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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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ASAP1 design and voltage response characteristics. (a) ASAP1 is a circularly permuted GFP inserted into the extracellular S3-S4 loop of a voltage-sensing domain. Depolarization leads to decreased fluorescence. (b) ASAP1 was localized to the plasma membrane in a 12-day-in-vitro dissociated rat hippocampal neuron imaged by confocal microscopy (top) and in a fixed brain slice from an 8-week-old mouse transfected in utero and imaged by two-photon microscopy (bottom). Right panels show details from the left panels. Scale bar, 10 μm. Quantification of membrane localization in 22 neurons is in Supplementary Fig. 5. (c) ASAP1 responses in a representative HEK293A cell (top) to voltage steps from −120 to 50 mV (bottom). Responses were measured at 5-ms intervals and were normalized to fluorescence at the −70 mV holding potential. (d) Mean ASAP1 response to transmembrane voltage in HEK293A cells (n = 10 cells). Error bars are standard error of the mean (SEM). (e) Comparison of activation and inactivation kinetics of ASAP1 (n = 4 cells) and ArcLight Q239 (n = 6) in HEK293A cells. Numbers are mean ± standard error of the mean. (f) Comparison of ASAP1 and ArcLight Q239 responses to representative single trial recordings of action potentials (APs) induced by current injection in cultured hippocampal neurons. AP full widths at half-maximum (FWHM) of the voltage traces (top) were 3.3 and 3.6 ms for ASAP1- and ArcLight Q239-expressing neurons, respectively. The corresponding FWHM of the fluorescence responses (bottom) were 3.7 ms and 6.5 ms for ASAP1 and ArcLight Q239, respectively. (g) ASAP1 produces larger responses to current-triggered APs in cultured hippocampal neurons than ArcLight Q239 (p = 0.001, n = 5 neurons from 3 litters for each sensor). Each data point is the average response of an individual neuron over 12–25 APs per neuron (91 APs total for ASAP1 and 87 APs total for ArcLight Q239). For each sensor, the mean response over all tested neurons is depicted using a horizontal bar.
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Figure 1: ASAP1 design and voltage response characteristics. (a) ASAP1 is a circularly permuted GFP inserted into the extracellular S3-S4 loop of a voltage-sensing domain. Depolarization leads to decreased fluorescence. (b) ASAP1 was localized to the plasma membrane in a 12-day-in-vitro dissociated rat hippocampal neuron imaged by confocal microscopy (top) and in a fixed brain slice from an 8-week-old mouse transfected in utero and imaged by two-photon microscopy (bottom). Right panels show details from the left panels. Scale bar, 10 μm. Quantification of membrane localization in 22 neurons is in Supplementary Fig. 5. (c) ASAP1 responses in a representative HEK293A cell (top) to voltage steps from −120 to 50 mV (bottom). Responses were measured at 5-ms intervals and were normalized to fluorescence at the −70 mV holding potential. (d) Mean ASAP1 response to transmembrane voltage in HEK293A cells (n = 10 cells). Error bars are standard error of the mean (SEM). (e) Comparison of activation and inactivation kinetics of ASAP1 (n = 4 cells) and ArcLight Q239 (n = 6) in HEK293A cells. Numbers are mean ± standard error of the mean. (f) Comparison of ASAP1 and ArcLight Q239 responses to representative single trial recordings of action potentials (APs) induced by current injection in cultured hippocampal neurons. AP full widths at half-maximum (FWHM) of the voltage traces (top) were 3.3 and 3.6 ms for ASAP1- and ArcLight Q239-expressing neurons, respectively. The corresponding FWHM of the fluorescence responses (bottom) were 3.7 ms and 6.5 ms for ASAP1 and ArcLight Q239, respectively. (g) ASAP1 produces larger responses to current-triggered APs in cultured hippocampal neurons than ArcLight Q239 (p = 0.001, n = 5 neurons from 3 litters for each sensor). Each data point is the average response of an individual neuron over 12–25 APs per neuron (91 APs total for ASAP1 and 87 APs total for ArcLight Q239). For each sensor, the mean response over all tested neurons is depicted using a horizontal bar.

Mentions: We obtained several protein constructs comprising cpGFP inserted into GgVSD that were well expressed at the plasma membrane of HEK293A human embryonic kidney cells (Supplementary Fig. 1b) and showed a fluorescence decrease in response to membrane depolarization (Supplementary Fig. 1c). Conveniently, because these construct were in their higher fluorescence state at resting membrane potentials, no additional fluorescent protein marker was required to detect transfected cells. Beginning with the brightest variant, where cpGFP was inserted between residues 147 and 148 of GgVSD, we tested substitutions of various fluorescent proteins (Supplementary Fig. 1d), and found that the OPT variant of circularly permuted superfolder GFP26 (cpsfGFP-OPT) improved both brightness and dynamic range while maintaining efficient expression at the membrane. We named this protein, whose basic design is depicted in Fig. 1a, Accelerated Sensor of Action Potentials 1 (ASAP1).


