Simultaneous Ca²⁺ Imaging and Optogenetic Stimulation of Cortical Astrocytes in Adult Murine Brain Slices

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. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Experimental preparation, setup, slice preparation and Rhod-2 AM staining Basic Protocol 2: Image acquisition and analysis.