Characterization of Astrocyte Morphology and Function Using a Fast and Reliable Tissue Clearing Technique.
Characterization of Astrocyte Morphology and Function Using a Fast and Reliable Tissue Clearing Technique.
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DOI：
10.1002/cpz1.279
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发表时间：
2021-10
期刊：
影响因子：
--
通讯作者：
Richards CI
中科院分区：
文献类型：
--
作者：
Aryal SP;Neupane KR;Masud AA;Richards CI
星形胶质细胞过程与整个大脑的突触相互作用，调节神经递质信号传导和突触通信。在暴露于滥用药物和神经系统疾病等条件下，星形胶质细胞通过改变其形态和功能特性作出反应。与静息星形胶质细胞相比，反应性星形胶质细胞表型表现出浓密的形态，索马体积改变，突起数量增加。反应性星形胶质细胞表型也过表达蛋白质，其中之一可以是胶质细胞酸性蛋白（GFAP）。荧光显微镜在薄组织切片（< 20 μm）上需要重建，通常通过多个切片，以描绘完整的星形胶质细胞形态。在脑电成像中，已经开发出组织清除方法，可以对包括整个大脑在内的更大切片进行成像，从而有机会看到单细胞结构的深入变化。在这个单元中，提供了一个详细的协议，用于研究星形胶质细胞形态学，使用组织清除和随后的全脑成像以及区域特异性切片。这种方法是理想的了解不同的生理条件对星形胶质细胞形态的影响。一个标准的生物化学实验室有资源来完成组织清除使用此协议和大多数大学有所需的成像设施。提供了在组织清除后使用抗体标记步骤研究来自含有星形胶质细胞特异性标志物的遗传修饰小鼠和来自野生型小鼠的脑的方案。我们还描述了一般协议进行荧光成像的星形胶质细胞在清除组织，以表征其形态。该协议可能是有用的研究人员在快速增长的领域工作的星形胶质细胞生物学。
Astrocytic processes interact with synapses throughout the brain modulating neurotransmitter signaling and synaptic communication. During conditions such as exposure to drugs of abuse and neurological diseases, astrocytes respond by altering their morphological and functional properties. Reactive astrocyte phenotypes exhibit a bushy morphology with altered soma volume and an increased number of processes compared to resting astrocytes. The reactive astrocytic phenotype also overexpresses proteins one of which can be glial fibrillary acidic protein (GFAP). Fluorescence microscopy on thin tissue sections (< 20 μm) requires reconstruction, often through multiple sections, to delineate the full astrocytic morphology. In constrast, tissue clearing methods have been developed that enable imaging of larger sections including the whole brain, providing an opportunity to see in-depth changes in single cell structure. In this unit a detailed protocol for studying astrocyte morphology using tissue clearing and subsequent imaging of whole brains as well as region-specific slices is provided. This method is ideal for understanding the effect of different physiological conditions on astrocyte morphology. A standard biochemistry laboratory has the resources to accomplish tissue clearing using this protocol and most universities have the required imaging facilities. Protocol to study brains from both genetically modified mice that contain an astrocyte-specific marker and from wild-type mice using antibody labeling steps after tissue clearing are provided. We also describe general protocols to conduct fluorescence imaging of astrocytes in cleared tissue to characterize their morphology. This protocol could be useful for researchers working in the rapidly growing field of astrocyte biology.