Badia-Soteras Aina, Octeau J Christopher, Verheijen Mark H G, Khakh Baljit S
Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam, Amsterdam, The Netherlands.
Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
Curr Protoc Neurosci. 2020 Mar;91(1):e91. doi: 10.1002/cpns.91.
Astrocytes are morphologically complex cells with numerous close contacts with neurons at the level of their somata, branches, and branchlets. The smallest astrocyte processes make discrete contacts with synapses at scales that cannot be observed by standard light microscopy. At such contact points, astrocytes are thought to perform both homeostatic and neuromodulatory roles-functions that are proposed to be determined by their close spatial apposition. To study such spatial interactions, we previously developed a Förster resonance energy transfer (FRET)-based approach, which enables observation and tracking of the static and dynamic proximity of astrocyte processes with synapses. The approach is compatible with standard imaging techniques such as confocal microscopy and permits assessment of the most proximate contacts between astrocytes and neurons in live tissues. In this protocol article we describe the approach to analyze the contacts between striatal astrocyte processes and corticostriatal neuronal projection terminals onto medium spiny neurons. We report the required protocols in detail, including adeno-associated virus microinjections, acute brain slice preparation, imaging, and post hoc FRET quantification. The article provides a detailed description that can be used to characterize and study astrocyte process proximity to synapses in living tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Förster resonance energy transfer imaging in cultured cells Basic Protocol 2: Förster resonance energy transfer imaging with the neuron-astrocyte proximity assay in acute brain slices.
星形胶质细胞是形态复杂的细胞，在其胞体、分支和小分支水平与神经元有大量紧密接触。最小的星形胶质细胞突起在标准光学显微镜无法观察到的尺度上与突触形成离散接触。在这些接触点，星形胶质细胞被认为具有稳态和神经调节作用，这些功能被认为是由它们紧密的空间毗邻关系所决定的。为了研究这种空间相互作用，我们之前开发了一种基于荧光共振能量转移（FRET）的方法，该方法能够观察和追踪星形胶质细胞突起与突触的静态和动态接近情况。该方法与共聚焦显微镜等标准成像技术兼容，并允许评估活组织中星形胶质细胞与神经元之间最接近的接触。在这篇方案文章中，我们描述了分析纹状体星形胶质细胞突起与投射到中等棘状神经元上的皮质纹状体神经元投射终末之间接触的方法。我们详细报告了所需的方案，包括腺相关病毒显微注射、急性脑片制备、成像以及事后FRET定量。本文提供了详细描述，可用于表征和研究活组织中星形胶质细胞突起与突触的接近情况。© 2020约翰威立国际出版公司。基本方案1：培养细胞中的荧光共振能量转移成像 基本方案2：急性脑片中神经元 - 星形胶质细胞接近度测定的荧光共振能量转移成像。
