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High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor.

St-Pierre F, Marshall JD, Yang Y, Gong Y, Schnitzer MJ, Lin MZ - Nat. Neurosci. (2014)

Bottom Line: Accurate optical reporting of electrical activity in genetically defined neuronal populations is a long-standing goal in neuroscience.We developed Accelerated Sensor of Action Potentials 1 (ASAP1), a voltage sensor design in which a circularly permuted green fluorescent protein is inserted in an extracellular loop of a voltage-sensing domain, rendering fluorescence responsive to membrane potential.With a favorable combination of brightness, dynamic range and speed, ASAP1 enables continuous monitoring of membrane potential in neurons at kilohertz frame rates using standard epifluorescence microscopy.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Pediatrics, Stanford University, Stanford, California, USA.

ABSTRACT
Accurate optical reporting of electrical activity in genetically defined neuronal populations is a long-standing goal in neuroscience. We developed Accelerated Sensor of Action Potentials 1 (ASAP1), a voltage sensor design in which a circularly permuted green fluorescent protein is inserted in an extracellular loop of a voltage-sensing domain, rendering fluorescence responsive to membrane potential. ASAP1 demonstrated on and off kinetics of ∼ 2 ms, reliably detected single action potentials and subthreshold potential changes, and tracked trains of action potential waveforms up to 200 Hz in single trials. With a favorable combination of brightness, dynamic range and speed, ASAP1 enables continuous monitoring of membrane potential in neurons at kilohertz frame rates using standard epifluorescence microscopy.

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Monitoring simulated hyperpolarizations and subthreshold potentials in voltage-clamped neurons. (a) ASAP1 can detect subthreshold potential and hyperpolarization waveforms in cultured hippocampal neurons. Subthreshold depolarizations and hyperpolarizations have peak amplitudes of 5, 10, 15, and 20 mV, and peak full width at half maximum of 17 ms (depolarizations) and 38 ms (hyperpolarizations). (b) Quantification of the fluorescence responses to subthreshold depolarizations (top) and hyperpolarizations (bottom). Asterisks identify statistically significant differences from pairwise two-tailed t-tests adjusted for multiple comparisons using the Holm-Bonferroni method (p = 0.006, −5 mV waveform; p = 0.0005, 0.008, 0.007 and 0.003 for the −5, −10, −15 and −20mV waveforms, respectively). Multiple comparisons adjustments were performed separately for depolarizations and hyperpolarizations. n = 6 (ASAP1) and 4 (ArcLight Q239) neurons from the same litter. Error bars, SEM. (c) ASAP1’s faster kinetics allow improved resolution of a 100-Hz, three-AP waveform sequence in cultured hippocampal neurons (ASAP1, n = 8 neurons; ArcLight Q239, n = 6 neurons; all cells from same litter). Command voltage spike FWHM is 1.8 ms. Additional examples are in Supplementary Fig. 7.
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Figure 3: Monitoring simulated hyperpolarizations and subthreshold potentials in voltage-clamped neurons. (a) ASAP1 can detect subthreshold potential and hyperpolarization waveforms in cultured hippocampal neurons. Subthreshold depolarizations and hyperpolarizations have peak amplitudes of 5, 10, 15, and 20 mV, and peak full width at half maximum of 17 ms (depolarizations) and 38 ms (hyperpolarizations). (b) Quantification of the fluorescence responses to subthreshold depolarizations (top) and hyperpolarizations (bottom). Asterisks identify statistically significant differences from pairwise two-tailed t-tests adjusted for multiple comparisons using the Holm-Bonferroni method (p = 0.006, −5 mV waveform; p = 0.0005, 0.008, 0.007 and 0.003 for the −5, −10, −15 and −20mV waveforms, respectively). Multiple comparisons adjustments were performed separately for depolarizations and hyperpolarizations. n = 6 (ASAP1) and 4 (ArcLight Q239) neurons from the same litter. Error bars, SEM. (c) ASAP1’s faster kinetics allow improved resolution of a 100-Hz, three-AP waveform sequence in cultured hippocampal neurons (ASAP1, n = 8 neurons; ArcLight Q239, n = 6 neurons; all cells from same litter). Command voltage spike FWHM is 1.8 ms. Additional examples are in Supplementary Fig. 7.

Mentions: We next tested the ability of ASAP1 to track various membrane potential waveforms. In voltage-clamped HEK293A cells, ASAP1 was able to track trains of up to 200 Hz while clearly discerning individual peaks in single trials without filtering. In contrast, ArcLight Q239 traces at 100 Hz appeared flattened with an elevated baseline and poor peak discrimination (Fig. 2a, b). In cultured hippocampal pyramidal neurons, ASAP1 was able to detect subthreshold depolarizations in the form of simulated excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) of 5–20 mV amplitude, comparing favorably to ArcLight Q239 (Fig. 3a, b). Importantly, ASAP1 was able to resolve spikes superimposed on a large EPSP, whereas ArcLight Q239 was not (Fig. 3c and Supplementary Fig. 7). ASAP1 also detected spontaneous spikes, spontaneous 10-Hz bursting, and subthreshold potential changes between spikes in neurons (Fig. 4a,b and Supplementary Fig. 8). As observed previously under voltage-clamp (Fig. 3b), ArcLight Q239 failed to detect or produced minimal responses to spontaneous APs superposed on large EPSPs (Fig. 4a and Supplementary Fig. 8). ASAP1 responses to slow low-amplitude changes were disproportionally larger than to the fast component of APs (Fig. 4a); this is expected from the relative steepness of the fluorescence response near the resting potential (Fig.1d) and a slow component in the ASAP1 response (Fig. 1e).


High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor.

