Zmuda J F, Rivas R J
Department of Biology, University of Maryland, College Park 20742-4415, USA.
Cell Motil Cytoskeleton. 1998;41(1):18-38. doi: 10.1002/(SICI)1097-0169(1998)41:1<18::AID-CM2>3.0.CO;2-B.
Cultured cerebellar granule neurons develop their characteristic axonal and dendritic morphologies in a series of discrete temporal steps highly similar to those observed in situ, initially extending a single process, followed by the extension of a second process from the opposite pole of the cell, both of which develop into axons to generate a bipolar morphology. A mature morphology is attained following the outgrowth of multiple, short dendrites [Powell et al., 1997: J. Neurobiol. 32:223-236]. To determine the relationship between the localization of the Golgi apparatus, the site of microtubule nucleation (the centrosome), and the sites of initial and secondary axonal extension, the intracellular positioning of the Golgi and centrosome was observed during the differentiation of postnatal mouse granule neurons in vitro. The Golgi was labeled using the fluorescent lipid analogue, C5-DMB-Ceramide, or by indirect immunofluorescence using antibodies against the Golgi resident protein, alpha-mannosidase II. At 1-2 days in vitro (DIV), the Golgi was positioned at the base of the initial process in 99% of unipolar cells observed. By 3 DIV, many cells began the transition to a bipolar morphology by extending a short neurite from the pole of the cell opposite to the initial process. The Golgi was observed at this site of secondary outgrowth in 92% of these "transitional" cells, suggesting that the Golgi was repositioned from the base of the initial process to the site of secondary neurite outgrowth. As the second process elongated and the cells proceeded to the bipolar stage of development, or at later stages when distinct axonal and somatodendritic domains had been established, the Golgi was not consistently positioned at the base of either axons or dendrites, and was most often found at sites on the plasma membrane from which no processes originated. To determine the location of the centrosome in relation to the Golgi during development, granule neurons were labeled with antibodies against gamma-tubulin and optically sectioned using confocal microscopy. The centrosome was consistently co-localized with the Golgi during all stages of differentiation, and also appeared to be repositioned to the base of the newly emerging axon during the transition from a unipolar to a bipolar morphology. These findings indicate that during the early stages of granule cell axonal outgrowth, the Golgi-centrosome is positioned at the base of the initial axon and is then repositioned to the base of the newly emerging secondary axon. Such an intracellular reorientation of these organelles may be important in maintaining the characteristic developmental pattern of granule neurons by establishing the polarized microtubule network and the directed flow of membranous vesicles required for initial axonal elaboration.
培养的小脑颗粒神经元在一系列与原位观察到的高度相似的离散时间步骤中形成其特征性的轴突和树突形态，最初延伸出一个单一的突起，随后从细胞的相对极延伸出第二个突起，这两个突起都发育成轴突以形成双极形态。在多个短树突长出后达到成熟形态[鲍威尔等人，1997年：《神经生物学杂志》32:223 - 236]。为了确定高尔基体的定位、微管成核位点（中心体）与初始和次级轴突延伸位点之间的关系，在体外培养的新生小鼠颗粒神经元分化过程中观察了高尔基体和中心体的细胞内定位。使用荧光脂质类似物C5 - DMB - 神经酰胺标记高尔基体，或使用针对高尔基体驻留蛋白α - 甘露糖苷酶II的抗体通过间接免疫荧光进行标记。在体外培养1 - 2天（DIV）时，在观察到的99%的单极细胞中，高尔基体位于初始突起的基部。到3 DIV时，许多细胞开始通过从与初始突起相对的细胞极延伸出一个短神经突而转变为双极形态。在这些“过渡”细胞的92%中，在次级长出的这个位点观察到了高尔基体，这表明高尔基体从初始突起的基部重新定位到了次级神经突长出的位点。随着第二个突起伸长且细胞进入双极发育阶段，或者在后期当明确的轴突和体树突区域已经建立时，高尔基体并不始终位于轴突或树突的基部，最常出现在没有突起发出的质膜位点。为了确定发育过程中中心体相对于高尔基体的位置，用针对γ - 微管蛋白的抗体标记颗粒神经元，并使用共聚焦显微镜进行光学切片。在分化的所有阶段，中心体始终与高尔基体共定位，并且在从单极形态转变为双极形态的过程中，似乎也重新定位到了新出现的轴突基部。这些发现表明，在颗粒细胞轴突长出的早期阶段，高尔基体 - 中心体位于初始轴突的基部，然后重新定位到新出现的次级轴突的基部。这些细胞器的这种细胞内重新定向可能通过建立极化微管网络和初始轴突细化所需的膜泡定向流动，在维持颗粒神经元的特征性发育模式方面很重要。
