@article{13a8a7f91c52474fbab1683b7328f179,
title = "Simultaneous Ca2+ Imaging and Optogenetic Stimulation of Cortical Astrocytes in Adult Murine Brain Slices",
abstract = "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 (Ca2+) 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 Ca2+ 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 Ca2+ indicator like Rhod-2 AM to avoid overlapping light issues between ChR2 and the Ca2+ 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 Ca2+ response to light stimulation. We have developed and standardized a protocol for brain extraction, sectioning, Rhod-2 AM loading, maintenance of slice health, and Ca2+ imaging during light stimulation. This has been successfully applied to optogenetically control adult cortical astrocytes, which exhibit synchronous patterns of Ca2+ activity upon light stimulation, drastically different from resting spontaneous activity.",
keywords = "Channelrhodopsin-2, Rhod-2 AM, astrocyte, calcium imaging, optogenetics",
author = "Lakshmini Balachandar and Montejo, {Karla A.} and Eleane Castano and Melissa Perez and Carolina Moncion and Chambers, {Jeremy W.} and Lujan, {J. Luis} and Diaz, {Jorge Riera}",
note = "Funding Information: This study was supported by various grants. The first grant was awarded by the Wallace Coulter Foundation, given to the Department of Biomedical Engineering, Florida International University. Karla Alejandra Montejo was supported by the National Institutes of Health NIGMS R25 GM75148 and NINDS R01 NS107336-02S1. The authors would like to sincerely thank Mr. Anders Asp, Dr. Shaohua Wang, and Mr. Chavier Laffitte for insightful discussions and feedback on the calibration of calcium imaging analysis. The authors would also like to thank Dr. James Schummers, Dr. Ranu Jung, Dr. Timothy Allen and Dr. Kalai Mathee for their help with equipment employed in this study and RIKEN, Japan, and JAX Labs, USA for their assistance with establishing the transgenic mice lines in the United States. Funding Information: This study was supported by various grants. The first grant was awarded by the Wallace Coulter Foundation, given to the Department of Biomedical Engineering, Florida International University. Karla Alejandra Montejo was supported by the National Institutes of Health NIGMS R25 GM75148 and NINDS R01 NS107336‐02S1. The authors would like to sincerely thank Mr. Anders Asp, Dr. Shaohua Wang, and Mr. Chavier Laffitte for insightful discussions and feedback on the calibration of calcium imaging analysis. The authors would also like to thank Dr. James Schummers, Dr. Ranu Jung, Dr. Timothy Allen and Dr. Kalai Mathee for their help with equipment employed in this study and RIKEN, Japan, and JAX Labs, USA for their assistance with establishing the transgenic mice lines in the United States. Publisher Copyright: {\textcopyright} 2020 Wiley Periodicals LLC",
year = "2020",
month = dec,
doi = "10.1002/cpns.110",
language = "English (US)",
volume = "94",
journal = "Current Protocols in Neuroscience",
issn = "1934-8584",
publisher = "John Wiley and Sons Inc.",
number = "1",
}