Balachandar Lakshmini, Montejo Karla A, Castano Eleane, Perez Melissa, Moncion Carolina, Chambers Jeremy W, Lujan J Luis, Diaz Jorge Riera
Department of Biomedical Engineering, Florida International University, Miami, Florida.
Department of Neurologic Surgery, Mayo Clinic, Rochester, Minnesota.
Curr Protoc Neurosci. 2020 Dec;94(1):e110. doi: 10.1002/cpns.110.
Astrocytes are actively involved in a neuroprotective role in the brain, which includes scavenging reactive oxygen species to minimize tissue damage. They also modulate neuroinflammation and reactive gliosis prevalent in several brain disorders like epilepsy, Alzheimer's, and Parkinson's disease. In animal models, targeted manipulation of astrocytic function via modulation of their calcium (Ca ) oscillations by incorporating light-sensitive cation channels like Channelrhodopsin-2 (ChR2) offers a promising avenue in influencing the long-term progression of these disorders. However, using adult animals for Ca imaging poses major challenges, including accelerated deterioration of in situ slice health and age- related changes. Additionally, optogenetic preparations necessitate usage of a red-shifted Ca indicator like Rhod-2 AM to avoid overlapping light issues between ChR2 and the Ca indicator during simultaneous optogenetic stimulation and imaging. In this article, we provide an experimental setting that uses live adult murine brain slices (2-5 months) from a knock-in model expressing Channelrhodopsin-2 (ChR2(C128S)) in cortical astrocytes, loaded with Rhod-2 AM to elicit robust Ca response to light stimulation. We have developed and standardized a protocol for brain extraction, sectioning, Rhod-2 AM loading, maintenance of slice health, and Ca imaging during light stimulation. This has been successfully applied to optogenetically control adult cortical astrocytes, which exhibit synchronous patterns of Ca activity upon light stimulation, drastically different from resting spontaneous activity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Experimental preparation, setup, slice preparation and Rhod-2 AM staining Basic Protocol 2: Image acquisition and analysis.
星形胶质细胞积极参与大脑中的神经保护作用，其中包括清除活性氧以将组织损伤降至最低。它们还调节神经炎症和反应性胶质增生，这些现象在癫痫、阿尔茨海默病和帕金森病等多种脑部疾病中普遍存在。在动物模型中，通过整合诸如通道视紫红质-2（ChR2）等光敏阳离子通道来调节星形胶质细胞的钙（Ca）振荡，从而有针对性地操纵星形胶质细胞功能，为影响这些疾病的长期进展提供了一条有前景的途径。然而，使用成年动物进行钙成像存在重大挑战，包括原位切片健康状况的加速恶化和与年龄相关的变化。此外，光遗传学实验需要使用红移钙指示剂，如罗丹明-2 AM，以避免在同时进行光遗传学刺激和成像时ChR2与钙指示剂之间出现光重叠问题。在本文中，我们提供了一种实验设置，使用来自在皮质星形胶质细胞中表达通道视紫红质-2（ChR2（C128S））的敲入模型的成年小鼠活体脑切片（2至5个月），加载罗丹明-2 AM以引发对光刺激的强烈钙反应。我们已经开发并标准化了一套用于脑提取、切片、罗丹明-2 AM加载、切片健康维护以及光刺激期间钙成像的方案。这已成功应用于光遗传学控制成年皮质星形胶质细胞，这些细胞在光刺激下表现出钙活性的同步模式，与静息自发活动截然不同。© 2020威利期刊有限责任公司。基本方案1：实验准备、设置、切片制备和罗丹明-2 AM染色 基本方案2：图像采集与分析。
