into saline-treated control group and heroin-treated induction group.The heroin-induced CPP model in rats was established by using the CPP system with small incremental doses.The rats in the induction group were injected subcutaneously with heroin in the morning and trained in the white box for 45 min,and injected with an equal amount of saline in the afternoon and trained in the black box for 45 min at a starting dose of 0.5 mg/kg for 7 days.The dose was increased by 0.5 mg/kg daily.The rats in the control group were treated with the same amount of saline instead of heroin,and the training method was the same as above.The CPP test was performed on the next day after training,and the behavioral parameters and movement trajectories of all rats were recorded.The success of the heroin-induced CPP model was judged based on the changes of the behavioral parameters before and after heroin administration.After the CPP model was constructed,the heroin-induced group was divided into heroin-induced CPP state and withdrawal state.The withdrawal state rats were discontinued and naturally withdrawn for 7 days.The CPP test was performed again and the relevant behavioral data were recorded.2.Detection of BDNF and GDNF expression levels in PL and IL regions: From heroin induced CPP model in rats,each group was randomly selected,and the rats were perfusion-dead,and the brains were removed and coronal frozen sections(20 μm)were made;the other rats were severed and executed,and the PL and IL regions were removed and placed in centrifuge tubes with reference to the rat brain stereotaxic atlas and stored at-80℃ in an ultra-low temperature refrigerator.The expression of BDNF and GDNF in PL and IL regions were detected by immunofluorescence histochemical staining and Western Bolt method,and the differences were compared between control group,heroin-addiction and withdrawal state.3.Observation of glial response changes of astrocytes and microglia in PL and IL areas: From heroin induced CPP model in rats,each group was randomly selected,the rats were executed by cardiac perfusion,the brains were taken,and coronal frozen sections(20 μm)were made,and the glial responses of astrocytes and microglia in PL and IL areas were observed by immunohistochemistry,and the response changes in control,heroin-addiction and withdrawal states were compared.Results:1.After 7 days of heroin-induced CPP training,the rats in the induction group showed a significant increase in the percentage of active time in the white box and the percentage of active distance traveled in the white box compared with those before the administration of saline(P < 0.05),suggesting that the heroin-induced CPP model was successfully established.There was no significant difference in the behavioral data of the control group before and after saline administration(P > 0.05),suggesting that saline could not induce the production of CPP.The percentage of time spent in the white box,the percentage of distance traveled,the total distance traveled and the number of shuttles in the black and white boxes were significantly increased(P < 0.05)in heroin withdrawal status rats compared with heroin CPP states and controls,suggesting significant drug foraging behavior in heroin withdrawal states rats.2.Immunofluorescence histochemical staining of rat brain slices showed that BDNF and GDNF were expressed in PL and IL regions of control,CPP state and withdrawal state rats.The Western Bolt results showed that the BDNF protein expression level in the PL region showed a gradient increase from control,CPP state to withdrawal state,and all of them were significantly different(P < 0.05);the IL region showed the highest expression in the CPP state,and the withdrawal state was lower than the CPP state(P < 0.05),but significantly higher than the control group(P < 0.05).GDNF protein expression in the PL region showed the same trend as BDNF;the IL region showed the lowest expression in the CPP state,and the withdrawal state increased relative to the CPP state(P < 0.05),but was significantly lower than the control group(P < 0.05).3.Immunohistochemical staining of rat brain slices showed that the expression of GFAP,a specific marker of astrocytes in the PL and IL regions,and Iba 1,a specific marker of microglia,were significantly enhanced in heroin CPP status rats compared with control rats(P < 0.05),and immunoreactive glial cells showed significant proliferative changes.After 7 days of withdrawal,GFAP and Iba 1 in the PL and IL regions of rats in the withdrawal state continued to be highly expressed,and the cells still maintained proliferative changes.Conclusion: In this study,using the CPP system,a heroin-addiction rat model was successfully established,and the percentage of time spent in white-box activity and the percentage of distance traveled in white-box activity increased in rats compared with those before drug administration,and the rats showed more significant positional preference after withdrawal.During the stages of heroin-induced CPP formation and withdrawal,BDNF and GDNF had differential expression changes in the PL and IL regions.In heroin-induced CPP formation and withdrawal state,astrocytes and microglia in PL and IL areas showed significant proliferative changes,and the expression of specific markers GFAP and Iba 1 were increased,suggesting that glial cells in both PL and IL areas can be activated during heroin-induced CPP process. |
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