| Objective: The key feature of opiate addiction is compelling and durative drug use. Persistent and stable changes in behavior and psychololgical function during drug addictin, are thought to be due to the reorganization of synaptic connections (synaptic plasticity) in relevant brain circuits. Because the vast majority of synaptic inputs onto neurons are on dendrites or dendritic spines, a focus of much research on structural plasticity has been on the morphology of dendrites and dendritic spines. Actin cytoskeleton plays a pivotal role in morphological plasticity of spines. However, it is not clear if morphine affects the rearrangement of actin, and the molecular mechanism still waits to be identified. The present researches were designed to determine whether morphine treatment rearranges actin at cellular and general levels, and to investigate the role of actin binding protein in rearranging of actin induced by morphine.Methods:1 The establishment of morphine dependence rat models: 42 male SD rats (200±20)g, provides by the Hebei Medical University Experimental Animal Center. Rats were randomly divided into 6 groups with 7 in each, including physiological saline control group, 1 week after morphine dependence, 2 weeks after morphine dependence and 4 weeks after morphine dependence group. Morphine dependence was established by ip injection increasing doses of morphine hydrochloride three times per day for 7 days. The daily dose of morphine was increased as 5, 10, 15, 20, 30, 40, 50mg.kg-1. Two rats were randomly selected in every group, injected with 5mg.kg-1 naloxone at d8 to precipitate withdrawal syndromes. 1 week after morphine denpend group and physiological saline control group were sacrificed by decapitation. The brain regions including frontal cortex, hippocampus, thalamus and striatum were separated and frozen until assay. 2 week after morphine denpend group and 4 week after morphine denpend group received continuously 50 mg.kg-1 morphine injection 1 time per day for 1 or 3 weeks. 5 hours after the last injection of morphine, rats were sacrificed by decapitation. The brain regions were separated. Rats in control group received the equal volume of saline.2 Determination of actin and actin binding protein levels in rat brain regions by Western-blot: brain tissues were homogenized in phosphate buffered saline. F-actin in cystoskeleton fraction was separated from G-actin in endochylema with different buffer; brain tissues were homogenized, then p-cofilin were detected from total protein; Drebrin is specific binding protein, so drebrin of suspension include two parts: the part of binding F-actin and free endochylema. The two parts of suspension were homogenized, then drebrin were detectived by western-blot; protein content was measured in the suspension by the BCA; Western blot analysis was performed on the homogenate. protein levels of F-actin,G-actin,p-cofilin,drebrinA/E were detective in morphine dependented rat brain regions by Western-blot.3 Primary hippocampal neuron cultures: Hippocampus from one-day-old Sprague Dawley rats were digested with trypsin (1.25g.L-1 in D-Hank's, 37℃for 15 min), washed and dissociated by passing the tissue through a Pasteur pipette with a fire-polished tip. Neurons were plated at a density of 2-5×10-3/cm-2 on coverslips. To ensure high-quality cell adhesion and growth, coverslips were first incubated in chlorhydric acid overnight and thoroughly washed with large amounts of water every 2h. Dried coverslips were then baked at 200℃for 2h, coated with poly-L-lysine (Sigma, 100μg.L-1) overnight and washed again before being incubated in plating medium for cell plating. The plating medium is DMEM containing 10% fetal bovine serum, 1% penicillin and streptomycin (1% P/S). After 24h of initial plating, the culture medium was completely replaced with feeding medium (Neurobasal medium supplemented with 2% B-27 and 1% P/S). Thereafter, neurons were fed twice a week with feeding medium until use.4 F-actin staining and immunocytochemistry: Hippocampal cultures were washed in PBS, fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% TritonX-100 for 5 min and blocked with 3% bovine serum albumin for 30 min. The cultures were incubated with 5ug/ml FITC-Phalloidin and P-cofilin rabbit mAb (1:100 dilution) overnight at 4℃. After washing with PBS for 30 min, the cultures were incubated with anti-rabbit Cy5 conjugated secondary antibody (1:1000 dilution) for 1 h at 37℃. Fluorescent images were visualized using a laser-scanning confocal microscope. Laser lines of 488 nm and 633 nm were used to excite FITC and Cy5, respectively. The intensity of staining was determined using Leica software. In order to allow for quantification of experimental data obtained from separate days, the data were normalized relative to the mean fluorescence intensity measured in the control neurons for each day.Results:1 Changes of expressions of F-actin,G-actin,p-cofilin and drebrinA\E protein in Hip of morphine dependent rats(1) Compared with the control group, there was no significant change of F-actin in Hip of morphine dependent 1 week and 2 weeks groups (Fig.1.1A), the expression of F-actin in Hip of morphine dependent 4 weeks group was up-regulated 90% (P<0.01) (Fig.1.1A).(2) Compared with the control group, there was no significant change of G-actin in Hip of morphine dependent 1 week, 2 weeks and 4 weeks groups (Fig.1.1B).(3) Compared with the control group, there were no significant changes of p-cofilin in Hip of morphine dependent 1 week group (Fig.1.1C), the expressions of p-cofilin in Hip of morphine dependent 2 weeks and 4 weeks group were up-regulated 45% (P<0.05) and 30% (P<0.05) respectively (Fig.1.1C).