Probing the neural circuit dynamics underlying behaviour would benefit greatly from improved genetically encoded voltage indicators. The proton pump Archaerhodopsin-3 (Arch), an optogenetic tool commonly used for neuronal inhibition, has been shown to emit voltage-sensitive fluorescence. Here we report two Arch variants with enhanced radiance (Archers) that in response to 655 nm light have 3–5 times increased fluorescence and 55–99 times reduced photocurrents compared with Arch WT. The most fluorescent variant, Archer1, has 25–40% fluorescence change in response to action potentials while using 9 times lower light intensity compared with other Arch-based voltage sensors.
Archer1 is capable of wavelength-specific functionality as a voltage sensor under red light and as an inhibitory actuator under green light. As a proof-of-concept for the application of Arch-based sensors in vivo, we show fluorescence voltage sensing in behaving Caenorhabditis elegans. Archer1’s characteristics contribute to the goal of all-optical detection and modulation of activity in neuronal networks in vivo. The study of brain circuitry encompasses three frames of reference: neuron-level spiking activity, circuit-level connectivity and systems-level behavioural output.
A pervasive goal in neuroscience is the ability to examine all three frames concurrently. Fluorescent sensors, which enable measurements of simultaneous changes in activity of specific populations of neurons, are envisioned to provide a solution,. Successful detection of both high-frequency trains of action potentials and subthreshold events in neuronal populations in vivo requires a genetically encoded voltage indicator (GEVI) with fast kinetics, high sensitivity and high baseline fluorescence. Recent developments of genetically encoded calcium and voltage sensors, have yielded progress towards achieving this goal.
The calcium sensor family GCaMP has been used to monitor populations of neurons in intact behaving organisms. However, the detection of fast-spiking activity, subthreshold voltage changes and hyperpolarization is difficult with GCaMP due to its relatively slow kinetics and reliance on calcium, a secondary messenger, flux into the cell. Newer iterations of voltage-sensitive fluorescent proteins based on fusions with circularly permuted GFP, for example, ASAP1 (ref. ), improve on both the speed and sensitivity of previous sensors, for example, Arclight, but are still limited by the ability to be combined with optogenetic actuators. This spectral overlap prohibits the combined use of these sensors with opsins for all-optical electrophysiology. Currently available sensors are not able to meet all of the needs for optical imaging of activity in vivo, calling for continued efforts to evolve GEVIs.Archaerhodopsin-3 (Arch), a microbial rhodopsin proton pump that has recently been introduced as a fluorescent voltage sensor, is fast and sensitive but suffers from low baseline fluorescence and strong inhibitory photocurrents.
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Previous optimizations of Arch successfully reduced photocurrents, for example, Arch D95N and Arch EEQ, and increased sensitivity and speed, for example, QuasArs, but have still to enable its use in vivo. All previous in vivo voltage sensing has been accomplished using lower power of fluorescence excitation light than is possible with reported Arch variants to date. For example, Arch WT uses × 3,600 higher intensity illumination than ASAP1 (ref. The high laser power used to excite Arch fluorescence causes significant autofluorescence in intact tissue and limits its accessibility for widespread use.Here we report two Arch mutants (‘Archers’: Arch with enhanced radiance), Archer1 (D95E and T99C) and Archer2 (D95E, T99C and A225M) with improved properties for voltage sensing. These mutants exhibit high baseline fluorescence ( × 3–5 over Arch WT), large dynamic range of sensitivity (85% Δ F/ F and 60% Δ F/ F per 100 mV for Archer1 and Archer2, respectively) that is stable over long illumination times, and fast kinetics, when imaged at × 9 lower light intensity (880 mW mm −2 at 655 nm) than the most recently reported Arch variants (and × 20.5 lower than Arch WT ). We demonstrate that Archer1’s improved characteristics enable its use to monitor rapid changes in membrane voltage throughout a single neuron and throughout a population of neurons in vitro.
