Two-photon Imaging of Microglial Processe's Attraction Toward ATP or Serotonin in Acute Brain Slices
Two-photon Imaging of Microglial Processe's Attraction Toward ATP or Serotonin in Acute Brain Slices
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DOI：
10.3791/58788
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发表时间：
2019-01-01
影响因子：
1.2
通讯作者：
Roumier, Anne
中科院分区：
文献类型：
--
作者：
Etienne, Fanny;Mastrolia, Vincenzo;Roumier, Anne
小胶质细胞是大脑的固有免疫细胞，它们不断地用它们的长过程扫描它们的环境，并且在体内平衡被破坏时，经历快速的形态变化。例如，激光损伤在几分钟内诱导小胶质细胞过程朝向损伤部位定向生长，也称为“定向运动”。通过局部递送ATP或血清素（5-羟色胺[5-HT]）可以获得类似的效果。在这篇文章中，我们描述了一个协议，以诱导小胶质细胞的过程中的局部应用ATP或5-HT在急性脑切片的年轻和成年小鼠的定向生长，并随着时间的推移，通过多光子显微镜成像这种吸引力。提出了一种使用免费开源图像分析软件进行量化的简单方法。急性脑切片仍然具有的一个挑战是有限的时间，随着年龄的增长而减少，在此期间细胞保持在生理状态。因此，该方案突出了一些技术改进（介质、气液界面室、具有双灌注的成像室），旨在优化几个小时内小胶质细胞的活力，特别是在成年小鼠的切片中。
Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called "directional motility", toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.