Laboratoire de Neurobiologie, Université d'Orléans, BP 6759, Rue de Chartres, F-45067 Orléans CEDEX 2, France.
Neuroscience. 2012 Jan 3;200:248-60. doi: 10.1016/j.neuroscience.2011.10.002. Epub 2011 Oct 18.
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™ 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.
很少有研究涉及原代纯培养神经元的超微结构和形态。因此，我们使用免疫组织化学和电子显微镜技术来研究体外长时间孵育培养神经元的超微结构。大鼠大脑皮质神经元在 Neurobasal™培养基中培养。根据接种密度，贴壁细胞发育成单个神经元或神经元簇的网络。几乎所有存活的细胞都是神经元，这可以通过神经丝免疫标记来证明。培养神经元的数量在体外培养 14-21 天（DIV）时显著增加，然后达到平台期，随后减少。从 DIV 1-10，神经元延伸出大的突起，随后发展出细而密集的突起，神经元存活到 DIV 30-50。值得注意的是，在突起内的纤维状物质中观察到大量线粒体，提示细胞内物质的活跃运输。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先前关于培养神经元电生理活性的报道。然而，许多神经元含有变形的线粒体和异常细胞器，包括多层膜囊泡和多泡髓样体。在添加抗生素的培养基中培养时，含有异常细胞器的神经元比例显著增加。在长期培养中，观察到神经元死亡和凋亡核。尽管存在异常细胞器，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有用的工具。