Although Archer1 has minimal pumping at wavelengths used for fluorescence excitation (655 nm), it maintains strong proton pumping currents at lower wavelengths (560 nm). We show that this single protein, Archer1, is a bi-functional tool that provides both voltage sensing with red light and inhibitory capabilities with green light. Finally, we demonstrate that Archer1 is capable of detecting small voltage changes in response to sensory stimulus in the context of intact multicellular organisms such as C. The combination of D95E, T99C and A225M mutations was first identified in a site-saturation mutagenesis library of the proton pump Gloeobacter violaceus rhodopsin (GR) designed to evolve for spectral shifts. Far-red shifted mutants of the GR library were then screened for fluorescence intensity in Escherichia coli, which revealed numerous hits with higher fluorescence than GR WT. The corresponding mutations found in the most intensely fluorescent variants can be transferred to the homologous residues of Arch WT and greatly improve its quantum efficiency and absolute brightness. The selected mutants were expressed in neurons to test if their improved characteristics were maintained in a mammalian system.
Characterization of two new mutant Arch voltage sensorsArch variants designed with TS and ER export domains for enhanced membrane localization ( and ) were screened in neurons for enhanced baseline fluorescence and decreased photocurrents at imaging wavelengths, compared with Arch WT. Of the Arch variants screened, Archer1 and Archer2 exhibited 5 × and 3 × increased fluorescence, respectively, over Arch WT. Archer1 and Archer2 also have × 55 and × 99 reduced photocurrents in response to 655 nm laser illumination, respectively, when compared with Arch WT ( and ). Archer1 exhibits a peak current on initial laser exposure, which then reaches a residual average steady state of 5.6 pA, while Archer2 produces no peak current, and an average steady state of 3.1 pA ( and ). Arch variants were also screened for increased voltage sensitivity and faster kinetics compared with previously reported variant Arch EEQ (ref.
Voltage sensitivity was measured as a fluorescence response to steps in membrane potential ranging from −100 mV to +50 mV. Due to Arch EEQ’s lower baseline fluorescence, its single-cell fluorescence traces show considerably more noise than those for Archer1 and Archer2. Archer1 shows the highest voltage-sensitive fluorescence, as depicted by single-cell sensitivity measurements ( and ), and by the averaged traces (, ). Facilitated by Archer1’s increased baseline fluorescence, imaging can be done with short 1 ms exposure times and at lower laser intensities (880 mW mm −2) than previously published Arch-based sensors. To characterize the stability of Archer1’s fluorescence, sensitivity was measured before and after prolonged laser illumination. Archer1 showed no reduction in voltage sensitivity over the 10–15 min timeframe measured.
( a) Quantification of Archer1 ( n=12) and Archer2 ( n=11) fluorescence compared with Arch WT ( n=13). Left—representative images of rhodopsin and fusion protein fluorescence; the published Arch EEQ–EYFP fusion is used, while all other sensors are fused to EGFP.
Right graph—summary data. Baseline rhodopsin fluorescence normalized to EGFP fluorescence. Arch EEQ not included in comparison, as it has a different fluorescent protein fusion.
Right construct—Arch-EGFP fusion vector design. Scale bar, 10 μm. ( b) Average steady-state photocurrents generated by Arch WT ( n=10) and different variants ( n=9, 10 and 9 respectively for Arch EEQ, Archer1, and Archer2) in neurons voltage clamped at V=−50 mV. Inset shows low levels of photocurrents expanded to indicate differences between variants. ( c) Fluorescent responses (imaged at 500 Hz) of single neurons expressing Arch EEQ, Archer1 and Archer2 to voltage-clamped steps in membrane potential. Neurons are held at −70 mV and stepped to voltages ranging from −100 mV to +50 mV in 10 mV increments. ( d) Sensitivity of Arch variants measured as the functional dependence of fluorescence to change in voltage.
