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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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Imaging neural activity in current-clamp from cortical slices and dissociated hippocampal cultures. (a) Fluorescence responses of ASAP1 (left) and ArcLight Q239 (right) to spontaneous subthreshold potentials and APs in cultured hippocampal neurons. From cell to cell, ASAP1 mean fluorescence responses ranged from −4.8 to −8.1 %, averaging −6.3 ± 0.6 % (n = 6 neurons from 5 litters, ≥ 10 APs per neuron). Arrow, AP not detected by ArcLight Q239. Additional examples are in Supplementary Figure 8. (b) ASAP1 followed a spontaneous AP train in a cultured hippocampal neuron (ΔF/F = −6.2 ± 0.5% mean ± SEM, n = 10 APs). Spontaneous bursts are rare events in cultured neurons and we made this observation only a single time in all our recordings. (c) ASAP1 responses to spontaneous activity in a cultured hippocampal neuron at the beginning (top) and end (bottom) of 15 min of continuous illumination (0.036 mW/mm2). Similar observations were made in 4 neurons from 3 litters; additional examples are shown in Supplementary Fig. 10. (d) In an acute cortical slice from a mouse brain transfected in utero, ASAP1 produced large responses to individual current-induced APs in a Layer-5 pyramidal cell (ΔF/F = −6.2 ± 0.2% mean ± sem, n = 10 spikes; single observation). (e) ASAP1 tracked APs and subthreshold depolarizations in a Layer-2/3 neuron injected with current pulses at 25 Hz. ΔF/F = −1.5 to −3.2 %, across 98 APs total from 4 neurons, each from a different slice from the same animal. All traces are from single trials, without filtering (a,b,d), with LOWESS smoothing (c), or with a 100-Hz 4th-order low-pass Butterworth filter (e).
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Figure 4: Imaging neural activity in current-clamp from cortical slices and dissociated hippocampal cultures. (a) Fluorescence responses of ASAP1 (left) and ArcLight Q239 (right) to spontaneous subthreshold potentials and APs in cultured hippocampal neurons. From cell to cell, ASAP1 mean fluorescence responses ranged from −4.8 to −8.1 %, averaging −6.3 ± 0.6 % (n = 6 neurons from 5 litters, ≥ 10 APs per neuron). Arrow, AP not detected by ArcLight Q239. Additional examples are in Supplementary Figure 8. (b) ASAP1 followed a spontaneous AP train in a cultured hippocampal neuron (ΔF/F = −6.2 ± 0.5% mean ± SEM, n = 10 APs). Spontaneous bursts are rare events in cultured neurons and we made this observation only a single time in all our recordings. (c) ASAP1 responses to spontaneous activity in a cultured hippocampal neuron at the beginning (top) and end (bottom) of 15 min of continuous illumination (0.036 mW/mm2). Similar observations were made in 4 neurons from 3 litters; additional examples are shown in Supplementary Fig. 10. (d) In an acute cortical slice from a mouse brain transfected in utero, ASAP1 produced large responses to individual current-induced APs in a Layer-5 pyramidal cell (ΔF/F = −6.2 ± 0.2% mean ± sem, n = 10 spikes; single observation). (e) ASAP1 tracked APs and subthreshold depolarizations in a Layer-2/3 neuron injected with current pulses at 25 Hz. ΔF/F = −1.5 to −3.2 %, across 98 APs total from 4 neurons, each from a different slice from the same animal. All traces are from single trials, without filtering (a,b,d), with LOWESS smoothing (c), or with a 100-Hz 4th-order low-pass Butterworth filter (e).

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)

Imaging neural activity in current-clamp from cortical slices and dissociated hippocampal cultures. (a) Fluorescence responses of ASAP1 (left) and ArcLight Q239 (right) to spontaneous subthreshold potentials and APs in cultured hippocampal neurons. From cell to cell, ASAP1 mean fluorescence responses ranged from −4.8 to −8.1 %, averaging −6.3 ± 0.6 % (n = 6 neurons from 5 litters, ≥ 10 APs per neuron). Arrow, AP not detected by ArcLight Q239. Additional examples are in Supplementary Figure 8. (b) ASAP1 followed a spontaneous AP train in a cultured hippocampal neuron (ΔF/F = −6.2 ± 0.5% mean ± SEM, n = 10 APs). Spontaneous bursts are rare events in cultured neurons and we made this observation only a single time in all our recordings. (c) ASAP1 responses to spontaneous activity in a cultured hippocampal neuron at the beginning (top) and end (bottom) of 15 min of continuous illumination (0.036 mW/mm2). Similar observations were made in 4 neurons from 3 litters; additional examples are shown in Supplementary Fig. 10. (d) In an acute cortical slice from a mouse brain transfected in utero, ASAP1 produced large responses to individual current-induced APs in a Layer-5 pyramidal cell (ΔF/F = −6.2 ± 0.2% mean ± sem, n = 10 spikes; single observation). (e) ASAP1 tracked APs and subthreshold depolarizations in a Layer-2/3 neuron injected with current pulses at 25 Hz. ΔF/F = −1.5 to −3.2 %, across 98 APs total from 4 neurons, each from a different slice from the same animal. All traces are from single trials, without filtering (a,b,d), with LOWESS smoothing (c), or with a 100-Hz 4th-order low-pass Butterworth filter (e).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Imaging neural activity in current-clamp from cortical slices and dissociated hippocampal cultures. (a) Fluorescence responses of ASAP1 (left) and ArcLight Q239 (right) to spontaneous subthreshold potentials and APs in cultured hippocampal neurons. From cell to cell, ASAP1 mean fluorescence responses ranged from −4.8 to −8.1 %, averaging −6.3 ± 0.6 % (n = 6 neurons from 5 litters, ≥ 10 APs per neuron). Arrow, AP not detected by ArcLight Q239. Additional examples are in Supplementary Figure 8. (b) ASAP1 followed a spontaneous AP train in a cultured hippocampal neuron (ΔF/F = −6.2 ± 0.5% mean ± SEM, n = 10 APs). Spontaneous bursts are rare events in cultured neurons and we made this observation only a single time in all our recordings. (c) ASAP1 responses to spontaneous activity in a cultured hippocampal neuron at the beginning (top) and end (bottom) of 15 min of continuous illumination (0.036 mW/mm2). Similar observations were made in 4 neurons from 3 litters; additional examples are shown in Supplementary Fig. 10. (d) In an acute cortical slice from a mouse brain transfected in utero, ASAP1 produced large responses to individual current-induced APs in a Layer-5 pyramidal cell (ΔF/F = −6.2 ± 0.2% mean ± sem, n = 10 spikes; single observation). (e) ASAP1 tracked APs and subthreshold depolarizations in a Layer-2/3 neuron injected with current pulses at 25 Hz. ΔF/F = −1.5 to −3.2 %, across 98 APs total from 4 neurons, each from a different slice from the same animal. All traces are from single trials, without filtering (a,b,d), with LOWESS smoothing (c), or with a 100-Hz 4th-order low-pass Butterworth filter (e).
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