Kapitein Lukas C, Yau Kah Wai, Hoogenraad Casper C
Department of Neuroscience, Erasmus Medical Center, 3015 GE, Rotterdam, The Netherlands.
Methods Cell Biol. 2010;97:111-32. doi: 10.1016/S0091-679X(10)97007-6.
Neuronal microtubules recently emerged as temporal and spatial regulators of dendritic spines, the major sites of excitatory synaptic input. By imaging microtubules in cultured mature primary hippocampal neurons using fluorescently tagged tubulin and microtubule plus-end binding (EB) protein EB3, dynamic microtubules were found to regularly depart from the dendritic shaft and enter dendritic spines. Evidence indicates that microtubule invasions into spines regulate spine actin dynamics and induce transient morphological changes, such as the formation of spine head protrusion and spine growth. Because alterations in spine morphology play an important role in synaptic plasticity and have been linked to learning and memory formation, it is possible that dynamic microtubules are engaged in adaptive processes in the adult brain. This chapter provides detailed methods for live imaging of dynamic microtubules in mature hippocampal neurons in culture. We describe protocols for culturing and transfecting mature hippocampal neurons and visualizing microtubules and microtubule plus-EB proteins by total internal reflection fluorescence microscopy and spinning disk confocal microscopy.
神经元微管最近成为树突棘的时空调节因子，树突棘是兴奋性突触输入的主要部位。通过使用荧光标记的微管蛋白和微管正端结合（EB）蛋白EB3对培养的成熟原代海马神经元中的微管进行成像，发现动态微管会定期从树突轴离开并进入树突棘。有证据表明，微管侵入棘突会调节棘突肌动蛋白动力学并诱导短暂的形态变化，例如棘突头部突起的形成和棘突生长。由于棘突形态的改变在突触可塑性中起重要作用，并且与学习和记忆形成有关，因此动态微管可能参与了成人大脑中的适应性过程。本章提供了在培养的成熟海马神经元中对动态微管进行实时成像的详细方法。我们描述了培养和转染成熟海马神经元以及通过全内反射荧光显微镜和转盘共聚焦显微镜观察微管和微管加EB蛋白的方案。
