ULTRASTRUCTURAL CHARACTERIZATION OF RAT NEURONS IN PRIMARY CULTURE
ULTRASTRUCTURAL CHARACTERIZATION OF RAT NEURONS IN PRIMARY CULTURE
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DOI：
10.1016/j.neuroscience.2011.10.002
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发表时间：
2012-01-03
期刊：
影响因子：
3.3
通讯作者：
Hevor, T.
中科院分区：
文献类型：
--
作者：
Robert, F.;Cloix, J-F.;Hevor, T.
对原代纯培养神经元的超微结构和形态的研究很少。因此，我们使用免疫组织化学和电子显微镜来研究体外培养神经元的超微结构。采用神经基底（Neurobasal， TM）培养基培养大鼠大脑皮层神经元。贴壁细胞发展为单个神经元或簇的网络，取决于镀密度。经神经丝免疫标记证实，几乎所有存活细胞均为神经元。体外培养的神经元数量在14-21天（DIV）内显著增加，然后趋于平稳，随后下降。从DIV 1-10开始，神经元延伸出大的神经突，随后发育成细而致密的神经突，神经元存活至DIV 30-50。值得注意的是，沿神经突内的纤维元件观察到许多线粒体，提示活跃的细胞内运输。电子显微镜还显示，神经元之间形成了多种类型的突触。这些超微结构结果证实了之前关于培养神经元电生理活动的报道。然而，许多神经元含有扭曲的线粒体和异常的细胞器，包括多层小泡和多泡髓样小体。在添加抗生素的培养基中，含有异常细胞器的神经元比例显著增加。长期培养观察到神经元死亡和细胞核凋亡。尽管细胞器存在异常，但培养神经元的超微结构与体内神经元非常相似；因此，体外培养为研究神经元发育、衰老和神经传递提供了一个有用的工具。(c) 2011 ibro。Elsevier Ltd.出版。版权所有。
Few studies have addressed the ultrastructure and morphology of neurons in primary pure culture. We therefore use immunohistochemistry and electron microscopy to investigate the ultrastructure of cultured neurons during extended incubation in vitro. Rat cerebral cortex neurons were cultured in Neurobasal (TM) medium. Adherent cells developed as networks of single neurons or clusters depending on the plating density. Almost all surviving cells were neurons as demonstrated by neurofilament immunolabeling. The number of cultured neurons increased substantially to 14-21 days in vitro (DIV) and then plateaued and subsequently declined. From DIV 1-10 neurons extended large neurites, followed by the development of fine and dense neurites, and neurones survived until DIV 30-50. Notably, numerous mitochondria were observed along fibrous elements within neurites, suggestive of active intracellular trafficking. Electron microscopy also revealed that multiple types of synapses were formed between neurons. These ultrastructural results confirm previous reports of electrophysiological activity in cultured neurons. However many neurons contained distorted mitochondria and abnormal organelles including multilamellar vesicles and multivesicular myeloid bodies. The proportion of neurons containing abnormal organelles increased significantly in culture medium supplemented with antibiotics. On long-term culture neuronal death and apoptotic nuclei were observed. Despite the presence of abnormal organelles, the ultrastructure of cultured neurons was very similar to that of in vivo neurons; in vitro culture therefore provides a useful tool for studies on neuronal development, aging, and neurotransmission. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.