van Lookeren Campagne M, Dotti C G, Verkleij A J, Gispen W H, Oestreicher A B
Rudolf Magnus Institute, Division of Molecular Neurobiology, University of Utrecht, The Netherlands.
Neuroscience. 1992 Dec;51(3):601-19. doi: 10.1016/0306-4522(92)90300-q.
Morphologically polarized hippocampal neurons, grown in culture for two days, contain immunoreactivity of the growth-associated protein B-50 along the plasma membrane of both dendrites and axons. In mature hippocampal neurons, both in vitro and in vivo, B-50 is located in the axon. In order to assess at which stage during neuronal differentiation B-50 is selectively located in the axon, an immuno-light and electron-microscopic study was performed on rat hippocampal neurons developing in vitro. B-50 immunofluorescence was detected in the axon, dendrites and soma of two-day-old polarized neurons. Simultaneously, microtubule-associated protein 2, a marker specific to dendritic microtubules, was predominantly found in the soma, the short dendritic processes and at the base of axonal growth cones. In hippocampal neurons cultured beyond seven days in vitro, microtubule-associated protein 2 immunofluorescence is restricted to the cell soma and dendrites. The spatial distribution of B-50, however, varies. In solitary neurons maturing without interneuronal contacts, B-50 immunofluorescence is observed in axons and in the dendrosomatic domain characterized by the presence of microtubule-associated protein 2. In contrast, in high-density cell cultures B-50 immunofluorescence is absent in the cell body and dendrites, but punctate in axons running along the dendrites. Electron microscopy was carried out on hippocampal neurons of eight to 21 days in vitro to study the process of redistribution of B-50 at the subcellular level. In neurons of eight days in vitro with prominent synapses, B-50 immunoreactivity is significantly elevated at the axonal plasma membrane compared to the plasma membrane of the dendrites and the soma. In neurons from the same culture without synapses, B-50 immunoreactivity is distributed rather densely along the plasma membrane of the soma, dendrites, and on the axonal plasma membrane. A similar B-50 distribution is observed in mature neurons cultured at low cell density without interneuronal cell contacts, for 15 days in vitro. In high-density cell cultures of 21 days in vitro, B-50 is virtually absent at the plasma membrane of the soma and dendrites, and heterogenously distributed along the plasma membrane of axon and axonal varicosities. Our results indicate that selective sorting of B-50 into axons occurs after initial morphological polarization of hippocampal neurons and is correlated with the formation of synapses and with the cessation of dendritic outgrowth.
在体外培养两天的形态学极化海马神经元中，树突和轴突的质膜上均含有生长相关蛋白B - 50的免疫反应性。在体外和体内的成熟海马神经元中，B - 50位于轴突中。为了评估在神经元分化的哪个阶段B - 50选择性地定位于轴突，对体外发育的大鼠海马神经元进行了免疫荧光和电子显微镜研究。在两天大的极化神经元的轴突、树突和胞体中检测到B - 50免疫荧光。同时，微管相关蛋白2，一种树突微管特异性标志物，主要存在于胞体、短树突突起和轴突生长锥基部。在体外培养超过七天的海马神经元中，微管相关蛋白2免疫荧光局限于细胞胞体和树突。然而，B - 50的空间分布有所不同。在没有神经元间接触而成熟的孤立神经元中，在轴突和以微管相关蛋白2存在为特征的树突 - 胞体区域观察到B - 50免疫荧光。相反，在高密度细胞培养中，B - 50免疫荧光在细胞体和树突中不存在，但在沿着树突延伸的轴突中呈点状。对体外培养8至21天的海马神经元进行电子显微镜检查，以研究B - 50在亚细胞水平的重新分布过程。在体外培养8天且有明显突触的神经元中，与树突和胞体的质膜相比，轴突质膜上的B - 50免疫反应性显著升高。在来自同一培养物但没有突触的神经元中，B - 50免疫反应性沿胞体、树突的质膜以及轴突质膜分布较为密集。在体外低细胞密度培养且没有神经元间细胞接触的情况下培养15天的成熟神经元中观察到类似的B - 50分布。在体外培养21天的高密度细胞培养物中，B - 50在胞体和树突的质膜上几乎不存在，并且沿轴突和轴突膨体的质膜呈异质性分布。我们的结果表明，B - 50选择性地分选到轴突中发生在海马神经元最初的形态极化之后，并且与突触的形成以及树突生长的停止相关。
