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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 AP trains in voltage-clamped HEK293A cells. (a) ASAP1 followed 200-Hz trains of AP waveforms, while ArcLight Q239 followed trains of 30 Hz but not 100 Hz or 200 Hz. For each frequency, simulated trains of APs (2.0-ms FWHM, 75-mV peak amplitude) were applied for 1 second. Traces shown are the fluorescence response to 5 AP waveforms at 500 ms from the start of each train. (b) We quantified the frequency response of ASAP1 and ArcLight-Q239 to 100 Hz and 200 Hz simulated spike trains described above. We first estimated the power spectra density of the response using fast Fourier transforms. For 100 Hz trains, we quantified the amplitude of the power density peak at 100 Hz; correspondingly, we quantified the amplitude of the 200 Hz peak for 200 Hz spike trains. Consistent with subpanel (a) (bottom row) ArcLight Q239 produced little or no response at these frequencies, with mean peak amplitudes of 0.012 ± 0.001 (100 Hz) and < 0.001 (200 Hz). ASAP1 showed greater mean response amplitudes to both 100 Hz and 200 Hz simulated AP trains (100 Hz, p = 0.021; 200 Hz, p = 0.031). Differences are statistically significant following Holm-Bonferroni correction for multiple comparisons. For each train, n = 5 HEK293A cells per construct. Error bars, SEM.
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Figure 2: Monitoring simulated AP trains in voltage-clamped HEK293A cells. (a) ASAP1 followed 200-Hz trains of AP waveforms, while ArcLight Q239 followed trains of 30 Hz but not 100 Hz or 200 Hz. For each frequency, simulated trains of APs (2.0-ms FWHM, 75-mV peak amplitude) were applied for 1 second. Traces shown are the fluorescence response to 5 AP waveforms at 500 ms from the start of each train. (b) We quantified the frequency response of ASAP1 and ArcLight-Q239 to 100 Hz and 200 Hz simulated spike trains described above. We first estimated the power spectra density of the response using fast Fourier transforms. For 100 Hz trains, we quantified the amplitude of the power density peak at 100 Hz; correspondingly, we quantified the amplitude of the 200 Hz peak for 200 Hz spike trains. Consistent with subpanel (a) (bottom row) ArcLight Q239 produced little or no response at these frequencies, with mean peak amplitudes of 0.012 ± 0.001 (100 Hz) and < 0.001 (200 Hz). ASAP1 showed greater mean response amplitudes to both 100 Hz and 200 Hz simulated AP trains (100 Hz, p = 0.021; 200 Hz, p = 0.031). Differences are statistically significant following Holm-Bonferroni correction for multiple comparisons. For each train, n = 5 HEK293A cells per construct. Error bars, SEM.

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 AP trains in voltage-clamped HEK293A cells. (a) ASAP1 followed 200-Hz trains of AP waveforms, while ArcLight Q239 followed trains of 30 Hz but not 100 Hz or 200 Hz. For each frequency, simulated trains of APs (2.0-ms FWHM, 75-mV peak amplitude) were applied for 1 second. Traces shown are the fluorescence response to 5 AP waveforms at 500 ms from the start of each train. (b) We quantified the frequency response of ASAP1 and ArcLight-Q239 to 100 Hz and 200 Hz simulated spike trains described above. We first estimated the power spectra density of the response using fast Fourier transforms. For 100 Hz trains, we quantified the amplitude of the power density peak at 100 Hz; correspondingly, we quantified the amplitude of the 200 Hz peak for 200 Hz spike trains. Consistent with subpanel (a) (bottom row) ArcLight Q239 produced little or no response at these frequencies, with mean peak amplitudes of 0.012 ± 0.001 (100 Hz) and < 0.001 (200 Hz). ASAP1 showed greater mean response amplitudes to both 100 Hz and 200 Hz simulated AP trains (100 Hz, p = 0.021; 200 Hz, p = 0.031). Differences are statistically significant following Holm-Bonferroni correction for multiple comparisons. For each train, n = 5 HEK293A cells per construct. Error bars, SEM.
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Figure 2: Monitoring simulated AP trains in voltage-clamped HEK293A cells. (a) ASAP1 followed 200-Hz trains of AP waveforms, while ArcLight Q239 followed trains of 30 Hz but not 100 Hz or 200 Hz. For each frequency, simulated trains of APs (2.0-ms FWHM, 75-mV peak amplitude) were applied for 1 second. Traces shown are the fluorescence response to 5 AP waveforms at 500 ms from the start of each train. (b) We quantified the frequency response of ASAP1 and ArcLight-Q239 to 100 Hz and 200 Hz simulated spike trains described above. We first estimated the power spectra density of the response using fast Fourier transforms. For 100 Hz trains, we quantified the amplitude of the power density peak at 100 Hz; correspondingly, we quantified the amplitude of the 200 Hz peak for 200 Hz spike trains. Consistent with subpanel (a) (bottom row) ArcLight Q239 produced little or no response at these frequencies, with mean peak amplitudes of 0.012 ± 0.001 (100 Hz) and < 0.001 (200 Hz). ASAP1 showed greater mean response amplitudes to both 100 Hz and 200 Hz simulated AP trains (100 Hz, p = 0.021; 200 Hz, p = 0.031). Differences are statistically significant following Holm-Bonferroni correction for multiple comparisons. For each train, n = 5 HEK293A cells per construct. Error bars, SEM.
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