Neuronal electrical activity is the central underpinning of nervous system function. While understood as essential for over a century, the tools to study circuit level neurophysiology have remained largely unchanged in 50 years. The advent of molecular biology has dramatically advanced neurobiology by allowing molecular characterization of the nervous system but has not translated into significant gains in neural electrophysiology. Opto-molecular methods have revolutionized our study of neuronal connectivity, development, gene distribution, calcium signaling and recently, targeted neuronal activation (i.e. optogenetics). A glaring exception to this light-based revolution is the use of optical methods to monitor electrical activity. Intracellular calcium levels and metabolic signals are often used as a surrogate marker of electrical activity, however they are temporally delayed, do not detect subthreshold events and more often than not fail to capture the relevant suprathreshold activity. The PIs laboratories, as part of a multi laboratory collaboration have been developing genetically encoded voltage sensors based on fusions of green fluorescent protein orthologs and voltage sensing domains. Our grant members have published most of the significant advances in genetically-encoded voltage sensors in recent years. Our most recent probes, Arclight and ElectricPK significantly improved the signal size and response speed of fluorescent voltage probes. The current application will continue this successful collaborative search for voltage probes. We are seeking probes which combine large F/ V signal sizes, a range of useful response speeds and red-shifted fluorescence spectra. During this previous funded period time, we discovered that by altering the voltage sensor domain, the linker length, the fluorescent protein and by introducing point mutations in the fluorescent protein, we could develop probes with vastly superior signal size and response kinetics. We also confirmed, however, that a purely empirical step (i.e. large scale screening of single, incrementally-modified constructs) is required to make dramatic improvements in response properties. We will employ a staged evolution approach involving successive rounds of directed and random sequence modification followed by direct testing in mammalian cells. The current experiments will be an advance over all previous studies in two important ways: i) we will create vastly greater numbers (20x) of potential probe (thousands) using domain swapping and site directed / random mutagenesis and ii) the larger numbers of constructs will be prescreened by an automated, robotic microfluorimetry method which evaluates the fluorescence signal size and speed in electrically-active mammalian cells. Finally, all successful candidates will be validated for in vio functionality in Drosophila circadian neurons and rodent somatosensory/barrel cortex.

Public Health Relevance

We will develop methods that will allow the electrical activity of brain cells to be determined using imaging techniques as opposed to electrodes as is commonly done. With further development these techniques can allow the neuronal activity in patients with high spinal cord damage to be read out allowing the patient to direct computers to perform tasks.

National Institute of Health (NIH)
Research Project (R01)
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Molecular Neurogenetics Study Section (MNG)
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Stewart, Randall R
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John B. Pierce Laboratory, Inc.
New Haven
United States
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Gruber, David F; Loew, Ellis R; Deheyn, Dimitri D et al. (2016) Biofluorescence in Catsharks (Scyliorhinidae): Fundamental Description and Relevance for Elasmobranch Visual Ecology. Sci Rep 6:24751
Abdelfattah, Ahmed S; Farhi, Samouil L; Zhao, Yongxin et al. (2016) A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices. J Neurosci 36:2458-72
Drobizhev, M; Callis, P R; Nifosì, R et al. (2015) Long- and Short-Range Electrostatic Fields in GFP Mutants: Implications for Spectral Tuning. Sci Rep 5:13223
Sarkisyan, Karen S; Goryashchenko, Alexander S; Lidsky, Peter V et al. (2015) Green fluorescent protein with anionic tryptophan-based chromophore and long fluorescence lifetime. Biophys J 109:380-9
Mehr, Shaadi; Verdes, Aida; DeSalle, Rob et al. (2015) Transcriptome sequencing and annotation of the polychaete Hermodice carunculata (Annelida, Amphinomidae). BMC Genomics 16:445
Stoltzfus, Caleb R; Barnett, Lauren M; Drobizhev, Mikhail et al. (2015) Two-photon directed evolution of green fluorescent proteins. Sci Rep 5:11968
Guan, Yinghua; Meurer, Matthias; Raghavan, Sarada et al. (2015) Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein. Mol Biol Cell 26:2054-66
Gruber, David F; Gaffney, Jean P; Mehr, Shaadi et al. (2015) Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment. PLoS One 10:e0140972
Klein, Mason; Afonso, Bruno; Vonner, Ashley J et al. (2015) Sensory determinants of behavioral dynamics in Drosophila thermotaxis. Proc Natl Acad Sci U S A 112:E220-9
Sparks, John S; Schelly, Robert C; Smith, W Leo et al. (2014) The covert world of fish biofluorescence: a phylogenetically widespread and phenotypically variable phenomenon. PLoS One 9:e83259

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