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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

Bicistronic constructs expressing Arch-EEQ with either ChR2 or GCaMP5 enabled voltage imaging in combination with either optogenetic control or calcium imaging.(a) Schematic of the Arch-2A-hChR2 construct. (b) The Arch-2A-hChR2 construct enabled all-optical stimulation and readout of neurons. We imaged neurons transfected with the Arch-2A-hChR2 construct and simultaneously measured the transmembrane potential (black trace) and Arch fluorescence (blue trace). Photoexcitation of ChR2 (horizontal cyan bars; λ = 488 nm, 4 mW/mm2) elicited spikes in both the optical and electrical measurements. (c) Schematic of the Arch-2A-GCaMP5 construct. (d) We observed neurons transfected with the Arch-2A-GCaMP5 construct by simultaneously imaging GCaMP5 fluorescence (green channel, cytosol localized) and Arch fluorescence (red channel, membrane localized). Scale bar is 20 µm. (e) The Arch-2A-GCaMP5 construct enabled simultaneous measurements of voltage (blue trace) and intracellular calcium (green trace). Although action potentials were apparent in both the Arch and GCaMP5 signals (black triangular markers in the GCaMP5 trace; λ = 488 nm excitation, I = 10 mW/mm2), Arch signals were far superior in reporting brief, sub-threshold depolarizations (orange circles in the Arch trace). The fluorescence imaging rate was 440 Hz, and the illumination intensity for Arch excitation (λ = 633 nm excitation) was 1400 mW/mm2 for all panels.
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pone-0066959-g005: Bicistronic constructs expressing Arch-EEQ with either ChR2 or GCaMP5 enabled voltage imaging in combination with either optogenetic control or calcium imaging.(a) Schematic of the Arch-2A-hChR2 construct. (b) The Arch-2A-hChR2 construct enabled all-optical stimulation and readout of neurons. We imaged neurons transfected with the Arch-2A-hChR2 construct and simultaneously measured the transmembrane potential (black trace) and Arch fluorescence (blue trace). Photoexcitation of ChR2 (horizontal cyan bars; λ = 488 nm, 4 mW/mm2) elicited spikes in both the optical and electrical measurements. (c) Schematic of the Arch-2A-GCaMP5 construct. (d) We observed neurons transfected with the Arch-2A-GCaMP5 construct by simultaneously imaging GCaMP5 fluorescence (green channel, cytosol localized) and Arch fluorescence (red channel, membrane localized). Scale bar is 20 µm. (e) The Arch-2A-GCaMP5 construct enabled simultaneous measurements of voltage (blue trace) and intracellular calcium (green trace). Although action potentials were apparent in both the Arch and GCaMP5 signals (black triangular markers in the GCaMP5 trace; λ = 488 nm excitation, I = 10 mW/mm2), Arch signals were far superior in reporting brief, sub-threshold depolarizations (orange circles in the Arch trace). The fluorescence imaging rate was 440 Hz, and the illumination intensity for Arch excitation (λ = 633 nm excitation) was 1400 mW/mm2 for all panels.

Mentions: First, we made an Arch-EEQ-2A-hChR2 construct, to enable simultaneous optical control and readout of neuronal voltage (Figure 5a). Under similar illumination as above, we simultaneously imaged the Arch sensor (633 nm excitation; 1400 mW/mm2) and depolarized the neuron (cyan bars; 488 nm excitation; 4 mW/mm2) (Figure 5b). Electrical recordings confirmed that action potentials occurred during the cyan stimulation. Second, we made an Arch-EEQ-2A-GCaMP5 construct, for simultaneous readouts of a neuron’s voltage and calcium dynamics (Figure 5c). Arch was membrane localized, whereas GCaMP5 expression was cytosolic, as expected with this construct (Figure 5d). This segregated localization pattern might facilitate separation of signals from the two fluorescence channels. We triggered action potentials by injecting brief current pulses and observed the ensuing voltage and calcium dynamics concurrently (Figure 5e). Since some of the current pulses failed to initiate action potentials, these induced only sub-threshold voltage depolarizations in the membrane potential (Methods). The Arch fluorescence trace reported these sub-threshold events (orange markers), but the GCaMP5 fluorescence trace did not.


Enhanced Archaerhodopsin Fluorescent Protein Voltage Indicators.

