Characterization of optimal optogenetic stimulation paradigms to evoke calcium events in cortical astrocytes

Understanding the roles of astrocytic calcium signaling in multiple brain regulatory mechanisms including metabolism, blood flow, neuromodulation and neuroinflammation has remained one of the enduring challenges in glial biology. To delineate astrocytic contribution from concurrent neuronal activity, it is vital to establish robust control and manipulate astrocytes using a technique like optogenetics due to its high cellular specificity and temporal resolution. Lack of an experimental paradigm to induce controlled calcium signaling in astrocytes has hindered progress in the field. To address this, i n this study, we systematically characterize and identify light stimulation paradigms for inducing regulated, on-demand increases in astrocytic calcium in cortical astrocytes in MlC1-ChR2(C128S)-EYFP mice. We identified paradigms 20%, 40% and 60% (of T = 100s) to elicit robust calcium responses upon multiple stimulations, while the 95% paradigm exhibited a response only during the first stimulation. We also quantified several parameters, including peak height, Full Width Half Maximum (FWHM), and latencies, and observe that the 20% paradigm has the highest peak ΔF/F 0 among the paradigms across all stimulations and the lowest FWHM during the first stimulation. Overall, the 20% paradigm is a favorable choice for eliciting robust astrocytic calcium responses in astrocytes while performing multiple stimulations.