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)

ASAP1 design and voltage response characteristics. (a) ASAP1 is a circularly permuted GFP inserted into the extracellular S3-S4 loop of a voltage-sensing domain. Depolarization leads to decreased fluorescence. (b) ASAP1 was localized to the plasma membrane in a 12-day-in-vitro dissociated rat hippocampal neuron imaged by confocal microscopy (top) and in a fixed brain slice from an 8-week-old mouse transfected in utero and imaged by two-photon microscopy (bottom). Right panels show details from the left panels. Scale bar, 10 μm. Quantification of membrane localization in 22 neurons is in Supplementary Fig. 5. (c) ASAP1 responses in a representative HEK293A cell (top) to voltage steps from −120 to 50 mV (bottom). Responses were measured at 5-ms intervals and were normalized to fluorescence at the −70 mV holding potential. (d) Mean ASAP1 response to transmembrane voltage in HEK293A cells (n = 10 cells). Error bars are standard error of the mean (SEM). (e) Comparison of activation and inactivation kinetics of ASAP1 (n = 4 cells) and ArcLight Q239 (n = 6) in HEK293A cells. Numbers are mean ± standard error of the mean. (f) Comparison of ASAP1 and ArcLight Q239 responses to representative single trial recordings of action potentials (APs) induced by current injection in cultured hippocampal neurons. AP full widths at half-maximum (FWHM) of the voltage traces (top) were 3.3 and 3.6 ms for ASAP1- and ArcLight Q239-expressing neurons, respectively. The corresponding FWHM of the fluorescence responses (bottom) were 3.7 ms and 6.5 ms for ASAP1 and ArcLight Q239, respectively. (g) ASAP1 produces larger responses to current-triggered APs in cultured hippocampal neurons than ArcLight Q239 (p = 0.001, n = 5 neurons from 3 litters for each sensor). Each data point is the average response of an individual neuron over 12–25 APs per neuron (91 APs total for ASAP1 and 87 APs total for ArcLight Q239). For each sensor, the mean response over all tested neurons is depicted using a horizontal bar.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: ASAP1 design and voltage response characteristics. (a) ASAP1 is a circularly permuted GFP inserted into the extracellular S3-S4 loop of a voltage-sensing domain. Depolarization leads to decreased fluorescence. (b) ASAP1 was localized to the plasma membrane in a 12-day-in-vitro dissociated rat hippocampal neuron imaged by confocal microscopy (top) and in a fixed brain slice from an 8-week-old mouse transfected in utero and imaged by two-photon microscopy (bottom). Right panels show details from the left panels. Scale bar, 10 μm. Quantification of membrane localization in 22 neurons is in Supplementary Fig. 5. (c) ASAP1 responses in a representative HEK293A cell (top) to voltage steps from −120 to 50 mV (bottom). Responses were measured at 5-ms intervals and were normalized to fluorescence at the −70 mV holding potential. (d) Mean ASAP1 response to transmembrane voltage in HEK293A cells (n = 10 cells). Error bars are standard error of the mean (SEM). (e) Comparison of activation and inactivation kinetics of ASAP1 (n = 4 cells) and ArcLight Q239 (n = 6) in HEK293A cells. Numbers are mean ± standard error of the mean. (f) Comparison of ASAP1 and ArcLight Q239 responses to representative single trial recordings of action potentials (APs) induced by current injection in cultured hippocampal neurons. AP full widths at half-maximum (FWHM) of the voltage traces (top) were 3.3 and 3.6 ms for ASAP1- and ArcLight Q239-expressing neurons, respectively. The corresponding FWHM of the fluorescence responses (bottom) were 3.7 ms and 6.5 ms for ASAP1 and ArcLight Q239, respectively. (g) ASAP1 produces larger responses to current-triggered APs in cultured hippocampal neurons than ArcLight Q239 (p = 0.001, n = 5 neurons from 3 litters for each sensor). Each data point is the average response of an individual neuron over 12–25 APs per neuron (91 APs total for ASAP1 and 87 APs total for ArcLight Q239). For each sensor, the mean response over all tested neurons is depicted using a horizontal bar.
Mentions: We obtained several protein constructs comprising cpGFP inserted into GgVSD that were well expressed at the plasma membrane of HEK293A human embryonic kidney cells (Supplementary Fig. 1b) and showed a fluorescence decrease in response to membrane depolarization (Supplementary Fig. 1c). Conveniently, because these construct were in their higher fluorescence state at resting membrane potentials, no additional fluorescent protein marker was required to detect transfected cells. Beginning with the brightest variant, where cpGFP was inserted between residues 147 and 148 of GgVSD, we tested substitutions of various fluorescent proteins (Supplementary Fig. 1d), and found that the OPT variant of circularly permuted superfolder GFP26 (cpsfGFP-OPT) improved both brightness and dynamic range while maintaining efficient expression at the membrane. We named this protein, whose basic design is depicted in Fig. 1a, Accelerated Sensor of Action Potentials 1 (ASAP1).

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