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

Bicistronic constructs expressing Arch-EEQ with either ChR2 or GCaMP5 enabled voltage imaging in combination with either optogenetic control or calcium imaging.(a) Schematic of the Arch-2A-hChR2 construct. (b) The Arch-2A-hChR2 construct enabled all-optical stimulation and readout of neurons. We imaged neurons transfected with the Arch-2A-hChR2 construct and simultaneously measured the transmembrane potential (black trace) and Arch fluorescence (blue trace). Photoexcitation of ChR2 (horizontal cyan bars; λ = 488 nm, 4 mW/mm2) elicited spikes in both the optical and electrical measurements. (c) Schematic of the Arch-2A-GCaMP5 construct. (d) We observed neurons transfected with the Arch-2A-GCaMP5 construct by simultaneously imaging GCaMP5 fluorescence (green channel, cytosol localized) and Arch fluorescence (red channel, membrane localized). Scale bar is 20 µm. (e) The Arch-2A-GCaMP5 construct enabled simultaneous measurements of voltage (blue trace) and intracellular calcium (green trace). Although action potentials were apparent in both the Arch and GCaMP5 signals (black triangular markers in the GCaMP5 trace; λ = 488 nm excitation, I = 10 mW/mm2), Arch signals were far superior in reporting brief, sub-threshold depolarizations (orange circles in the Arch trace). The fluorescence imaging rate was 440 Hz, and the illumination intensity for Arch excitation (λ = 633 nm excitation) was 1400 mW/mm2 for all panels.
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Related In: Results  -  Collection

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pone-0066959-g005: Bicistronic constructs expressing Arch-EEQ with either ChR2 or GCaMP5 enabled voltage imaging in combination with either optogenetic control or calcium imaging.(a) Schematic of the Arch-2A-hChR2 construct. (b) The Arch-2A-hChR2 construct enabled all-optical stimulation and readout of neurons. We imaged neurons transfected with the Arch-2A-hChR2 construct and simultaneously measured the transmembrane potential (black trace) and Arch fluorescence (blue trace). Photoexcitation of ChR2 (horizontal cyan bars; λ = 488 nm, 4 mW/mm2) elicited spikes in both the optical and electrical measurements. (c) Schematic of the Arch-2A-GCaMP5 construct. (d) We observed neurons transfected with the Arch-2A-GCaMP5 construct by simultaneously imaging GCaMP5 fluorescence (green channel, cytosol localized) and Arch fluorescence (red channel, membrane localized). Scale bar is 20 µm. (e) The Arch-2A-GCaMP5 construct enabled simultaneous measurements of voltage (blue trace) and intracellular calcium (green trace). Although action potentials were apparent in both the Arch and GCaMP5 signals (black triangular markers in the GCaMP5 trace; λ = 488 nm excitation, I = 10 mW/mm2), Arch signals were far superior in reporting brief, sub-threshold depolarizations (orange circles in the Arch trace). The fluorescence imaging rate was 440 Hz, and the illumination intensity for Arch excitation (λ = 633 nm excitation) was 1400 mW/mm2 for all panels.
Mentions: First, we made an Arch-EEQ-2A-hChR2 construct, to enable simultaneous optical control and readout of neuronal voltage (Figure 5a). Under similar illumination as above, we simultaneously imaged the Arch sensor (633 nm excitation; 1400 mW/mm2) and depolarized the neuron (cyan bars; 488 nm excitation; 4 mW/mm2) (Figure 5b). Electrical recordings confirmed that action potentials occurred during the cyan stimulation. Second, we made an Arch-EEQ-2A-GCaMP5 construct, for simultaneous readouts of a neuron’s voltage and calcium dynamics (Figure 5c). Arch was membrane localized, whereas GCaMP5 expression was cytosolic, as expected with this construct (Figure 5d). This segregated localization pattern might facilitate separation of signals from the two fluorescence channels. We triggered action potentials by injecting brief current pulses and observed the ensuing voltage and calcium dynamics concurrently (Figure 5e). Since some of the current pulses failed to initiate action potentials, these induced only sub-threshold voltage depolarizations in the membrane potential (Methods). The Arch fluorescence trace reported these sub-threshold events (orange markers), but the GCaMP5 fluorescence trace did not.

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