Fluorescence changes are averaged over 1,000 ms voltage steps and plotted against voltage. Results exhibit linear dependence with R 2 values of 0.98, 0.95, and 0.99 for Archer1 ( n=10), Archer2 ( n=3) and Arch EEQ ( n=5) respectively. ( e) On/Off kinetics in response to a 100 mV step (−70 mV to +30 mV) for Archer1 ( n=10) compared with Arch WT ( n=6).%Δ F/ F for each time point is normalized to the maximum step response (%Δ F/ F averaged over the whole step) (imaged at 1,000 Hz). Laser illumination for Arch WT, Archer1 and Archer2 ( λ=655 nm; I=880 mW mm −2) is lower than that used for Arch EEQ ( λ=655 nm; I=1,500 mW mm −2). Error bars represent s.e.m. P0.05, unpaired student’s t-test. Sensitivity kinetics enables comparison across sensorsThe choice of a specific voltage sensor for a given experimental application depends on whether the sensor will yield a significant fluorescence change in response to a given voltage change within the timeframe of interest.
Traditionally, sensitivity is quantified by measuring the steady-state fluorescence change for a step in voltage, but the steady-state value does not provide information about the initial dynamics of the fluorescence response (sensor kinetics). The methods for kinetic analysis vary with different types of sensors. Following a previously used method for Arch-based sensor kinetics, we compared Archer1 with Arch WT by normalizing the fluorescent responses of each sensor during a 1 s voltage step (−70 mV to +30 mV) to the steps maximum fluorescence. These results indicate very similar kinetics between the two , without addressing Archer1’s × 35 larger change in fluorescence. The large time scale of these voltage steps is not relevant for neuronal applications. However, normalizing over a shorter time scale produces variable results depending on the time point used for normalization. A method that takes into account the sensitivity of a sensor on the time scale relevant to an action potential is necessary.
( a) Overview of the method used to quantify SKi. Step 1: averaged fluorescence responses (imaged at 500 Hz) of neurons expressing Archer1 ( n=10) to voltage-clamped steps in membrane potential.
Neurons are held at −70 mV and then stepped to voltages ranging from −100 mV to +50 mV in increments of 10 mV. Step 2: voltage sensitivity of fluorescence is plotted for each time point and a linear fit is calculated. This step assumes a linear dependence of fluorescence on voltage. Step 3: the slope for each linear fit is plotted over time. This measure allows one to calculate%Δ F/ F for a desired voltage change over any time scale. ( b) Averaged change in fluorescence due to a 100 mV step (−70 mV to +30 mV) of Archer1 ( n=10) compared with Arch WT ( n=6) shows significant differences in response magnitude ( × 25–30). To compare the kinetics of the two sensors, normalization across the step is necessary.
The maximum value within three different regions (I, II and III) is used as a normalization factor, resulting in different apparent kinetics and prompting the need for a different method for kinetic analysis. ( c) Plotting the voltage sensitivity for each time point with linear best fits for Arch EEQ ( n=5) and Archer2 ( n=3) shows a slower rise to the steady-state value than Archer1 ( n=10). ( d) Summarizing the SKi comparison of Archer1, Arch EEQ and Archer2. Inset expands the first 40 ms. Laser illumination for Arch WT, Archer1 and Archer2 ( λ=655 nm; I=880 mW mm −2) and for Arch EEQ ( λ=655 nm; I=1,500 mW mm −2). Our proposed method for analysis, sensitivity kinetics ( SKi), expands on the traditional method by providing%Δ F/ F for any given voltage change over time.
With this method, both the sensitivity and kinetics can be compared simultaneously among sensors. SKi is calculated by evaluating the slope of the fluorescence response to steps in voltage for each time point after the step’s initiation.
The sensitivity slopes are then plotted over time. Characterization of the SKi for Arch variants reveals that Archer1 produces the largest changes in fluorescence of the sensors we tested , within any timeframe. Tracking action potentials in primary neuronal culturesAction potentials were evoked in cultured rat hippocampal neurons expressing Archer1 through current injection. Archer1 fluorescence is capable of tracking action potentials in both individual processes and the cell body ( and ).
