State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, China.
Neurosci Bull. 2011 Jun;27(3):163-72. doi: 10.1007/s12264-011-1007-7.
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.
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.
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.
These results suggest that excitatory stimulation can induce the distribution change of Munc18-1 in neuron, which may subsequently modulate neuronal functions in brain.
Munc18-1 在神经递质释放中具有重要作用，并控制中枢神经系统胞吐途径的每一个步骤。本研究分析了癫痫发作是否会导致神经元核内 Munc18 定位发生变化。
通过向 Sprague-Dawley (SD) 大鼠海马内注射海人酸 (KA) 溶液或昆明小鼠腹腔内注射 KA 建立癫痫模型。从胚胎第 18 天的 SD 大鼠中制备海马神经元，并在神经基底培养基中培养，然后用谷氨酸处理 3 小时。通过蔗糖密度梯度离心分离神经元和神经胶质核。用 0.1% Cresyl Violet 染色核丰富部分进行形态学分析。用抗 Munc18-1 抗体进行免疫化学和免疫电镜检查，以确定 Munc18-1 的核定位。用免疫印迹法检测 Munc18-1 的蛋白水平。
通过神经元核部分的免疫化学、免疫电镜和免疫印迹检测，证实了 Munc18-1 在大鼠海马神经元核内的定位。在接受海马内或腹腔内注射 KA 的动物中，免疫染色显示 CA 区锥体细胞层以及海马齿状回的门区和颗粒细胞层中 Munc18-1 的表达减少。此外，免疫印迹分析显示 KA 处理动物海马核部分 Munc18-1 的表达水平显著降低。还在原代培养神经元中检测了 Munc18-1 在神经元核内表达变化与神经元过度激活之间的关系。用 50 μmol/L 谷氨酸处理 3 小时后，原代培养神经元的核部分 Munc18-1 水平降低，细胞质部分增加。
这些结果表明，兴奋性刺激可诱导 Munc18-1 在神经元中的分布发生变化，从而可能随后调节大脑中的神经元功能。
