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Enhanced Archaerhodopsin Fluorescent Protein Voltage Indicators.

Gong Y, Li JZ, Schnitzer MJ - PLoS ONE (2013)

Bottom Line: Here we present Arch-derived voltage sensors with trafficking signals that enhance their localization to the neural membrane.We benchmarked these voltage sensors regarding their spike detection fidelity by using a signal detection theoretic framework that takes into account the experimentally measured photon shot noise and optical waveforms for single action potentials.This analysis revealed that by combining the sequence mutations and enhanced trafficking sequences, the new sensors improved the fidelity of spike detection by nearly three-fold in comparison to Arch-D95N.

View Article: PubMed Central - PubMed

Affiliation: James H. Clark Center, Stanford University, Stanford, California, United States of America ; CNC Program, Stanford University, Stanford, California, United States of America.

ABSTRACT
A longstanding goal in neuroscience has been to develop techniques for imaging the voltage dynamics of genetically defined subsets of neurons. Optical sensors of transmembrane voltage would enhance studies of neural activity in contexts ranging from individual neurons cultured in vitro to neuronal populations in awake-behaving animals. Recent progress has identified Archaerhodopsin (Arch) based sensors as a promising, genetically encoded class of fluorescent voltage indicators that can report single action potentials. Wild-type Arch exhibits sub-millisecond fluorescence responses to trans-membrane voltage, but its light-activated proton pump also responds to the imaging illumination. An Arch mutant (Arch-D95N) exhibits no photocurrent, but has a slower, ~40 ms response to voltage transients. Here we present Arch-derived voltage sensors with trafficking signals that enhance their localization to the neural membrane. We also describe Arch mutant sensors (Arch-EEN and -EEQ) that exhibit faster kinetics and greater fluorescence dynamic range than Arch-D95N, and no photocurrent at the illumination intensities normally used for imaging. We benchmarked these voltage sensors regarding their spike detection fidelity by using a signal detection theoretic framework that takes into account the experimentally measured photon shot noise and optical waveforms for single action potentials. This analysis revealed that by combining the sequence mutations and enhanced trafficking sequences, the new sensors improved the fidelity of spike detection by nearly three-fold in comparison to Arch-D95N.

No MeSH data available.


Related in: MedlinePlus

Fluorescence spectra and membrane localization of enhanced Arch constructs.(a) We expressed the enhanced Arch constructs (EEQ and EEN versions) as a fusion with eYFP under the control of the Camk2a promoter and targeted the fusion protein to the cell membrane using the localization sequences TS and ER. (b) Fluorescence signals from a labeled neuron, as seen in the YFP (left) and Arch-EEN (middle) fluorescence channels, along with the normalized spatial map (right) of the fluorescence response to a voltage step depolarization. Regions in which YFP fluorescence was visible also generally showed responses by Arch to voltage depolarization, which is indicative of successful membrane targeting. Scale bar is 20 µm. (c) Absorption and fluorescence spectra of Arch-EEN. The spectra of Arch-EEQ are similar.
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pone-0066959-g001: Fluorescence spectra and membrane localization of enhanced Arch constructs.(a) We expressed the enhanced Arch constructs (EEQ and EEN versions) as a fusion with eYFP under the control of the Camk2a promoter and targeted the fusion protein to the cell membrane using the localization sequences TS and ER. (b) Fluorescence signals from a labeled neuron, as seen in the YFP (left) and Arch-EEN (middle) fluorescence channels, along with the normalized spatial map (right) of the fluorescence response to a voltage step depolarization. Regions in which YFP fluorescence was visible also generally showed responses by Arch to voltage depolarization, which is indicative of successful membrane targeting. Scale bar is 20 µm. (c) Absorption and fluorescence spectra of Arch-EEN. The spectra of Arch-EEQ are similar.

Mentions: We generated all of the Arch variants discussed in this paper within a similar construct containing the CaMKIIα (Camk2a) promoter and the woodchuck hepatitis post-transcriptional regulatory element (WPRE) (Figure 1a). In neurons transfected with constructs containing the trafficking signals, YFP, and Arch fluorescence signals were generally membrane-localized, indicative of minimal intracellular aggregation (Figure 1b).


