Abstracts

Rationale: Understanding how the brain works at the circuit level requires sensors that can track various types of neuronal activity with sufficient resolution in space and time. Genetically-encoded voltage indicators (GEVIs) offer great potential in achieving this goal, but still require a lot of optimization to be routinely used in vivo. Development of GEVIs has been hampered by a lack of a high-throughput screening methods, which have been instrumental for rapid improvement of calcium indicators. GEVI screening is challenging because one must induce a change in membrane potential while accurately monitoring fluorescence output from the sensor on a fast timescale. Methods: Here we report the development of a new electrical screening platform for improving GEVI variants based on a novel concept for inducing rapid membrane voltage changes. We used the system to optimize ASAP-family GEVIs, which are constructed by inserting a circularly permuted superfolding green fluorescent protein within the four-helix voltage sensing domain of G. gallus voltage-sensing phosphatase. With ability to rapidly screen hundreds of GEVI variants, and using mechanism-guided approach, we obtained an improved sensor, ASAP3. Results: ASAP3 demonstrates steady-state responsivity of –50% in the physiological range, fast activation kinetics that enable accurate AP timing, and moderate inactivation kinetics that improve detectability of action potential responses.  Conclusions: With the dramatic improvement of ASAP3 over the previous generation of voltage indicators, and retention of its voltage sensitivity under two-photon microscopy, ASAP3 will be useful for imaging voltage in genetically defined neurons in the brain.   Funding: NIH BRAIN Initiative Grant 1U01NS090600