Genetically encoded fluorescent voltage indicators: are we there yet?

Abstract

In order to understand how brain activity produces adaptive behavior we need large-scale, high-resolution recordings of neuronal activity. Fluorescent genetically encoded voltage indicators (GEVIs) offer the potential for these recordings to be performed chronically from targeted cells in a minimally invasive manner. As the number of GEVIs successfully tested for in vivo use grows, so has the number of open questions regarding the improvements that would facilitate broad adoption of this technology that surpasses mere 'proof of principle' studies. Our aim in this review is not to provide a status check of the current state of the field, as excellent publications covering this topic already exist. Here, we discuss specific questions regarding GEVI development and application that we think are crucial in achieving this goal.

Figure 1

Design of genetically-encoded voltage sensors (GEVIs) that were successfully used for studying electrical brain activity in fruit fly and/or mouse. Left side panels in all are schematic representation of GEVI design. Right side panels in all are examples of electrical and optical traces simultaneously recorded from mammalian neurons in vitro expressing respective GEVI. Optical traces in all are corrected for photobleaching. In A., B., and C. GEVIs based on voltage-sensitive domain derived from voltage-phosphatase and fluorescent protein(s). In these GEVIs conformational change in voltage sensitive domain causes change in fluorescence intensity of FP(s), (a) ArcLight, based on a fusion of Ciona intestinalis VSD and super ecliptic pHluorin GFP (227D) has been used for recording electrical activity in flies and mice. Example traces modified from [28^••]. (b) VSFP-Butterfly 1.2, based on a fusion of Ciona intestinalis VSD and FRET pair of fluorescent proteins mCitrine/mKate2 has been used for recording electrical activity in mice. Example traces modified from [27] (c) ASAP1/ASAP2f/ASAP2s, several variants of probe with same general design based on a fusion of Gallus gallus VSD and circularly permuted superfolder GFP have been used for recording electrical activity in flies. Example traces modified from [29^••] (d) Ace2N-mNeon, based on a fusion of Acetabularia acetabulum rhodopsin and GFP mNeon-Green has been used for recording of activity in flies and mice. In this probe voltage dependent change in photophysical state of opsin causes quenching of fluorescence in spectrally compatible FP, Example traces modified from [31^••]

Figure 2

Examples of genetically encoded voltage sensors (GEVIs) successfully used for recording neuronal activity in fruit fly (Drosophila melanogaster). Sophisticated genetic toolbox and tractability that allow for targeted expression made fruit fly a model of choice for functional testing of current generation of indicators. (a) Expression of GEVI ArcLight in fruit fly allows for recording of spontaneous electrical activity (subthreshold and action potentials) in intact neuronal circuits using single-photon excitation. Synchronous membrane activity was recorded in somata of multiple neurons (C1–3) and one neurite (N1) of wild-type clock neurons (ILNVs). Color coding of ROIs corresponds to the optical traces, The simultaneous whole-cell patch-clamp recording of the cell in the red ROI shown in black. Images were recorded at 500 Hz using 488 nm 50 mW laser with ~5 W/cm² light power at the preparation. Scale bar is 10 μm, Taken from [38^•], (b) Two-photon exciton was used in comparative study of several GEVIs expressed in L2 neurons in Drosophila visual system. Flies expressing VSD-based GEVIs (ASAP1, ASAP2s and ArcLight), opsin-based GEVIs (MacQ-mCitrine) and GECI GCaMP6f were tested with visual stimulus alternating between 300-ms dark (black bar) and light (white bar) flashes. Top: mean response across multiple cells (n = 44 cells/3 flies for ASAP1,111 cells/5 flies for ASAP2s, 65 cells/5 flies for ArcLight, 64 cells/3 flies for MacQ-mCitrine, 23 cells/4 flies for Ace2N-2AA-mNeon, and 232 cells/10 flies for GCaMP6f). For each cell recordings from 100 trials were averaged. Bottom: 5 examples of singe-trial responses from single L2 cell (in grey). Colored trace is mean response over 100 trials from the same cell. Cells were imaged at a frame rate of 38.9 Hz. Modified from [58^••].

Figure 3

Examples of genetically encoded voltage sensors (GEVIs) successfully used for recording neuronal activity in mice in vivo. (a) Single- (left) and two-photon (right) microscopy was used for recording of cell populations responses in vivo in mouse olfactory bulb using GEVI ArcLight. In all, responses are evoked with ethyl tiglate. For wide-field imaging preparation was illuminated with 485 ± 25nm light from either tungsten halogen lamp or a 150 W Xenon arc lamp. Images were recorded at 125 Hz frame rate. Single and averaged traces recorded with wide-field microscopy are unfiltered. In averaged trace single-trail traces were aligned to the first sniff of odorant. The optical traces using 2-photon microscopy are recorded with 920 nm laser light using either 8 Hz or 100 Hz sampling rate as indicated in the figure. In all, traces were filtered with Gausssian low-pass filter at 2 or 4 Hz. Modified from [53]. (b) Sub-millisecond speed of opsin-FP GEVI Ace2N-4AA-mNeon allows for electrical transients (subthreshold and action potentials) recordings in layer 2/3 visual cortical neurons of awake mice. Example optical traces were recorded from a cortical V1→LM neuron responding to visual drifting gratings. Orientations and motion directions are marked above each trace. The images were recorded at 1 kHz using illumination intensity of 20 mW/mm². Modified from [58^••].