Enhanced genetically-encoded probes for voltage imaging

The combination of optical imaging methods with targeted expression of protein-based fluorescent probes enables the functional analysis of selected cell populations within intact neuronal circuitries. We previously demonstrated optogenetic monitoring of electrical activity in isolated cells, brain slices and living animals using voltage-sensitive fluorescent proteins (VSFPs). However, several properties of these voltage reporters remained suboptimal, limiting the spatiotemporal resolution of VSFP-based voltage imaging. A major limitation of VSFPs had been a reduced signal-to-noise ratio arising from intracellular aggregation and poor membrane targeting upon long-term expression in vivo. Here we report the generation of enhanced genetically-encoded sensors for membrane voltage (named VSFP-Butterflies) based on a novel molecular design that combines the advantageous features of VSFP2s and VSFP3s with molecular trafficking strategies. The new sensors exhibit faster response kinetics at subthreshold membrane potentials and enhanced localization to neuronal plasma membranes after long-term expression in vivo, enabling the optical recording of action potentials from individual neurons in single sweeps. VSFP-Butterflies provide optical readouts of population activity such as sensory-evoked responses and neocortical slow-wave oscillations with signal amplitudes exceeding 1% ΔR/R0 in anesthetized mice. VSFP-Butterflies will empower optogenetic electrophysiology by enabling new type of experiments bridging cellular and systems neuroscience and illuminating the function of neural circuits across multiple scales.