| Objective: The morphological changes of dendritic spines are related toexcitatory synaptic activity, and especially dependent on the actincytoskeleton rearrangement. The study confirmed the skeleton actin which indendritic spine formation, disappear, stability, shape and size, playing a keyrole. It happens for actin that were reversible polymerization (formation ofF-actin) and depolymerization (formation of the dynamic changes in G-actin),the reversible process called actin dynamics. Regulates actin dynamics notonly affected the morphology of dendritic spines but also associated withchanges of synaptic efficacy. Specific actin regulation mechanism was thebasic of dendritic spine plasticity, learning and memory. Recent studies haveshown that the actin rearrangement of amygdala is the key to the offensivememory formation which associated with morphine withdrawal, suggestingthat actin rearrangement may play an important role in the formation ofmorphine addiction. However, the molecular mechanism of actinrearrangement induced by morphine in dendrites spine still waits to beidentified. The present experiments were designed to explore themorphine-induced actin rearrangement and regulatory mechanisms indendritic spines. The role of Calcineurin/SSH-1L signaling pathway onmorphine-induced actin rearrangement is investigated. To further deepen themechanisms of cellular and molecular biology of opiate addiction.Methods:1Primary hippocampal neuron cultures:Hippocampus from one-day-old SD rats were digested with papain(2mg/L in DMEM,37℃for30min), washed with DMEM plus serum anddissociated by passing the tissue through imported pipette tip, then made cellsuspension culture fluid with planting the cell concentration was adjusted to 7×10~5/ml. Then transferred to a6-well or24-well cell culture plate which wascoated with poly-L-lysine, the plating medium is DMEM containing10%fetalbovine serum,1%penicillin and streptomycin(1%P/S). After6h to8h of initialplating, the culture medium was completely replaced with feedingmedium(Neurobasal medium supplemented with2%B27) and changedculture medium every3days. There after, neurons were fed with11daysfeeding medium until use.2Immunocytochemistry:Hippocampal cultures were washed in PBS, fixed with4%paraformaldehyde for30min, permeabilized with0.1%TritonX-100for5minand blocked with3%bovine serum albumin for30min. The cultures wereincubated with anti-NeuN (1:100dilution) overnight at4℃. After washingwith PBS for three times, the cultures were incubated with biotinylatedsecondary antibody solution at37℃for1h. Under an optical microscope,count100cells per field and statistics percentage of NeuN positive cells.3Determination of protein levels in rat hippocampal neurons by Western-blot:Collected the cells, according to that F-actin in cystoskeleton fractionwas separated from G-actin in endochylema with different buffer, detect thechanges of expression of drebrin, cortactin. In the same way collected the cells,extracted total protein, with BCA kit for protein quantification and then detectthe changes of expression of actin, actin binding protein by morphinetreatment at different times by western blot.Results:1Primary culture and pure of hippocampal neurons:Hippocampal neurons were adherent and round or oval after inoculationfor1or2hours, then the cells grow a few of protrusions from the cell body at3days, after7to10days the cells begen to mature, the cell body graduallygrow to40μm, halation was significant, nucleus and nucleolus were visible,protrusion became thicken, longer and intertwined to form a network underLight microscope. Immunocytochemistry staining showed that the purity ofhippocampal neurons was up to93%. 2Effects of morphine treatment on F-actin/G-actin ratio in hippocampalneuronsCompared with the control group, F-actin/G-actin the ratio weredecreased by25%(P<0.01) and32%(P<0.001) and15%(P<0.001) aftermorphine treatment for1h,24h and72h, indicating that morphine inducedactin reconstruction.3Effects of morphine treatment on drebrin in hippocampal neurons3.1Changes of drebrin in total proteinCompared with the control group, the results showed a significantdecrease of drebrin in total protein by25%(P<0.05),41%(P<0.001) and43%(P<0.001) by morphine treatment for1h,24h and72h.3.2Changes of F-actin binding drebrinCompared with the control group, there was no significant change ofdrebrin binding F-actin in hippocampal neurons after morphine dependent1h;the results showed a significant decrease of drebrin binding F-actin by57%(P<0.001) and74%(P<0.001) by morphine for24h and72h.3.3Changes of the ratio of F-drebrin to G-drebrinCompared with the control group, the results showed a significantdecrease of drebrin of the actin cytoskeleton and cytoplasm ratio by25%(P<0.001),74%(P<0.001) and67%(P<0.001) by morphine treatment for1h,24h and72h. It is suggested that morphine induced drebrin shift and fall fromF-actin.4Effects of morphine treatment on cortactin in hippocampal neurons4.1Changes of cortactin in total proteinCompared with the control group, there was no significant change ofcortactin in total protein of hippocampal neurons by morphine treatment for1h,24h and72h.4.2Changes of F-actin binding cortactinCompared with the control group, there was no significant change ofcortactin binding F-actin of hippocampal neurons after morphine dependent1h; the results showed a significant decrease of cortactin binding F-actin by 25%(P<0.05) and46%(P<0.001) by morphine for24h and72h.4.3Changes of the ratio of F-cortactin to G-cortactinCompared with the control group, there was no significant change ofcortactin of the cytoskeleton and cytoplasm in hippocampal neurons bymorphine treatment for1h; the results showed a significant decrease ofcortactin of the cytoskeleton and cytoplasm of hippocampal neurons by71%(P<0.001) and52%(P<0.001) after morphine treatment for24h and72h.5Effects of morphine treatment on cofilin/p-cofilin in hippocampal neuronsCompared with the control