Balachandar Lakshmini, Moncion Carolina, Suarez Alejandro, Diaz Jorge Riera
NMD Laboratory at the Department of Biomedical Engineering, Florida International University, Miami, Florida 33174.
NMD Laboratory at the Department of Biomedical Engineering, Florida International University, Miami, Florida 33174
eNeuro. 2025 Sep 15;12(9). doi: 10.1523/ENEURO.0220-25.2025. Print 2025 Sep.
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 = 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 Δ/ 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.
了解星形胶质细胞钙信号在包括代谢、血流、神经调节和神经炎症在内的多种脑调节机制中的作用，一直是神经胶质生物学中持久的挑战之一。为了区分星形胶质细胞与同时发生的神经元活动的贡献，由于光遗传学具有高细胞特异性和时间分辨率，因此使用光遗传学等技术建立对星形胶质细胞的强大控制和操作至关重要。缺乏在星形胶质细胞中诱导可控钙信号的实验范式阻碍了该领域的进展。为了解决这个问题，在本研究中，我们系统地表征和识别了用于诱导MlC1-ChR2(C128S)-EYFP小鼠（雌雄不限）急性脑片皮质星形胶质细胞中星形胶质细胞钙按需调节性增加的光刺激范式。我们确定了20%、40%和60%（共100秒）的范式，在周期性刺激时可引发强烈的钙反应，而95%的范式仅在第一次刺激时出现反应。我们还量化了几个参数，包括峰值高度、半高宽（FWHM）和潜伏期，并观察到在所有刺激的范式中，20%的范式/占空比在第一次刺激时具有最高的峰值Δ/，且半高宽最低。为了说明我们研究的影响，我们使用激光多普勒血流仪在体内观察到，由于20%的光遗传学刺激，脑血流发生了强烈变化。总体而言，在进行周期性刺激时，20%的范式是在星形胶质细胞中引发强烈星形胶质细胞钙反应的有利选择。
