The pattern of activity in the circuits of the brain and their experience-dependent changes underlie the processing of sensory information, perception, and motor control. Much has been learned about the anatomical wiring of brain circuits and about the properties of individual neurons in the intact brain, but considerable mystery remains about how the properties of individual neurons emerge from their connectivity and how multiple groups of neurons are activated during behaviors. Part of the problem has been that high precision electrical recording is usually obtained from only one or a few neurons at a time, when salient events are actually processed by large assemblies of neurons. To provide for a fast high-resolution recording from mammalian neurons, this proposal seeks to improve fluorescent protein (FP) based voltage sensors. These probes will be self-contained, not requiring any exogenous factors to function, and thus will be genetically-encodable. We are seeking probes that are readily expressed on the cell's surface, show maximum changes in intensity with membrane potential alterations, respond rapidly to changes in membrane potential, and are minimally disruptive to cells. Members of the project have been involved in the development of first generation FP-voltage sensors, including Fluorescent Shaker (FlaSh), Voltage-Sensitive Fluorescent Protein (VSFP) and Sodium channel Protein Activity Reporting Construct (SPARC). These constructs have demonstrated the feasibility of creating channel-FP constructs that alter fluorescence intensity with changes in cell membrane potential. Significant improvements in the response characteristics may come from a pseudo-saturating examination of the ion channel/transporter and fluorescent protein space. This proposal will create large libraries of membrane protein / FP fusion constructs varying the membrane protein, the location of the inserted FP and the isoform of the inserted FP. These libraries will be created by a novel transposon-based FP insertion process. Constructs will be screened for surface expression in hippocampal neurons and tested for voltage-dependent fluorescence changes using fast fluorescence measurements combined with voltage-clamp electrophysiology. We will express the most promising FP-voltage sensors in brain slices and in vivo using viral infection followed by the production of transgenic mice.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Resource-Related Research Projects--Cooperative Agreements (U24)
Project #
3U24NS057631-03S1
Application #
7912355
Study Section
National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
Program Officer
Stewart, Randall R
Project Start
2007-09-01
Project End
2011-09-29
Budget Start
2009-09-30
Budget End
2011-09-29
Support Year
3
Fiscal Year
2009
Total Cost
$240,162
Indirect Cost
Name
Yale University
Department
Physiology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Akemann, Walther; Song, Chenchen; Mutoh, Hiroki et al. (2015) Route to genetically targeted optical electrophysiology: development and applications of voltage-sensitive fluorescent proteins. Neurophotonics 2:
Sung, Uhna; Sepehri-Rad, Masoud; Piao, Hong Hua et al. (2015) Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions. PLoS One 10:e0141585
Reiner, Andreas; Isacoff, Ehud Y (2014) Tethered ligands reveal glutamate receptor desensitization depends on subunit occupancy. Nat Chem Biol 10:273-80
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
Cavanaugh, Daniel J; Geratowski, Jill D; Wooltorton, Julian R A et al. (2014) Identification of a circadian output circuit for rest:activity rhythms in Drosophila. Cell 157:689-701
Han, Zhou; Jin, Lei; Chen, Fuyi et al. (2014) Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight. PLoS One 9:e113873
Patti, Jordan; Isacoff, Ehud Y (2013) Measuring membrane voltage with fluorescent proteins. Cold Spring Harb Protoc 2013:606-13
Cao, Guan; Platisa, Jelena; Pieribone, Vincent A et al. (2013) Genetically targeted optical electrophysiology in intact neural circuits. Cell 154:904-13
Han, Zhou; Jin, Lei; Platisa, Jelena et al. (2013) Fluorescent protein voltage probes derived from ArcLight that respond to membrane voltage changes with fast kinetics. PLoS One 8:e81295
Jin, Lei; Han, Zhou; Platisa, Jelena et al. (2012) Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe. Neuron 75:779-85

Showing the most recent 10 out of 23 publications