Zhou Shihao, Zhu Qiyu, Eom Minho, Fang Shilin, Subach Oksana M, Ran Chen, Alvarado Jonnathan Singh, Sunkavalli Praneel S, Dong Yuanping, Wang Yangdong, Hu Jiewen, Zhang Hanbin, Wang Zhiyuan, Sun Xiaoting, Yang Tao, Mu Yu, Yoon Young-Gyu, Guo Zengcai V, Subach Fedor V, Piatkevich Kiryl D
School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China.
Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, China.
PLoS Biol. 2025 Apr 29;23(4):e3003048. doi: 10.1371/journal.pbio.3003048. eCollection 2025 Apr.
Recent advancements in genetically encoded calcium indicators, particularly those based on green fluorescent proteins, have optimized their performance for monitoring neuronal activities in a variety of model organisms. However, progress in developing red-shifted GECIs, despite their advantages over green indicators, has been slower, resulting in fewer options for end users. In this study, we explored topological inversion and soma-targeting strategies, which are complementary to conventional mutagenesis, to re-engineer a red genetically encoded calcium indicator, FRCaMP, for enhanced in vivo performance. The resulting sensors, FRCaMPi and soma-targeted FRCaMPi (SomaFRCaMPi), exhibit up to 2-fold higher dynamic range and peak ΔF/F0 per single AP compared to widely used jRGECO1a in neurons both in culture and in vivo. Compared to jRGECO1a and FRCaMPi, SomaFRCaMPi reduces erroneous correlation of neuronal activity in the brains of mice and zebrafish by two- to 4-fold due to diminished neuropil contamination without compromising the signal-to-noise ratio. Under wide-field imaging in primary somatosensory and visual cortices in mice with high labeling density (80-90%), SomaFRCaMPi exhibits up to 40% higher SNR and decreased artifactual correlation across neurons. Altogether, SomaFRCaMPi improves the accuracy and scale of neuronal activity imaging at single-neuron resolution in densely labeled brain tissues due to a 2-3-fold enhanced automated neuronal segmentation, 50% higher fraction of responsive cells, up to 2-fold higher SNR compared to jRGECO1a. Our findings highlight the potential of SomaFRCaMPi, comparable to the most sensitive soma-targeted GCaMP, for precise spatial recording of neuronal populations using popular imaging modalities in model organisms such as zebrafish and mice.
基因编码钙指示剂，尤其是基于绿色荧光蛋白的指示剂，其近期进展已优化了它们在多种模式生物中监测神经元活动的性能。然而，尽管红移基因编码钙指示剂相较于绿色指示剂具有优势，但其开发进展较慢，这使得终端用户的选择较少。在本研究中，我们探索了拓扑反转和靶向胞体策略，这些策略与传统诱变互补，用于对红色基因编码钙指示剂FRCaMP进行重新设计，以增强其体内性能。所得的传感器FRCaMPi和靶向胞体的FRCaMPi（SomaFRCaMPi），与广泛使用的jRGECO1a相比，在培养和体内的神经元中，单次动作电位的动态范围和峰值ΔF/F0高达两倍。与jRGECO1a和FRCaMPi相比，SomaFRCaMPi在不影响信噪比的情况下，由于神经纤维污染减少，将小鼠和斑马鱼大脑中神经元活动的错误相关性降低了2至4倍。在高标记密度（80 - 90%）的小鼠初级体感皮层和视觉皮层的宽场成像中，SomaFRCaMPi的信噪比高达40%，且神经元间的伪相关性降低。总体而言，由于自动神经元分割增强了2至3倍、反应性细胞比例提高了50%、与jRGECO1a相比信噪比高达两倍，SomaFRCaMPi提高了在密集标记脑组织中以单神经元分辨率进行神经元活动成像的准确性和规模。我们的研究结果突出了SomaFRCaMPi的潜力，其与最灵敏的靶向胞体的GCaMP相当，可使用斑马鱼和小鼠等模式生物中常用的成像方式对神经元群体进行精确的空间记录。