In addition, the magnitude and shape of dendritic fluorescence changes closely mimic that of the cell body in response to the same event. As predicted by the SKi, Archer1 fluorescence, with a 6 × increase in signal-to-noise ratio (SNR), more closely follows the electrical recording of action potentials than Arch EEQ at similar frequencies.
Archer1 exhibits a large percentage change in fluorescence in response to action potentials (25–40% Δ F/ F), and can track 40 Hz firing rate as well as simulated changes in membrane voltage occurring at 100 and 150 Hz (50% Δ F/ F). The ability to follow action potential throughout neurons by imaging with significantly lower laser intensity (880 mW mm −2) is enabling for monitoring voltage-sensitive fluorescence in vivo. ( a) Fluorescence of Archer1 expressing rat hippocampal neuron. Cell body and individual processes are outlined. Scale bar, 10 μm.
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( b) Fluorescence (imaged at 500 Hz) from single-trial optical and electrophysiological recordings of action potentials induced by a step current injection (800 ms, 50 pA) analysed for the color-matched somatic and dendritic areas outlined in a. ( c) Fluorescence (imaged at 500 Hz) from single-trial recordings of action potentials in neurons expressing Archer1 and Arch EEQ. Firing of 20 and 22.5 Hz, respectively is generated through a step current injection (800 ms, 50 pA) in current-clamped cells. Fluorescence change is measured in absolute terms, as opposed to a percentage change, due to the lower baseline fluorescence of Arch EEQ. ( d) Expanded regions of action potentials from c. Archer1 shows 2 × higher change in fluorescence and 6 × increase in SNR (24.03 versus 3.75) when compared with Arch EEQ, allowing it to better track action potential waveforms. Each fluorescent point is 2 ms apart.
( e) Archer1 fluorescence (imaged at 1,000 Hz) successfully tracks action potentials in cultured rat hippocampal neurons at 40 Hz: higher limit for such cultures, generated through a succession of brief, large amplitude current pulses (5 ms, 500 pA). Individual action potentials at 40 Hz show 40% change in Δ F/ F. ( f) Single-trial recording of high frequency (100 Hz and 150 Hz) voltage steps (−70 mV to +30 mV) are generated in neurons to test Archer1’s ability to detect fast trains of depolarization and hyperpolarization.
Fluorescence changes (imaged at 1,000 Hz) exhibited by Archer1 are 50% Δ F/ F for both frequencies and return near baseline between each pulse. Each fluorescent point is 1 ms apart. Laser illumination for Archer1 ( λ=655 nm; I=880 mW mm −2) and Arch EEQ ( λ=655 nm; I=1,500 mW mm −2). Fluorescence traces in b– e have undergone background subtraction and Gaussian averaging. Archer1 functions as a voltage sensor and inhibitory actuatorAll-optical electrophysiology requires an optical method for both sensing and perturbing cells. Recent work presented a construct with dual capabilities: voltage sensing and neuronal activation at distinct wavelengths through co-expression of a sensor and a light-gated channel.
Archer1 also provides two useful functionalities, both in a single protein. While minimally active with high intensity 655 nm laser illumination (880 mW mm −2), Archer1 is significantly more active at low intensity 560 nm light-emitting diode (LED) illumination (3 mW mm −2) ( × 51 at peak and × 35 at steady state). The hyperpolarizing photocurrents generated by Archer1 in response to green light successfully inhibit action potentials, while red light does not. Thus, Archer1 is capable of inducing inhibitory currents with green light and sensing activity with red. ( a) Normalized steady-state activation spectrum of Archer1 spanning wavelengths between 386–650 nm ( n=11). ( b) Currents induced by low intensity green LED illumination ( n=8, λ=560±25 nm; I=3 mW mm −2) are significantly larger than those induced by high intensity red laser illumination ( n=16, λ=655 nm; I=880 mW mm −2).
