Effect of neuronal excitotoxicity on Munc18-1 distribution in nuclei of rat hippocampal neuron and primary cultured neuron
Effect of neuronal excitotoxicity on Munc18-1 distribution in nuclei of rat hippocampal neuron and primary cultured neuron
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DOI：
10.1007/s12264-011-1007-7
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发表时间：
2011
影响因子：
5.6
通讯作者：
Yan-Ping Zhang 张彦平;Ping Wan 万萍;Hong-Quan Wang 王洪权;Hong Zhao 赵红;Yu-Xia Xu 许玉霞;Ru Yang 杨茹;Cui-Qing Zhu 朱粹青
中科院分区：
文献类型：
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作者：
Yan-Ping Zhang 张彦平;Ping Wan 万萍;Hong-Quan Wang 王洪权;Hong Zhao 赵红;Yu-Xia Xu 许玉霞;Ru Yang 杨茹;Cui-Qing Zhu 朱粹青
目的：Munc 18 -1在中枢神经系统神经递质释放中起重要作用，控制着胞吐通路的各个环节。方法：采用海人酸（KA）溶液注入SD大鼠海马和昆明种小鼠腹腔建立癫痫模型。取胎龄18 d的SD大鼠海马神经元，在神经基础培养基中培养，然后用谷氨酸处理3 h。采用蔗糖密度梯度离心法分离海马神经元和胶质细胞核。用0.1%甲酚紫对核富集级分进行染色，用于形态学测定。用免疫化学和免疫电镜技术检测Munc 18 - 1的核定位。免疫印迹法检测Munc 18 - 1的蛋白水平。结果：免疫组化、免疫电镜及免疫印迹法检测均证实Munc 18 -1在大鼠海马神经元胞核中的定位。海马内或腹腔注射KA后，Munc 18 -1在CA区锥体细胞层、海马门区和齿状回颗粒细胞层表达减少。此外，免疫印迹分析显示，KA处理的动物海马核组分中Munc 18 -1的表达水平显著降低。在原代培养的神经元中，还测试了神经元核中Munc 18 -1表达的变化与神经元过度激活之间的关系。50 μmol/L谷氨酸处理3 h后，原代培养神经元胞核Munc 18 -1水平降低，胞浆Munc 18 -1水平升高。结论：兴奋性刺激可引起Munc 18 -1在神经元内分布的改变，进而影响神经元的功能。
Objective:Munc18-1 has an important role in neurotransmitter release, and controls every step in the exocytotic pathway in the central nervous system. In the present study, whether epileptic seizure causes a change of Munc18 localization in neuronal nuclei was analyzed.Methods:Epilepsy models were established by injection of kainic acid (KA) solution into hippocampus of Sprague-Dawley (SD) rats or intraperitoneal injection of KA in Kunming mice. The hippocampal neurons were prepared from embryonic day 18 SD rats, and cultured in neurobasal medium, followed by treatment with glutamate for 3 h. Neuronal and glial nuclei of hippocampus were separated by sucrose density gradient centrifugation. The nucleus-enriched fractions were stained with 0.1% Cresyl Violet for morphological assay. Immunochemistry and immunoelectron microscopy with anti-Munc18-1 antibody were used to determine the nuclear localization of Munc18-1. Immunoblotting was used to detect the protein level of Munc18-1.Results:The localization of Munc18-1 in nucleus of rat hippocampal neuron was confirmed by immunochemistry, immunoelectron microscopy, and immunoblotting detection of neuronal nucleus fraction. In animals receiving intrahippocampal or intraperitoneal injection of KA, immunostaining revealed that the expression of Munc18-1 decreased in pyramidal cell layer of CA regions, as well as in hilus and granular cell layer of dentate gyrus in hippocampus. Moreover, immunoblotting analysis showed that the expression level of Munc18-1 in nucleus fraction of hippocampus significantly decreased in KA-treated animals. The relationship between the change of Munc18-1 expression in neuronal nuclei and neuronal over-activation was also tested in primary cultured neurons. After treatment with 50 μmol/L glutamate acid for 3 h, Munc18-1 level was decreased in nucleus fraction and increased in cytoplasmic fraction of primary cultured neurons.Conclusion:These results suggest that excitatory stimulation can induce the distribution change of Munc18-1 in neuron, which may subsequently modulate neuronal functions in brain.