Three-Dimensional Nanostructure of an Intact Microglia Cell
Three-Dimensional Nanostructure of an Intact Microglia Cell
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DOI：
10.3389/fnana.2018.00105
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发表时间：
2018-12-05
影响因子：
2.9
通讯作者：
Gross, Cornelius T.
中科院分区：
文献类型：
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作者：
Bolasco, Giulia;Weinhard, Laetitia;Gross, Cornelius T.
小胶质细胞是髓系的非神经元细胞，在发育过程中侵入并长期居住在大脑中（Ginhoux等人，2010），并越来越多地参与神经元成熟、稳态和病理（Bessis等人，2007；Paolicelli等人，2011；Li等人，2012；Aguzzi等人，2013;Cunningham, 2013； Cunningham等人，2013）。自二十世纪初以来，已经开发了几种染色和可视化小胶质细胞的方法。Ramon y Cajal小组的科学家（Achucarro, 1913; Rio-Hortega, 1919）开创了这些方法，他们的工作导致小胶质细胞被命名为神经系统的第三个元素，不同于星形胶质细胞和神经元。最近，影像学、遗传学和免疫学工具的结合已被用于可视化活脑中的小胶质细胞（Davalos等人，2005；Nimmerjahn等人，2005）。研究发现，小胶质细胞在静息条件下具有高度的运动能力，对损伤反应迅速（Kettenmann et al., 2011），这表明小胶质细胞在大脑稳态和病理中都有作用。透射电子显微镜（TEM）提供了关于小胶质细胞形态学和生理学的重要补充信息，但直到最近，EM分析还仅限于单个或有限的连续切片研究（Tremblay等人，2010年，2012年；Paolicelli等人，2011年；Schafer等人，2012年；Sipe等人，2016年）。透射电镜研究成功地定义了小胶质细胞的一组形态学标准：多边形核，周围染色质凝聚，相对较小的细胞质，大量存在粗内质网（RER），核周有大量溶酶体和包涵体。体积电子显微镜技术的最新进展允许在纳米分辨率下对大样本进行三维重建，从而为理解完整组织中的细胞生物学和结构开辟了新的途径。与此同时，相关的光学和电子显微镜（CLEM）技术已经扩展到3D脑样本，以帮助在大EM体积内导航和识别关键的分子标记（Briggman和Denk, 2006； Maco等人，2013；Blazquez-Llorca等人，2015；Bosch等人，2015）。在这里，我们使用连续块面扫描电子显微镜（SBEM）对几乎完整的小鼠海马小胶质细胞进行了首次体积超微结构重建。在CA1放射层对小鼠进行了产后早期（P15）的影像学检查。使用CLEM，我们确保了大的、小的和丝状小胶质细胞过程的包含。数据集的分割使我们能够对小胶质细胞结构进行全面的盘点，包括囊泡、细胞器、膜突起和突起。本研究为研究小胶质细胞生物学提供了数据挖掘资源。
Microglia are non-neuronal cells of themyeloid lineage that invade and take up long-term residence in the brain during development (Ginhoux et al., 2010) and are increasingly implicated in neuronal maturation, homeostasis, and pathology (Bessis et al., 2007; Paolicelli et al., 2011; Li et al., 2012; Aguzzi et al., 2013; Cunningham, 2013; Cunningham et al., 2013). Since the early twentieth century several methods for staining and visualizing microglia have been developed. Scientists in Ramon y Cajal's group (Achucarro, 1913; Rio-Hortega, 1919) pioneered these methods and their work led to the christening of microglia as the third element of the nervous system, distinct from astrocytes and neurons. More recently, a combination of imaging, genetic, and immunological tools has been used to visualize microglia in living brain (Davalos et al., 2005; Nimmerjahn et al., 2005). It was found that microglia are highly motile under resting conditions and rapidly respond to injuries (Kettenmann et al., 2011) suggesting a role for microglia in both brain homeostasis and pathology. Transmission Electron microscopy (TEM) has provided crucial complementary information on microglia morphology and physiology but until recently EM analyses have been limited to single or limited serial section studies (Tremblay et al., 2010, 2012; Paolicelli et al., 2011; Schafer et al., 2012; Sipe et al., 2016). TEM studies were successful in defining a set of morphological criteria for microglia: a polygonal nucleus with peripheral condensed chromatin, a relatively small cytoplasm with abundant presence of rough endoplasmic reticulum (RER), and a large volume of lysosomes and inclusions in the perikaryon. Recent advances in volumetric electron microscopy techniques allow for 3D reconstruction of large samples at nanometer-resolution, thus opening up new avenues for the understanding of cell biology and architecture in intact tissues. At the same time, correlative light and electron microscopy (CLEM) techniques have been extended to 3D brain samples to help navigate and identify critical molecular landmarks within large EM volumes (Briggman and Denk, 2006; Maco et al., 2013; Blazquez-Llorca et al., 2015; Bosch et al., 2015). Here we present the first volumetric ultrastructural reconstruction of a nearly complete mouse hippocampal microglia using serial block face scanning electronmicroscopy (SBEM). Imaging was performed on amouse at early postnatal stage (P15) in CA1 stratumradiatum. Using CLEM we have ensured the inclusion of both large, small, and filopodial microglia processes. Segmentation of the dataset allowed us to carry out a comprehensive inventory of microglia cell structures, including vesicles, organelles, membrane protrusions, and processes. This study provides a reference that can serve as a data mining resource for investigating microglia cell biology.