St-Pierre F et al. (2014), High-fidelity optical reporting of neuronal ele...

XB-ART-49798

Nat Neurosci 2014 Jun 01;176:884-9. doi: 10.1038/nn.3709.

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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 .

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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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Species referenced: Xenopus laevis
Genes referenced: asap1 pnma2 sh2b2

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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 Â± 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.
	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, â5 mV waveform; p = 0.0005, 0.008, 0.007 and 0.003 for the â5, â10, â15 and â20mV 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) ASAP1âs 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.
	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 â4.8 to â8.1 %, averaging â6.3 Â± 0.6 % (n = 6 neurons from 5 litters, â¥ 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 (ÎF/F = â6.2 Â± 0.5% mean Â± 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 (ÎF/F = â6.2 Â± 0.2% mean Â± 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. ÎF/F = â1.5 to â3.2 %, 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).

References [+] :

Akemann, Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein. 2012, Pubmed, Xenbase