An immunofluorescence study of neurofilament protein expression by developing hippocampal neurons in tissue culture.
An immunofluorescence study of neurofilament protein expression by developing hippocampal neurons in tissue culture.
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发表时间：
1985-11
影响因子：
6.6
通讯作者：
G. Shaw;G. Banker;K. Weber
中科院分区：
文献类型：
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作者：
G. Shaw;G. Banker;K. Weber
我们用单抗和多克隆抗体的单标和双标免疫荧光法研究了培养的海马神经元中中间丝蛋白的发育。众所周知，这些培养中的神经元分化的方式与原位培养的神经元相似：特别是它们发育的轴突和树突在形态、超微结构和突触极性上彼此不同。在培养的最初几天，发育中的神经元不能被任何神经丝蛋白的抗体染色，尽管许多细胞与抗波形蛋白发生反应。在第一周晚些时候，抗体染色显示L(68000道尔顿)和M(145000道尔顿)神经丝亚单位有明显的丝状染色，尽管M反应在发育早期阶段要强得多。许多细胞中的一些神经丝阳性轮廓也可被波形蛋白染色，尽管波形蛋白免疫反应在进一步发育过程中逐渐变得不明显，并在培养约两周后消失。此外，在体外培养约两周时，我们注意到神经丝H蛋白(200000道尔顿)首次出现免疫反应，定位于一组长轴突，从形态学角度可以将其识别为轴突。这些突起缺少树突状标记物微管相关蛋白2(MAP2)的染色。它们倾向于靠近神经胶质细胞的孤岛，这表明H的诱导可能需要复杂的神经元-神经胶质相互作用。这些结果与H蛋白免疫反应是轴突生长的标志的说法是一致的。除了明显的丝状染色外，我们还能够将神经丝抗原定位于一类有趣的小环状结构，随着培养年龄的增长，这种结构越来越频繁。我们还提出了证据表明，酪氨酸化的α-微管蛋白在这些培养的神经元的树突和轴突中都存在。
We have studied the development of intermediate filament proteins in the neurons found in hippocampal cell cultures using single and double label immunofluorescence with both monoclonal and polyclonal antibodies. Neurons in these cultures are known to differentiate in a manner similar to their counterparts in situ: in particular they develop axonal and dendritic processes which differ from each other in form, in ultrastructure, and in synaptic polarity. During the first days in culture, developing neurons could not be stained with antibodies against any of the neurofilament proteins, although many cells reacted with anti-vimentin. Later in the first week, antibody staining revealed clearly filamentous staining for the L (68 000 daltons) and the M (145 000 daltons) neurofilament subunits, though M reactivity was much stronger at this earlier stage of development. Some neurofilament positive profiles in many cells could also be stained with vimentin, though the vimentin immunoreactivity became progressively less pronounced during further development, and disappeared after about two weeks in culture. Also at about two weeks in vitro we noted the first appearance of neurofilament H protein (200 000 daltons) immunoreactivity, which was localized to a subset of long neurites which could be identified on morphological grounds as axons. These processes lacked staining for microtubule associated protein 2 (MAP2), a dendritic marker. They tended to be close to islands of glial cells, suggesting that H induction may require complex neuron-glial interactions. These results are consistent with the suggestion that H protein immunoreactivity is a marker for axonal outgrowth. In addition to obvious filamentous staining, we were able to localize neurofilament antigens to an interesting class of small ring-like structures, found increasingly frequently as the cultures aged. We also present evidence that tyrosinated alpha-tubulin is present both within dendrites and axons of neurons in these cultures.