Imaging Neuronal Voltage and Interrogationg Biochemical Network with Fluorescent Proteins

Neuronal circuits are remarkable in the sophisticated computations they perform, and in their ability to be restructured by learning. By revealing spiking and subthreshold electrical activity, genetically encoded voltage indicators (GEVIs) offer the ability to understand computation and plasticity in the brain. However, with a finite amount of GEVI expression per cell, improving throughput necessarily depends on improving GEVI brightness, speed, and responsivity. We established a high-throughput bacteria-free pipeline to improve GFP-based ASAP-family GEVIs by multiparameter screening, resulting most recently in ASAP4. Similarly to commonly used calcium indicators, ASAP4 brightens in response to depolarization, allowing for improved photostability in 1-photon and 2-photon voltage imaging in vivo. Most recently, a new ASAP variant further enhances relative fluorescence changes and signal-to-noise ratios in response to action potentials in vivo, and enables detection of miniature excitatory post-synaptic potentials in neurons in vitro. Finally, I will present ongoing work on an entirely different use of GFP – as domains whose association and dissociation are controlled by light, enabling the creation of single-chain photoswitchable proteins for optogenetic interrogation of biochemical signaling networks.