Enhanced Archaerhodopsin Fluorescent Protein Voltage Indicators.

Gong Y, Li JZ, Schnitzer MJ - PLoS ONE (2013)

Fluorescence spectra and membrane localization of enhanced Arch constructs.(a) We expressed the enhanced Arch constructs (EEQ and EEN versions) as a fusion with eYFP under the control of the Camk2a promoter and targeted the fusion protein to the cell membrane using the localization sequences TS and ER. (b) Fluorescence signals from a labeled neuron, as seen in the YFP (left) and Arch-EEN (middle) fluorescence channels, along with the normalized spatial map (right) of the fluorescence response to a voltage step depolarization. Regions in which YFP fluorescence was visible also generally showed responses by Arch to voltage depolarization, which is indicative of successful membrane targeting. Scale bar is 20 µm. (c) Absorption and fluorescence spectra of Arch-EEN. The spectra of Arch-EEQ are similar.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3686764&req=5

pone-0066959-g001: Fluorescence spectra and membrane localization of enhanced Arch constructs.(a) We expressed the enhanced Arch constructs (EEQ and EEN versions) as a fusion with eYFP under the control of the Camk2a promoter and targeted the fusion protein to the cell membrane using the localization sequences TS and ER. (b) Fluorescence signals from a labeled neuron, as seen in the YFP (left) and Arch-EEN (middle) fluorescence channels, along with the normalized spatial map (right) of the fluorescence response to a voltage step depolarization. Regions in which YFP fluorescence was visible also generally showed responses by Arch to voltage depolarization, which is indicative of successful membrane targeting. Scale bar is 20 µm. (c) Absorption and fluorescence spectra of Arch-EEN. The spectra of Arch-EEQ are similar.
Mentions: We generated all of the Arch variants discussed in this paper within a similar construct containing the CaMKIIα (Camk2a) promoter and the woodchuck hepatitis post-transcriptional regulatory element (WPRE) (Figure 1a). In neurons transfected with constructs containing the trafficking signals, YFP, and Arch fluorescence signals were generally membrane-localized, indicative of minimal intracellular aggregation (Figure 1b).

Bottom Line: Here we present Arch-derived voltage sensors with trafficking signals that enhance their localization to the neural membrane.We benchmarked these voltage sensors regarding their spike detection fidelity by using a signal detection theoretic framework that takes into account the experimentally measured photon shot noise and optical waveforms for single action potentials.This analysis revealed that by combining the sequence mutations and enhanced trafficking sequences, the new sensors improved the fidelity of spike detection by nearly three-fold in comparison to Arch-D95N.

View Article: PubMed Central - PubMed

Affiliation: James H. Clark Center, Stanford University, Stanford, California, United States of America ; CNC Program, Stanford University, Stanford, California, United States of America.

ABSTRACT
A longstanding goal in neuroscience has been to develop techniques for imaging the voltage dynamics of genetically defined subsets of neurons. Optical sensors of transmembrane voltage would enhance studies of neural activity in contexts ranging from individual neurons cultured in vitro to neuronal populations in awake-behaving animals. Recent progress has identified Archaerhodopsin (Arch) based sensors as a promising, genetically encoded class of fluorescent voltage indicators that can report single action potentials. Wild-type Arch exhibits sub-millisecond fluorescence responses to trans-membrane voltage, but its light-activated proton pump also responds to the imaging illumination. An Arch mutant (Arch-D95N) exhibits no photocurrent, but has a slower, ~40 ms response to voltage transients. Here we present Arch-derived voltage sensors with trafficking signals that enhance their localization to the neural membrane. We also describe Arch mutant sensors (Arch-EEN and -EEQ) that exhibit faster kinetics and greater fluorescence dynamic range than Arch-D95N, and no photocurrent at the illumination intensities normally used for imaging. We benchmarked these voltage sensors regarding their spike detection fidelity by using a signal detection theoretic framework that takes into account the experimentally measured photon shot noise and optical waveforms for single action potentials. This analysis revealed that by combining the sequence mutations and enhanced trafficking sequences, the new sensors improved the fidelity of spike detection by nearly three-fold in comparison to Arch-D95N.

No MeSH data available.


Related in: MedlinePlus