Rats Chronic Mophine-dependent Behaviors And Palmitoylation On PSD-95 In Hippocampus And Nucleus Accumbens

BackgroundPalmitoylation is a reversible and regulatable posttranslational modification, regulated by synaptic activity. PSD-95 was identified as one of the candidate genes underlying addiction and involved in regulating dopamine-mediated synaptic and behavioral plasticity. Palmitoylation on PSD-95 contributes to the modulation of receptors function and synaptic plasticity. Here we investigated whether palmitoylation of PSD-95 is involved in morphine-induced drug-dependent, withdrawal and re-instatement in vivo.Objective(1) To establish the morphine conditioned place preference (CPP) rats model; and to study their behaviors at different time points of drug-dependent, withdrawal and re-instatement.(2) To demonstrate protein expression of PSD-95 in both hippocampus and nucle accumbens in different time points of morphine-induced drug-dependent, withdrawal and re-instatement.(3) To explore the relationship of morphine conditioned place preference (CPP) and dynamic change of palmitoylation on PSD-95 in hippocampus and nucleus accumbens at different time points of drug-dependent, withdrawal and re-instatement.Methods(1) Thirty male Sprague-Dawley rats were randomly assigned to morphine exposure group (n=24) and control group (n=6). Rats in morphine exposure group were injected morphine hydrochloride (5mg/kg, i.p.) twice per day at 6 h intervals for 10 days. The incremental dose (5 mg/kg per day) was from 5 mg/kg on day 1 to 50 mg/kg on day 10. Rats in control group received saline injection. The natural non-preferred chamber was chosen as drug-paired side, CPP was test as basline. After 10 days CPP training, the time spent in drug-paired chamber was record two hours after final injection (withdrawal 0 day), two days (withdrawal 2 days), eight days (withdrawal 8 days), ten days after final injection (withdrawal 10 days). After withdrawal twelve days, rats received the priming injection of morphine (5mg/kg, i.p.) before the final CPP test. We observed the Withdrawal Signs, including teeth chattering (TC), wet dog shakes (WS), rearing, stretching, penis licking (PL), diarrhea, piloerection during withdrawal phase and weight loss were recorded daily. The results of each group at different time points were analyzed by students' t test and one-way ANOVA, followed by LSD.(2) Based on results of CPP tests on the last day of conditioning training, rats in CPP group were randomly assigned into 4 subgroups: withdrawal dayO group (WDO:n=6), withdrawal day2 group (WD2: n=6), withdrawal day 10 group (WD10:n=6), and on day 13 of withdrawal morphine re-exposure (RE:n=6) groups, CPP tests on the day0, day2, day8, day 10 and day 13 of withdrawal. Within 2 hours after behavior tests on day0, day2, day10 and morphine re-exposure day, hippocampus and nucleus accumbens dissections were obtained from decapitated rats in each group, respectively. Whole proteins were extracted and parts of them were used for Western blot. PSD-95 expression levels of six rats in each group were summarized and compared with control group.(3) Immunoprecipitation (IP)-based Acyl-biotin exchange labeling assays (ABE) were performed on the remaining whole proteins to detect palmitoylation of PSD-95. The results were normalized by total PSD-95 after IP and record the dynamics of palmitoylation on PSD-95 in rat hippocampus and nucleus accumbens at different time points.Result(1) With 10 days of morphine-conditioned training, there is a significant increase of the time spent in drug-paired compartment as compared to the pertreatment baseline. Rats showed higher CPP at the first two days of withdrawal, but CPP was waned and physical signs were gradually extinct during long-term withdrawal. Morphine re-injection restored significant preference of drug-paired compartment.(2) The expression levels of PSD-95 decreased both in the hippocampus and nucleus accumbens and maintained at a low level during withdrawal. However, there were no significant differences in PSD-95 levels of morphine-conditioned group, compared to control group.(3) Rats developed significant CPP had higher level of palmitoylation on PSD-95 increased in the hippocampus and nucleus accumbens. Furthermore, palmitoylation on PSD-95 was significantly decreased in hippocampus, but increased in nucleus accumbens during the beginning of withdrawal, with long-term withdrawal, palmitoylated PSD-95 in these regions recovered while CPP was waned and physical signs were gradually disappeared. However, morphine re-injection restored high CPP without producing any significant change of palmitoylation on PSD-95.Conclusion(1) The chronic morphine treatment developed significant conditioned place preference, and rats expressed remarkable withdrawal symptoms at the first two days after drug extinction. Morphine re-injection restored high CPP after long-term withdrawal. It suggested that the rat models of morphine dependence, withdrawal and reinstatement were successfully established.(2) Reduction of PSD-95 protein expression in hippocampus and nucleus accumbens during withdrawal might be one of the biomolecular mechanisms underlying morphine-dependent behavior, and might involve in surface expression of dopamine receptor and NMDA/AMPA receptors, which contribute to the drug-induced synaptic plasticity.(3) Dynamic change of palmitoylation on PSD-95 in hippocampus and nucleus accumbens at different time points indicated that palmitoylation on PSD-95 might be one of mechanisms of morphine-induced neural plasticity and addictive behaviors.