Dual-polarity voltage imaging of the concurrent dynamics of multiple neuron types

Abstract

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between targeted cell populations. However, current indicators lack the requisite sensitivity for in vivo multipopulation imaging. We describe next-generation green and red voltage sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants pAce and pAceR. Our indicators enable 0.4- to 1-kilohertz voltage recordings from >50 spiking neurons per field of view in awake mice and ~30-minute continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered brain state–dependent antagonism between neocortical somatostatin-expressing (SST + ) and vasoactive intestinal peptide–expressing (VIP + ) interneurons and contributions to hippocampal field potentials from cell ensembles with distinct axonal projections. By combining three mutually compatible indicators, we performed simultaneous triple-population imaging. These approaches will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution.

Bright and sensitive red voltage indicators for imaging action potentials in brain slices and pancreatic islets

“…Upon membrane depolarization from -70 mV to 30 mV, both indicators exhibited millisecond kinetics with 1.9 ± 0.1 ms and 1.5 ± 0.2 ms (Figure S7). The voltage sensitivity of Cepheid1b and Cepheid1s compare favorably against several recently published red GEVIs, including VARNAM 6 , VARNAM2 13 , and Ace2N-7aa-mScarlet 21 , which are constructed by fusing mRuby3 and mScarlet to the C-terminus of Ace rhodopsin mutants. When the membrane potential is altered step-wise from -100 mV to +40 mV (Figure S8), the whole-cell fluorescence of VARNAM, VARNAM2, and Ace2N-7aa-mScarlet change by -17.9 ± 0.6%, -26.7 ± 1.7% and -16.0 ± 1.0% ΔF/F0, respectively (Figure 1b).…”

“…To improve molecular brightness, a red fluorescent protein (RFP) donor is C-terminally fused to a voltage-sensing rhodopsin, which serves as the electrochromic Förster resonance energy transfer (eFRET) quencher. The resulting eFRET GEVIs, including QuasAr2-mRuby2 12 and VARNAMs 6, 13 , exhibit substantially improved brightness while maintaining fast response kinetics. However, their absolute sensitivities are limited to ~20% ΔF/F 0 per 100 mV 6, 12, 13 owing to low FRET efficiencies 14, 15 .…”

“…The resulting eFRET GEVIs, including QuasAr2-mRuby2 12 and VARNAMs 6, 13 , exhibit substantially improved brightness while maintaining fast response kinetics. However, their absolute sensitivities are limited to ~20% ΔF/F 0 per 100 mV 6, 12, 13 owing to low FRET efficiencies 14, 15 .…”

Single Photon Kilohertz Frame Rate Imaging of Neural Activity

“…For example, while the SPAD we used here was designed for acquisition of binary frames to allow for compact pixels built into a relatively large array, introduction of multi‐bit photon counting will increase the dynamic range of the sensor, potentially enabling lower magnification objectives to be used, and more neurons to be captured in the field of view. Further improvement in brightness and sensitivity of voltage indicators [ 21–23 ] could further enhance detection of voltage signals in subcellular compartments and more densely‐labelled samples.…”

Virtual-scanning light-field microscopy for robust snapshot high-resolution volumetric imaging

“…To demonstrate its advantages over traditional LFM and sLFM, we constructed an upright sLFM system with a high-speed scientific camera to observe the 3D voltage transients of sparsely labeled dopamine neurons across the whole brain of awake behaving Drosophila (MB06 5B-GAL4>20×UAS-pAce) 46,47 at 500 vps. We imaged the same sample by LFM and sLFM sequentially for comparison.…”