(4) Compared with the control group, there were no significant changes of drebrin in Hip of morphine dependent 1 week group(Fig.1.1D), the expressions of drebrin in Hip of morphine dependent 2 weeks and 4 weeks group were down-regulated 30% (P<0.05) and 28% (P<0.05) respectively (Fig.1.1D).2 Changes of expressions of F-actin,G-actin,p-cofilin and drebrinA\E protein in FPc of morphine dependent rats(1) Compared with the control group, there were no significant changes of F-actin in FPc of morphine dependent 1 week group (Fig.1.2A), the expressions of F-actin in FPc of morphine dependent 2 weeks and 4 weeks group were up-regulated 58% (P<0.05) and 101% (P<0.01) respectively (Fig.1.2A).(2) Compared with the control group, the expressions of G-actin were unchanged in FPc of morphine dependent 1 week, 2 weeks and 4 weeks groups (Fig.1.2B).(3) Compared with the control group, there were no significant changes of p-cofilin in FPc of morphine dependent 1 week group (Fig.1.2C), the expressions of p-cofilin in FPc of morphine dependent 2 weeks acd 4 weeks group were up-regulated 93% (P<0.01) and 88% (P<0.01) respectively (Fig.1.2C).(4) Compared with the control group, the expressions of drebrin were unchanged in FPc of morphine dependent 1 week, 2 weeks and 4 weeks groups (Fig.1.2D).3 No significant changes of F-actin,G-actin,p-cofilin and drebrinA\E were detected in thalamus of morphine dependent 1 week, 2 weeks and 4 weeks groups (Fig.1.3A,Fig.1.3B,Fig.1.3C,Fig.1.3D).4 Changes of expressions of F-actin,G-actin,p-cofilin and drebrinA\E protein in striatum of morphine dependent rats(1) Compared with the control group, there were no significant changes of F-actin,G-actin and drebrinA\E in striatum of morphine dependent 1 week, 2 weeks and 4 weeks groups (Fig.1.4A,Fig.1.4B,Fig.1.4D).(2) Compared with the control group, there were no significant changes of p-cofilin in striatum of morphine dependent 1 week group (Fig.1.4C), the expressions of p-cofilin in striatum of morphine dependent 2 weeks and 4 weeks group were down-regulated 25% (P<0.05) and 30% (P<0.05) respectively (Fig.1.4C).5 Effects of morphine on the morphologies of hippocampal neurons At immature stage (7-10days), the dendritic protrusions gradually shrunk after 2h treatment with morphine, and eventually collapsed after 24h treatment with morphine. The dendritic shaft showed rosary. The branches of dendrites became more and complicated. Exposure to morphine (24h) induced collapse of dendritic spines at mature stage (18-21days). The branches of dendrites became more and complicated after 72h treatment with morphine, and abnomal spines such as spines with multiple heads (branched spines) appeared.6 Effects of morphine on the reorganization of F-actin in hippocampal neurons(1) Fluorescent intensity changes of F-actin in hippocampal neurons at immature stage (7~10days) after treatment with morphine. Compared with the control, there were no significant changes of the fluorescent intensity of F-actin in hippocampal neurons plasma membrane and dendritic shaft afer 30min treatment with morphine; 2h after treatment with morphine, the fluorescent intensity of F-actin in dendritic shaft decreased by 25%(P<0.01); 24h after treatment with morphine, the fluorescent intensity of F-actin in plasma membrane and dendritic shaft decreased by 34.56% (P<0.01)and 25.74% (P<0.01)respectively; Compared with the control group, the fluorescent intensity of F-actin in plasma membrane and dendritic shaft did not change significantly after 48h treatment with morphine, but compared with Mor24h group, the fluorescent intensity of F-actin in plasma membrane elevated obviously(P<0.01). 72h after treatment with morphine, the fluorescent intensity of F-actin in plasma membrane increased by 15.08%(P<0.05). Compared with Mor24h group, the fluorescent intensity of F-actin in plasma membrane elevated obviously (P<0.01)(Fig.2.2,2.4).(2) Fluorescent intensity changes of F-actin in hippocampal neurons at mature stage (18~21days) after treatment with morphine.Compared with the control, there were no significant changes of the fluorescent intensity of F-actin in hippocampal neurons plasma membrane and dendritic shaft afer 30min treatment with morphine; 1h after treatment with morphine, the fluorescent intensity of F-actin in dendritic shaft increased by 15.79%(P<0.01); 24h after treatment with morphine, the fluorescent intensity of F-actin in hippocampal neurons plasma membrane and dendritic shaft decreased by 31.32%(P<0.01)and 19.51%(P<0.05)respectively; 72h after treatment with morphine, the fluorescent intensity of F-actin in plasma membrane did not change significantly, but the fluorescent intensity of F-actin in dendritic shaft increased by 16%(P<0.01). Compared with Mor24h group, the fluorescent intensity of F-actin in hippocampal neurons plasma membrane elevated obviously(P<0.01)(Fig.2.3,Fig.2.5).7 Effects of morphine on the actin binding protein p-cofilin in hippocampal neuronsResults showed that the fluorescent intensity of p-cofilin decreased by 22.57% after 2h treatment with morphine. There was no significant difference compared with the control group(P>0.05); 24h after treatment with morphine, the fluorescent intensity of p-cofilin decreased by 43.06% (P<0.01). Compared with the control group, there were no significant changes of the fluorescent intensity of p-cofilin after 48h treatment with morphine (P>0.05), however, the fluorescent intensity of p-cofilin elevated obviously contrast with Mor24h group (P<0.01)(Fig.2.6).Conclusion: In conclusion, our studies indicated that chronic morphine treatment could induce the development of morphine dependence, which accompanied with marked changes of actin and actin binding proteins in time-dependent and brain region-dependent. Morphologies of hippocampal neuron dendritic spines were changed by morphine treatment. Morphine treatment increased actin cycling of hippocampal neuron. Morphine treatment induced the actin cycling of hippocampal neuron, which might be correlated with the expression of actin binding protein p-cofilin.
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