St-Pierre F, Marshall JD, Yang Y, Gong Y, Schnitzer MJ, Lin MZ - Nat. Neurosci. (2014)

Monitoring simulated hyperpolarizations and subthreshold potentials in voltage-clamped neurons. (a) ASAP1 can detect subthreshold potential and hyperpolarization waveforms in cultured hippocampal neurons. Subthreshold depolarizations and hyperpolarizations have peak amplitudes of 5, 10, 15, and 20 mV, and peak full width at half maximum of 17 ms (depolarizations) and 38 ms (hyperpolarizations). (b) Quantification of the fluorescence responses to subthreshold depolarizations (top) and hyperpolarizations (bottom). Asterisks identify statistically significant differences from pairwise two-tailed t-tests adjusted for multiple comparisons using the Holm-Bonferroni method (p = 0.006, −5 mV waveform; p = 0.0005, 0.008, 0.007 and 0.003 for the −5, −10, −15 and −20mV waveforms, respectively). Multiple comparisons adjustments were performed separately for depolarizations and hyperpolarizations. n = 6 (ASAP1) and 4 (ArcLight Q239) neurons from the same litter. Error bars, SEM. (c) ASAP1’s faster kinetics allow improved resolution of a 100-Hz, three-AP waveform sequence in cultured hippocampal neurons (ASAP1, n = 8 neurons; ArcLight Q239, n = 6 neurons; all cells from same litter). Command voltage spike FWHM is 1.8 ms. Additional examples are in Supplementary Fig. 7.
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Related In: Results  -  Collection

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Figure 3: Monitoring simulated hyperpolarizations and subthreshold potentials in voltage-clamped neurons. (a) ASAP1 can detect subthreshold potential and hyperpolarization waveforms in cultured hippocampal neurons. Subthreshold depolarizations and hyperpolarizations have peak amplitudes of 5, 10, 15, and 20 mV, and peak full width at half maximum of 17 ms (depolarizations) and 38 ms (hyperpolarizations). (b) Quantification of the fluorescence responses to subthreshold depolarizations (top) and hyperpolarizations (bottom). Asterisks identify statistically significant differences from pairwise two-tailed t-tests adjusted for multiple comparisons using the Holm-Bonferroni method (p = 0.006, −5 mV waveform; p = 0.0005, 0.008, 0.007 and 0.003 for the −5, −10, −15 and −20mV waveforms, respectively). Multiple comparisons adjustments were performed separately for depolarizations and hyperpolarizations. n = 6 (ASAP1) and 4 (ArcLight Q239) neurons from the same litter. Error bars, SEM. (c) ASAP1’s faster kinetics allow improved resolution of a 100-Hz, three-AP waveform sequence in cultured hippocampal neurons (ASAP1, n = 8 neurons; ArcLight Q239, n = 6 neurons; all cells from same litter). Command voltage spike FWHM is 1.8 ms. Additional examples are in Supplementary Fig. 7.
Mentions: We next tested the ability of ASAP1 to track various membrane potential waveforms. In voltage-clamped HEK293A cells, ASAP1 was able to track trains of up to 200 Hz while clearly discerning individual peaks in single trials without filtering. In contrast, ArcLight Q239 traces at 100 Hz appeared flattened with an elevated baseline and poor peak discrimination (Fig. 2a, b). In cultured hippocampal pyramidal neurons, ASAP1 was able to detect subthreshold depolarizations in the form of simulated excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) of 5–20 mV amplitude, comparing favorably to ArcLight Q239 (Fig. 3a, b). Importantly, ASAP1 was able to resolve spikes superimposed on a large EPSP, whereas ArcLight Q239 was not (Fig. 3c and Supplementary Fig. 7). ASAP1 also detected spontaneous spikes, spontaneous 10-Hz bursting, and subthreshold potential changes between spikes in neurons (Fig. 4a,b and Supplementary Fig. 8). As observed previously under voltage-clamp (Fig. 3b), ArcLight Q239 failed to detect or produced minimal responses to spontaneous APs superposed on large EPSPs (Fig. 4a and Supplementary Fig. 8). ASAP1 responses to slow low-amplitude changes were disproportionally larger than to the fast component of APs (Fig. 4a); this is expected from the relative steepness of the fluorescence response near the resting potential (Fig.1d) and a slow component in the ASAP1 response (Fig. 1e).

Bottom Line: Accurate optical reporting of electrical activity in genetically defined neuronal populations is a long-standing goal in neuroscience.We developed Accelerated Sensor of Action Potentials 1 (ASAP1), a voltage sensor design in which a circularly permuted green fluorescent protein is inserted in an extracellular loop of a voltage-sensing domain, rendering fluorescence responsive to membrane potential.With a favorable combination of brightness, dynamic range and speed, ASAP1 enables continuous monitoring of membrane potential in neurons at kilohertz frame rates using standard epifluorescence microscopy.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Pediatrics, Stanford University, Stanford, California, USA.

ABSTRACT
Accurate optical reporting of electrical activity in genetically defined neuronal populations is a long-standing goal in neuroscience. We developed Accelerated Sensor of Action Potentials 1 (ASAP1), a voltage sensor design in which a circularly permuted green fluorescent protein is inserted in an extracellular loop of a voltage-sensing domain, rendering fluorescence responsive to membrane potential. ASAP1 demonstrated on and off kinetics of ∼ 2 ms, reliably detected single action potentials and subthreshold potential changes, and tracked trains of action potential waveforms up to 200 Hz in single trials. With a favorable combination of brightness, dynamic range and speed, ASAP1 enables continuous monitoring of membrane potential in neurons at kilohertz frame rates using standard epifluorescence microscopy.

Show MeSH
Related in: MedlinePlus