| Objective:(1) To study the development of morphine conditioned place preference (CPP) after chronic stress in rats; and to study the interaction of glucocorticoid (GC) in chronic stress and drug addiction.(2) To demonstrate the effects of chronic stress and GC on morphine induced fosB transcription in striatum.(3) To explore the relationship of histone modification and fosB transcription and the influence of GR on histone modification at fosB promoter.Methods:(1) Fifty-four male Sprague-Dawley rats were randomly assigned to footshock (FS) group, mifepristone group, and control group. Rats in mifepristone group were injected mifepristone (20mg/kg, s.c.). Rats in FS group and control group were injected vehicle (20% DMSO in saline). After 30min of injection, rats in FS group and mifepristone group were received foot shock (2mA, 1sec on, 9～12 off, for 15min) for 7 days. Rats in control group stayed in their home cages. After 7 days, rats in each group were injected morphine (5mg/kg, s.c.) and were received CPP training for 2 days. The natural non-preferred chamber was chosen as drug-paired side. The time spent in drug-paired chamber in CPP test after the treatment substracts that before the treatment is CPP score. CPP scores of each group were analyzed using student's t test.(2) Within 2 hours of last morphine administration, striatal dissections were obtained from decapitated rats. The striatal dissenctions obtained from six rats in each group were used for isolating total RNA (remaining striatal dissections were used for ChIP assays). Total RNA was purified by Trizol, processed, reverse-transcribed to cDNA using a first-strand synthesis kit, and quantified using real-time PCR. PCR reactions were repeated at least two times independently. The relative level of fosB mRNA in striatal dissection was normalized by compared with that of GAPDH.(3) ChIP assays were performed on the basis of a protocol from Millipore ChIP kit. The following antibodies were used: anti-acetylated H3; anti-acetylated H4; anti-dimethylated H3K4; and normal rabbit IgG. Quantification of Chromatin Immunoprecipitation by Real-Time PCR: Levels of specific histone modifications at fosB and GAPDH gene promoter were determined by measuring the amount of that gene in chromatin immunoprecipitates by use of real-time PCR. Input or total DNA (nonimmunoprecipitated) and immunoprecipitated DNA were PCR amplified in the presence of SYBR Green. Ct values from each sample were obtained. Relative quantification of amplified template was performed. Each real-time PCR reaction was repeated at least twice independently.Result:(1) The FS rats (359.4±132.1, n=12) showed significant increase of morphine conditioned place preference compared to the rats in mifepristone group (68.2±83.6, n=12) and control group (21.7±67.8, n=12). There is no significant difference in CPP score for rats in mifepristone group and control group.(2) The rats in the FS group (3.35±0.51, n=6) had a higher level of fosB mRNA in striatal than the rats in mifepristone group (1.86±0.58, n=5) and control group (1.01±0.22, n=6), which is parallel to the CPP score.(3) There was no significant difference in levels of acetylated histone H3 and histone H4 on fosB promoter in striatal of rats in FS group and control group. However, dimethylated histone H3 lysine 4 were much higher on fosB promoter in striatal of rats in FS group (3.58±1.01, n=5) than mifepristone group (1.23±0.70, n=5) and control group (1.01±0.18, n=5).Conclusion:(1) Chronic stress promotes formation of morphine CPP in rats, which appears to be dependent upon HPA-activated corticosterone secretion and could be partially blocked by GR antagonist mifepristone.(2) In chronic stressed rats, GR appears to be evolved in regulation of morphine-induced fosB expression in striatum. The increase of fosB expression in striatum after morphine exposure potentiates morphine CPP.(3) Histone H3 lysine 4 dimethylation facilitates the expression of fosB in striatum, which may be regulated by GR.
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