group, there was no significant change ofcofilin in total protein of hippocampal neurons after morphine treatment for1h,24h and72h. However, the results showed a significant decrease ofp-cofilin by48%(P<0.01) and70%(P<0.001) after morphine treatment for1h and24h, as well there was no significant change of p-cofilin aftermorphine treatment for72h.6Effects of morphine treatment on LIMK/P-LIMK, SSH-1L in hippocampalneurons6.1Changes of LIMK in total proteinCompared with the control group, there was no significant change ofLIMK in hippocampal neurons after morphine treatment for1h; the resultsshowed a significant increase of LIMK by30%(P<0.001) after morphinetreatment for24h.6.2Changes of P-LIMK in total proteinCompared with the control group, there was no significant change ofP-LIMK in hippocampal neurons after morphine treatment for1h and24h.6.3Changes of SSH-1L in total proteinCompared with the control group, there was no significant change ofSSH-1L in hippocampal neurons after morphine treatment for1h; the resultsshowed a significant increase of SSH-1L by64%(P<0.01) after morphinetreatment for24h.7Effects of MK801on morphine-induced actin rearrengment in hippocampalneurons 7.1Effects of MK801on morphine-induced changes of F-actin/G-actin ratioin hippocampal neuronsCompared with the control group, F-actin/G-actin ratio inmorphine-treated group was significantly reduced by32%(P<0.001).Compared with the morphine group, F-actin/G-actin ratio in MK801groupwas significantly increased by45%(P<0.01).7.2Effects of MK801on morphine-induced changes of drebrin inhippocampal neuronsCompared with the control group, drebrin levels of the total protein inmorphine-treated group reduced by41%(P<0.001)，F-actin binding drebrinlevel in morphine-treated group reduced by56%(P<0.001) and drebrin in theactin cytoskeleton and cytoplasmic ratio reduced by74%(P<0.001) inmorphine-treated group. Compared with the morphine-treated group, drebrinlevel of total protein in MK801group raised24%(P<0.01); F-actin bindingdrebrin level in MK801group upregulated41%(P<0.001); drebrin of theactin cytoskeleton and cytoplasm ratio raised to65%(P<0.001) in MK801group.7.3Effects of MK801on morphine-induced changes of cortactin inhippocampal neuronsThere was no significant difference of cortactin levels of total proteinamong three groups. Compared with the control group, the level of F-actinbinding cortactin reduced by25%(P<0.05) and cortactin in actin cytoskeletonand cytoplasm ratio reduced by71%(P<0.001) in morphine group. Comparedwith morphine group, cortactin in actin cytoskeleton and cytoplasm ratioincreases to62%(P<0.001) in MK801group.7.4Effects of MK801on morphine-induced changes of cofilin/p-cofilin inhippocampal neuronsThere was no significant difference of cofilin among three groups.However, compared with the control group, p-cofilin levels in morphine groupreduced by70%(P<0.001); Compared with morphine, p-cofilin in MK801group raised87%(P<0.001). 7.5Effects of MK801on morphine-induced changes of SSH-1L inhippocampal neuronsCompared with the control group, SSH-1L level in morphine groupraised to64%(P<0.01); Compared with morphine group, SSH-1L level inMK801group down to30%(P<0.05).8Effects of FK506on morphine-induced actin rearrengment in hippocampalneurons8.1Effects of FK506on morphine-induced changes of F-actin/G-actin ratio inhippocampal neuronsCompared with the control group, F-actin/G-actin ratio inmorphine-treated group was significantly reduced by32%(P<0.001);Compared with the morphine group, F-actin/G-actin ratio in FK506groupsignificantly increased by57%(P<0.01).8.2Effects of FK506on morphine-induced changes of drebrin in hippocampalneuronsCompared with the control group, drebrin levels of total protein reducedby41%(P<0.001), the F-actin binding drebrin level reduced by56%(P<0.001), and drebrin ratio of the actin cytoskeleton and cytoplasm inmorphine-treated group reduced by74%(P<0.001) in morphine-treated group.Compared with morphine group, drebrin levels of total protein has nosignificant change, drebrin level of F-actin binding increases33%(P<0.01),and drebrin ratio of the actin cytoskeleton and cytoplasm significantlyincreased by60%(P<0.001) in FK506group.8.3Effects of FK506on morphine-induced changes of cortactin inhippocampal neuronsThere was no significant difference of cortactin of total protein amongthree groups. Compared with the control group, the level of F-actin bindingcortactin in morphine group was down25%(P<0.05), however, there was nosignificant difference between morphine group and FK506group. Comparedwith the control group, cortactin in actin cytoskeleton and cytoplasm ratio inmorphine group reduced by71%(P<0.001); Compared with the morphine group, cortactin in actin cytoskeleton and cytoplasm ratio increases to63%(P<0.001) in FK506group.8.4Effects of FK506on morphine-induced changes of cofilin/p-cofilin inhippocampal neuronsThere was no significant difference of cofilin among three groups.Compared with the control group, p-cofilin levels in morphine group reducedby70%(P<0.001); compared with the morphine group, p-cofilin levels inFK506group increased by64%(P<0.001).8.5Effects of SSH-1L of hippocampal neurons by FK506treatmentCompared with the control group, the SSH-1L level in morphine groupraised to64%(P<0.01); compared with the morphine group, the SSH-1L levelin FK506group down to50%(P<0.05).Conclusions:1The result indicates that morphine induced actin rearrangement ofhippocampal neurons.2Morphine treatment not only affects the content of the actin-bindingprotein drebrin and cortactin, but also affects the combination of F-actin.3Morphine treatment affected the cofilin phosphorylation process.4Morphine may affect cofilin phosphorylation state through calcineurin/SSH-1L pathway, reducing the combination of F-actin between drebrin andcortactin, leading to actin remodeling of the dendritic spines in neurons,dendritic spine instability. |
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