The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Allen Institute for Brain Science Seattle WA USA

Department of Biomedical Engineering Boston University Boston MA USA

Department of Physiology 2nd Faculty of Medicine Charles University Prague Czech Republic

George W Woodruff School of Mechanical Engineering Georgia Institute of Technology Atlanta GA USA

Institute of Neuroscience National Yang Ming Chiao Tung University Taipei Taiwan

Janelia Research Campus Howard Hughes Medical Institute Ashburn VA USA

Janelia Research Campus Howard Hughes Medical Institute Ashburn VA USA; Departments of Molecular and Cell Biology and Physics Howard Hughes Medical Institute Helen Wills Neuroscience Institute University of California Berkeley Berkeley CA USA; Molecular Biophysics and Integrated Bioimaging Division Lawrence Berkeley National Laboratory Berkeley CA USA

Janelia Research Campus Howard Hughes Medical Institute Ashburn VA USA; GENIE Project Team Janelia Research Campus Howard Hughes Medical Institute Ashburn VA USA

Portland OR USA

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The role of electroencephalography in epilepsy research-From seizures to interictal activity and comorbidities

Seeing the Spikes: The Future of Targetable Synthetic Voltage Sensors