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. The lack of an experimental paradigm to induce controlled calcium signaling in astrocytes has hindered progress in the field. To address this, in this study, we systematically characterize and identify light stimulation paradigms for inducing regulated, on-demand increases in astrocytic calcium in acute brain slice cortical astrocytes from MlC1-ChR2(C128S)-EYFP mice (of either sex). We identified paradigms 20, 40 and 60% (of T = 100 s) to elicit robust calcium responses upon periodic stimulations, while the 95% paradigm exhibited a response only during the first stimulation. We also quantified several parameters, including peak height, full-width at half-maximum (FWHM), and latencies, and observe that the 20% paradigm/duty cycle has the highest peak ΔF/F ₀ among the paradigms across all stimulations and the lowest FWHM during the first stimulation. To illustrate the impact of our study, we observed robust changes in cerebral blood flow, because of 20% optogenetic stimulation, in vivo, using laser Doppler flowmetry. Overall, the 20% paradigm is a favorable choice for eliciting robust astrocytic calcium responses in astrocytes while performing periodic stimulations.