( c) Archer1 exposed to green light successfully inhibits action potentials induced by step current injections (at 20, 30 and 40 pA) when compared with non-illuminated current injections in the same cell. ( d) Action potentials induced by a 100 pA current injection (900 ms) are inhibited by a pulse of green light (300 ms; I=3 mW mm −2), while no inhibition of action potentials is observed with a pulse of red laser at the power used to excite fluorescence (300 ms; I=880 mW mm −2). In addition, with no current injection, hyperpolarization is observed with exposure to green, but not red light. Error bars represent s.e.m. Optical monitoring of cultured neuronal networksFluorescent voltage sensors should enable the detection of spiking activity across all neurons in a population. Original Arch variants require the use of high optical magnification combined with binning and heavy pixel weighing to detect modest changes in fluorescence, due to low baseline.
Until recently, these stringent imaging requirements had prevented microbial rhodopsin-based voltage sensors from being used to monitor multiple cells simultaneously. Archer1, similar to QuasAr, by virtue of its increased fluorescence and higher SKi, allows simultaneous imaging of activity for a population of cells while perturbing only one of them through current injection (, schematic). Within the same optical field, we tracked the fluorescence of three cells with different behaviours: one showed a step change (due to an induced voltage step), one had spontaneous spikes that increased concurrently with the step and one remained unchanged (, traces). ( a) Monitoring fluorescence in three Archer1 expressing cultured neurons with electrical stimulation of one cell. Cell A undergoes a voltage clamped 100 mV step and fluorescence changes in the population are measured simultaneously.
Cell A exhibits a step-like increase in fluorescence corresponding to the voltage step. Cell B, whose fluorescence indicates spontaneous firing previous to the step, shows an increase in firing rate concurrent with the voltage step in Cell A, with continued firing after the step is completed. Fluorescence of Cell C appears not responsive to the voltage step in Cell A. Asterisks indicate action potential-like changes in fluorescence (35–40% Δ F/ F increase within 10 ms). Scale bar, 20 μm. Elegans expressing Archer1 in one AWC neuron shows opsin fluorescence ( λ=655 nm; I=880 mW mm −2, 100 ms exposure) co-localizing with fused EGFP fluorescence ( λ=485±20 nm; I=0.05 mW mm −2, 100 ms exposure).
Scale bar, 20 μm. ( c) Top: behavioural paradigm: worms are stimulated with odorant (Isoamyl alcohol, IAA) for 5 min, flow is switched to buffer (S Basal) for 30 s, and then odorant flow is restored. On the same worm, a control is performed where odorant is replaced with buffer.
Bottom traces: imaging of Archer1 fluorescence (250 Hz) is performed continuously for 40 s, starting 5 s before flow switch. Averaged Δ F traces for two worms are shown. ( d) Mean fluorescence of the 4-s time window after switch shows a significant increase with stimulus compared with no-stimulus controls ( n=4 worms). Fluorescence traces imaged at λ=655 nm; I=880 mW mm −2.
Fluorescence traces in a and b have undergone background subtraction and Gaussian averaging. Error bars represent s.e.m. Optical monitoring of sensory neurons in behaving C. ElegansA major application for voltage sensors is all-optical neuronal activity monitoring in model organisms in which electrophysiological recordings are inherently difficult, for example, C. The aforementioned improved fluorescence and SKi of Archer1 have enabled us to extend its use from cultured cells to live, behaving nematodes.
To test whether Archer1 will work in C. Elegans, we examined the olfactory neuron AWC-ON (WormBase cell WBbt:0005832), one of the pair of C-type Amphid Wing cells. Previously, sensory-evoked Ca 2+ transients that were monitored using GCaMP show fluorescence increase on odour removal, which peaks within 10 s and gradually decreases over minutes post stimulation. To monitor the small voltage changes underlying this effect, we expressed Archer1 in AWC-ON, and observed fluorescence changes in response to turning off the odorant stimulus (isoamyl alcohol) in anesthetized and non-anesthetized animals. According to Kato et al., the chemosensory responses in AWC neurons are not affected by the application of cholinergic agonist. As shown in, Archer1’s fluorescence indicates that voltage transients peak within 2 s and end 10 s after turning off stimulus ( and ).
