Kosik K S, Finch E A
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115.
J Neurosci. 1987 Oct;7(10):3142-53. doi: 10.1523/JNEUROSCI.07-10-03142.1987.
We sought to determine whether the strict segregation of MAP2 and tau into somatodendritic and axonal compartments in situ was maintained in dissociated neuronal cultures of the rat cerebrum. Cultures grown under serum-free conditions were immunolabeled with monoclonal antibodies specific for MAP2 and tau. At 14 d after plating, a clear distinction between MAP2- and tau-immunoreactive neurites was apparent. MAP2-immunoreactive neurites were relatively short, thick, tapering, and branched. Tau-immunoreactive neurites formed a crisscrossing meshwork of long, fine-caliber neurites, which, in more densely plated cultures, had a tendency to form thick, ropelike fascicles. Unlike the MAP2 pattern, tau antibodies labeled somata only lightly. Since distinct populations of neurites were labeled with the 2 antibodies, we sought to observe the development of the topographically distinct compartments by double-labeled immunocytochemistry with both polyclonal and monoclonal antibodies to MAP2 and tau. Cells observed within the first 8 hr after plating demonstrated equally intense MAP2 and tau immunoreactivity in a coextensive distribution throughout the cell body and initial neurites. By 16 hr, some neurites began to assume dendritic and axonal features; however, many such processes contained reaction product for both MAP2 and tau. Beginning at this time, neurites that appeared axonal showed a progressively weaker reaction with MAP2 antibodies, and neurites that appeared dendritic showed a progressively weaker reaction with tau antibodies. In most neurites the diminution appeared to occur uniformly over the entire extent of the neurite. During this transformation period there were occasional axon-like neurites that contained MAP2 immunoreactivity proximally, while tau immunoreactivity extended over the entire length of the neurite. We conclude that neurons in culture are able to compartmentalize MAP2 and tau into their appropriate processes and only attain an apparently homogeneous population of one of these MAPs after the neuron has assumed dendritic and axonal features. The analysis also lends indirect support to the hypothesis that microtubule-associated proteins (MAPs) form this association at the distal extent of the growing neurite.
我们试图确定在大鼠大脑的解离神经元培养物中，原位MAP2和tau在树突体和轴突区室中的严格分隔是否得以维持。在无血清条件下培养的细胞用针对MAP2和tau的单克隆抗体进行免疫标记。接种后14天，MAP2免疫反应性神经突和tau免疫反应性神经突之间有明显区别。MAP2免疫反应性神经突相对较短、较粗、逐渐变细且有分支。tau免疫反应性神经突形成了由长而细的神经突组成的纵横交错的网络，在接种密度更高的培养物中，这些神经突倾向于形成粗大的索状束。与MAP2模式不同，tau抗体仅轻微标记胞体。由于两种抗体标记了不同群体的神经突，我们试图通过用针对MAP2和tau的多克隆和单克隆抗体进行双重标记免疫细胞化学来观察地形上不同区室的发育。接种后最初8小时内观察到的细胞在整个细胞体和初始神经突中显示出同样强烈的MAP2和tau免疫反应性，且分布范围相同。到16小时时，一些神经突开始呈现树突和轴突特征；然而，许多这样的突起同时含有MAP2和tau的反应产物。从此时开始，呈现轴突样的神经突与MAP2抗体的反应逐渐减弱，呈现树突样的神经突与tau抗体的反应逐渐减弱。在大多数神经突中，这种减弱似乎在神经突的整个长度上均匀发生。在这个转变期，偶尔会有轴突样神经突，其近端含有MAP2免疫反应性，而tau免疫反应性延伸至神经突的全长。我们得出结论，培养中的神经元能够将MAP2和tau分隔到它们各自合适的突起中，并且只有在神经元呈现出树突和轴突特征后，才会出现其中一种MAP的明显同质群体。该分析也间接支持了微管相关蛋白（MAPs）在生长神经突的远端形成这种关联的假说。
