Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, 'Voltron', that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 minutes of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.Animal behavior is produced by patterns of neuronal activity that span a wide range of spatial and temporal scales. To understand how neural circuits mediate behavior thus requires high-speed recording from ensembles of neurons for long periods of time.Although the activity of large numbers of neurons can now be routinely recorded using genetically encoded calcium indicators (GECIs) (1), the slow kinetics of calcium signals complicate the measurement of action potentials, and sub-threshold voltage signals are missed entirely (1-3). Voltage imaging using genetically encoded voltage indicators (GEVIs) can overcome these challenges, enabling imaging of fast spikes and subthreshold dynamics in genetically defined neurons (4, 5). The high imaging speed and excitation intensity required for voltage imaging, combined with the smaller volume of the cellular membrane, place increased demands on voltage indicators relative to GECIs.Extant GEVIs rely on fluorescence from either microbial rhodopsins (6-8) or fluorescent proteins (FPs) (9-13). These fluorophores lack the brightness and photostability to allow in vivo voltage imaging from large fields of view over timescales of many behavioral events, precluding the millisecond-timescale interrogation of neural circuits. Recent development of improved rhodamine dyes such as the Janelia Fluor ® (JF) dyes enable their use in complex biological experiments due to their high brightness and photostability (14), compatibility with self-labeling protein tags (15, 16), and the ability to traverse the blood-brain barrier for in vivo delivery (17). Here we describe a 'chemigenetic' GEVI scaffold-termed 'Voltron'-which incorporates these synthetic fluorophore dyes. Voltron provides an increased photon yield that enables in vivo imaging of neuronal spiking and sub-threshold voltage signals in model organisms with order-of-magnitude improvement in the number of neurons imaged simultaneously over substantially longer durations.Our design for a chemigenetic voltage indicator combines a voltage-sensitive microbial rhodopsin domain (6, 7, 11) with a self-labeling protein tag domain (F...