These observed fluorescence changes, which correspond to small reported changes in AWC membrane voltage, validate the sensor’s in vivo utility. A combination of results from Archer1 and GCaMP experiments can be used to better understand the dynamics of C. Elegans voltage-gated calcium channels. All-optical methods for in vivo recording will require a GEVI with fast kinetics, high sensitivity, high baseline fluorescence and compatibility with optical methods for controlling neuronal activity.
Here we report an Arch mutant, Archer1, in which these combined improvements enable the accurate tracking of action potentials at high speed, the detection of simultaneous activity within populations of neurons, wavelength-specific inhibition of neuronal activity and the real-time observation of voltage changes in response to a stimulus in live nematodes. Fluorescence measurements of Archer1 and Archer2 were achieved at lower intensity of laser illumination than has been possible in experiments using previously reported Arch variants. Reduction in excitation light intensity required for fluorescent measurements increases the accessibility of Arch-based voltage sensors and their potential use in vivo.Archer1 is an enhanced voltage sensor under red light and it also enables inhibition of action potentials under green light. Recent work has been done to generate an all-optical system for neuronal excitation and voltage sensing (Optopatch ). Archer1, on the other hand, provides the first example of a combination of wavelength-specific sensing and hyperpolarization with a single protein. This wavelength-specific bi-functionality can enable all-optical dissection of a neural network through targeted inhibition and global fluorescence monitoring.
Tools like Archer1 and Optopatch could be used for all-optical loss and gain of function circuit analysis, respectively.Voltage sensors can also provide insights into neuronal response to stimuli in organisms in which electrophysiology is challenging, such as Caenorhabditis elegans and Drosophila melanogaster. Archer1 represents the first genetically encoded voltage sensor that has been used in live, behaving nematodes.
This work provides a foundation for more detailed characterization of cell types with unknown voltage dynamics as well as fast-spiking muscle cells in C. Additional applications of this tool likely include other transparent organisms, such as, fly larvae and zebrafish, where a fluorescent voltage sensor could be used to dissect neural circuitry.Until recently, due to their low baseline fluorescence, Arch-based sensors were not compatible with in vivo applications. This work on Archer1, as well as recent work on QuasArs, demonstrates that Arch-based sensors are not fundamentally limited, but can be used for a variety of neuronal applications, including in vivo. Our data show that variants of Arch are capable of increased fluorescence, enabling practical detection, while retaining Arch WT's superior speed and dynamic range compared to XFP-based sensors. Even though this work uses the lowest excitation intensity for an Arch-based sensor (.
Ethics statementAll experiments using animals in this study were approved by Institutional Animal Care and Use Committee (IACUC) at the California Institute of Technology. Sensor constructsArch variant constructs were generated by first amplifying EGFP from FCK-Arch-GFP (Accession codes listed in ) and adding the ER export domain using GFPfwdoverlapTSend and FCK-GFPrevERexport primers to make EGFP-ER. Arch-TS was then amplified from pLenti-CaMKIIa-eArch3.0-EYFP using Archfwd and TSrevintoGFPstart primers , assembled with EGFP-ER using Archfwd and ERrev primers , and subsequently cloned back into pLenti-CaMKIIa-eArch3.0-EYFP cut with BamHI and EcoRI restriction enzymes, to make pLenti-CaMKIIa-eArch3.0-EGFP. To make pLenti-CaMKIIa-Archer1-EGFP and pLenti-CaMKIIa-Archer2-EGFP, the D95E, T99C, and A225M mutations were introduced in the pLenti-CaMKIIa-eArch3.0-EGFP vector through overlap assembly PCR using Archfwd, ERrev, Arch3.0D95ET99Cfwd, Arch3.0D95ET99Crev, Arch3.0A225Mfwd, and Arch3.0A225Mrev primers (.