Zhou Guanwei, Li Ruonan, Bartolik Ola, Ma Yuqian, Wan Wei Wei, Meng Jennifer, Hu Yujia, Ye Bing, Wang Wenjing
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.
Program in Chemical Biology, University of Michigan, Ann Arbor, MI, USA.
bioRxiv. 2025 Jan 18:2025.01.13.632875. doi: 10.1101/2025.01.13.632875.
Recording and manipulating neuronal ensembles that underlie cognition and behavior is challenging. FLARE is a light- and calcium-gated transcriptional reporting system for labeling activated neurons on the order of minutes. However, FLARE is limited by its sensitivity to prolonged neuronal activities. Here, we present an improved version of FLARE, termed cytoFLARE. cytoFLARE incorporates cytosolic expression of the transcription factor and a more sensitive pair of calcium sensing domains. We showed that cytoFLARE provides more calcium- and light- dependent signals in HEK293T cells and higher signal-to-background ratios in neuronal cultures. We further established cytoFLARE transgenic models and applied cytoFLARE to label activated neurons upon sensory or optogenetic stimulation within a defined time window. Notably, through cytoFLARE-driven expression of an optogenetic actuator, we successfully reactivated neurons involved in the larval nociceptive system. Our findings demonstrate the first characterization and application of time-gated calcium integrators in .
记录和操纵构成认知与行为基础的神经元集群具有挑战性。FLARE是一种光控和钙控转录报告系统，用于在数分钟量级上标记激活的神经元。然而，FLARE受限于其对长时间神经元活动的敏感性。在此，我们展示了一种改进版的FLARE，称为细胞FLARE（cytoFLARE）。细胞FLARE整合了转录因子的胞质表达以及一对更敏感的钙感应结构域。我们表明，细胞FLARE在HEK293T细胞中提供更多的钙和光依赖信号，在神经元培养物中具有更高的信号背景比。我们进一步建立了细胞FLARE转基因模型，并应用细胞FLARE在定义的时间窗口内标记感觉或光遗传学刺激时激活的神经元。值得注意的是，通过细胞FLARE驱动的光遗传学致动器表达，我们成功地重新激活了参与幼虫伤害感受系统的神经元。我们的研究结果展示了时间门控钙整合器在……中的首次表征和应用。
