New genetically-encoded voltage-sensitive probes: ArcLight and ElectricPk

There has been almost two decades of research in the field of genetically-encoded voltage indicators (GEVIs). Early probes carried channel-based voltage sensors (e.g. derived from potassium and sodium channels) but showed poor membrane localization. Membrane localization of GEVIs was greatly improved by replacing channel-based sensors with the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSD). Despite this major improvement, the goal to record single action potentials and subthreshold electrical events in mammalian neurons with adequate temporal and spatial resolution still presents a challenge. The latest step toward this goal was taken by the Pieribone lab which came up with two Ci-VSD-based monochromatic GEVIs.

The first one, called ArcLight and published in Neuron, was obtained by combining the Ci-VSD and a super ecliptic pHluorin that carries a critical point mutation (A227D). Based on ArcLight, five probes were engineered with super ecliptic pHluorin A227D relocated closer to the S4 domain of the Ci-VSD, after amino acids Q239, M240, K241, A242, or S243. Out of these 5 variants, ArcLight A242 emerged as the best, exhibiting -35% change in ΔF/F in response to 100mV depolarizing steps in HEK293 cells and up to -5% change in ΔF/F for single action potentials in neurons. ArcLight A242 however, won’t amaze with its kinetics which remains in the 10 ms range like most of its predecessors.

Detection of Subthreshold Depolarizations in Neurons with the ArcLight Q239 Probe. (A) Sample traces of single trial recordings of spontaneous subthreshold and action potential activity from the neuron shown in (B). All optical traces have double exponential subtraction of the bleach and are low pass filtered with a Kaiser-Bessel 30 filter (200 Hz cut off). The arrowheads indicate the depolarizations (likely EPSPs) that triggered visible changes in the optical recordings.(B) An 80 × 80 depixelated image of the neuron presented in (A). Scale bar: 50 μm.

The second probe, called ElectricPk and published in PLOS ONE, succeeds where ArcLight fails (and reversely). ElectricPk is a fusion between Ci-VSD and a circularly permutated eGFP (cp-eGFP). ElectricPk was identified by screening many Ci-VSD::cp-eGFP constructs with different fusion sites and Ci-VSD truncations. While two constructs exhibited a slow on and off rate (τ~69 ms), 13 constructs had on and off rates that were dominated by extremely fast components (τ~2 ms). Although ElectricPk is the probe with the greatest response magnitude amongst these 13 variants, its fluorescence change remains modest (around 1.5% ΔF/F per 100 mV). Still, ElectricPk was able to report individual action potentials in cultured hippocampal neurons, even in single trials, through a modest (0.7% ΔF/F) but sharp fluorescent change.

Detection of action potentials in hippocampal neurons in vitro using ElectricPK. A) Wide field image of an in vitro hippocampal neuron expressing ElectricPk. Bar = 15 µm. B) Single (light red trace) and averaged (red trace) optical response to action potentials evoked in the neuron seen in (A) taken using wide field microscopy and a RedShirtImaging NeuroCCD camera. The red trace is an average of 32 action potentials. All responses captured at 2000 fps. C) Fluorescence change (light red trace-unfiltered, red trace-filtered) to a single train of evoked action potentials recorded from an in vitro hippocampal neuron expressing ElectricPk. Lower black trace is the voltage recording made from the patch electrode. All fluorescence traces are bleach subtracted and where indicated, low pass filtered (Bessel) at 350 Hz. From Barnett et al, 2012.

These recent results clearly raise the bar in the field of GEVI engineering, with great improvements in both dynamic range and speed, although obtained separately in two different probes. ArcLight and ElectricPk represent a significant advance towards the dream voltage-sensitive probe affording high fidelity optical recordings of neuronal activity.

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