Villette Vincent, Yang Shang, Valenti Rosario, Macklin John J, Bradley Jonathan, Mathieu Benjamin, Lombardini Alberto, Podgorski Kaspar, Dieudonné Stéphane, Schreiter Eric R, Abdelfattah Ahmed S
Institut de Biologie de l'École Normale Supérieure (IBENS), École Normale Supérieure, CNRS, INSERM, PSL Research University, Paris 75005, France.
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
bioRxiv. 2024 Nov 17:2024.11.15.623698. doi: 10.1101/2024.11.15.623698.
Genetically encoded voltage indicators (GEVIs) allow optical recording of membrane potential from targeted cells . However, red GEVIs that are compatible with two-photon microscopy and that can be multiplexed with green reporters like GCaMP, are currently lacking. To address this gap, we explored diverse rhodopsin proteins as GEVIs and engineered a novel GEVI, 2Photron, based on a rhodopsin from the green algae . 2Photron, combined with two photon ultrafast local volume excitation (ULoVE), enabled multiplexed readout of spiking and subthreshold voltage simultaneously with GCaMP calcium signals in visual cortical neurons of awake, behaving mice. These recordings revealed the cell-specific relationship of spiking and subthreshold voltage dynamics with GCaMP responses, highlighting the challenges of extracting underlying spike trains from calcium imaging.
基因编码电压指示剂（GEVIs）能够对靶向细胞的膜电位进行光学记录。然而，目前缺乏与双光子显微镜兼容且可与诸如GCaMP等绿色报告基因复用的红色GEVIs。为了填补这一空白，我们探索了多种视紫红质蛋白作为GEVIs，并基于绿藻中的一种视紫红质设计了一种新型GEVI——2Photron。2Photron与双光子超快局部体积激发（ULoVE）相结合，能够在清醒行为小鼠的视觉皮层神经元中与GCaMP钙信号同时对动作电位发放和阈下电压进行复用读出。这些记录揭示了动作电位发放和阈下电压动态与GCaMP反应之间的细胞特异性关系，突出了从钙成像中提取潜在动作电位序列的挑战。
