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| Item Type: | Article |
|---|---|
| Title: | Visualization of gap junction-mediated astrocyte coupling in acute mouse brain slices |
| Creators Name: | Kompier, N.F., Siemonsmeier, G., Meyer, N., Kettenmann, H. and Rathjen, F.G. |
| Abstract: | Gap junctions are transmembrane protein channels that enable the exchange of small molecules such as ions, second messengers, and metabolites between adjacent cells. Gap junctions are found in various mammalian organs, including skin, endothelium, liver, pancreas, muscle, and central nervous system (CNS). In the CNS, they mediate coupling between neural cells including glial cells, and the resulting panglial networks are vital for brain homeostasis. Tracers of sufficiently small molecular mass can diffuse across gap junctions, and are used to visualize the extent of cell-to-cell coupling in situ by delivering them to a single cell through sharp electrodes or patch clamp micropipettes. Here, we describe a protocol for pre-labeling and identification of astrocytes in acute mouse forebrain slices using Sulforhodamine 101 (SR101). Fluorescent cells can then be targeted for whole-cell patch clamp, which allows for further confirmation of astroglial identity by assessing their electrophysiological properties, as well as for passive dialysis with a tracer such as biocytin. Slices can then be subjected to chemical fixation and immunostaining to detect dye-coupled networks. This protocol provides a method for the identification of astrocytes in live tissue though SR101 labeling. Alternatively, transgenic reporter mice can also be used to identify astrocytes. While we illustrate the use of this protocol for the study of glial networks in the mouse brain, the general principles are applicable to other species, tissues and cell types. |
| Keywords: | Astrocytes, Panglial Networks, Cell-to-Cell Coupling, Gap Junctions, Patch Clamp, Biocytin, Dialysis, Sulforhodamine 101, Immunohistochemistry, Animals, Mice |
| Source: | Bio-protocol |
| ISSN: | 2331-8325 |
| Publisher: | Bio-protocol LLC |
| Volume: | 15 |
| Page Range: | 1365 |
| Date: | 20 February 2025 |
| Official Publication: | https://doi.org/10.21769/BioProtoc.5220 |
| PubMed: | View item in PubMed |
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