Effect of neuronal excitotoxicity on Munc18-1 distribution in nuclei of rat hippocampal neuron and primary cultured neuron

Abstract

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.

摘要

目的

Munc18-1在中枢神经系统递质释放过程中具有重要作用, 控制着突触囊泡释放步骤的每一个环节。 Munc18-1功能异常与癫痫发病相关。 本文主要探讨癫痫是否会引起神经元胞核内Munc18-1定位的改变。

方法

通过海马内注射海人藻酸建立Sprague-Dawley (SD)大鼠癫痫模型, 腹腔注射海人藻酸建立昆明小鼠癫痫模型。 分离胎龄18天SD大鼠海马神经元, 用Neurobasal培养基培养7天后, 用谷氨酸处理3 h。 用8517糖密度梯度离心法分离神经元和神经胶质细胞的细胞核组份, 通过甲酚紫染色对上述富含细胞核的组份进行形态学鉴别。 用免疫组化和免疫电镜分析法确定Munc18-1的细胞核定位。 免疫印迹法检测不同细胞组分的Munc18-1蛋白的表达水**。

结果

免疫组化、 免疫电镜以及对神经元细胞核组份的免疫印迹证实了Munc18-1在海马神经元细胞核的分布定位。 在海人藻酸诱导癫痫的动物海马内, 免疫组化染色显示Munc18-1在海马CA区9525体细胞层、 9F7F状回9897粒细胞层和门区的多型细胞表达减少。 同时, 免疫印迹分析表明, Munc18-1在海马神经元胞核中的表达水**明显下降。 免疫印迹检测显示, 原代培养神经元经50 μmol/L谷氨酸处理3 h后, Munc18-1在神经元胞核中的分布减少, 而胞浆组份中含量增加。 免疫荧光的形态学检测也显示部分神经元明显失去了Munc18-1在细胞核聚集分布的特征。

结论

兴奋性刺激能够使Munc18-1在神经元的表达分布发生改变, 这种变化可能参与调节脑神经元的功能。