Bright and photostable chemigenetic indicators for extended in vivo voltage imaging

Abdelfattah, Ahmed S.; Kawashima, Takashi; Singh, Amrita; Novák, Ondřej; Liu, Hui; Shuai, Yichun; Huang, Yi Chieh; Campagnola, Luke; Seeman, Stephanie C.; Yu, Jianing; Zheng, Jihong; Grimm, Jonathan B.; Patel, Ronak; Friedrich, Johannes; Mensh, Brett D.; Paninski, Liam; Macklin, John J.; Murphy, Gabe J.; Podgorski, Kaspar; Lin, Bei Jung; Chen, Tsai Wen; Turner, Glenn; Liu, Zhe; Koyama, Minoru; Svoboda, Karel; Ahrens, Misha B.; Lavis, Luke D.; Schreiter, Eric R.

doi:10.1126/science.aav6416

Cited by 411 publications

(332 citation statements)

References 62 publications

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“…S10-11). Detailed structural studies in rats have shown 20 that each barrel contains ~40-50 L1 interneurons, consistent with our estimates based on published micrographs from mice (19). The lateral extent of the axonal and dendritic trees is ~200 -250 µm, comparable to the size of the barrel.…”

Section: Numerical Model Of L1 Microcircuitsupporting

confidence: 89%

“…Due to their multimodal, temporally precise inputs (16) and temporally precise outputs, 15 one would like to measure the sub-threshold dynamics and spike timing of L1 neurons with high precision in voltage and time. Recent advances in genetically encoded voltage indicators (GEVIs) enabled voltage imaging with single-neuron, single-spike resolution in hippocampus (17)(18)(19)(20) and in superficial cortex (18,19) in vivo, opening the possibility for optical explorations of L1 circuit function in vivo. 20 Voltage alone does not distinguish the relative contributions of E and I synaptic inputs, yet this distinction is critical for understanding circuit mechanisms.…”

Section: Main Textmentioning

confidence: 99%

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All-optical electrophysiology reveals excitation, inhibition, and neuromodulation in cortical layer 1

Fan

Kheifets

Böhm

et al. 2019

Preprint

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The stability of neural dynamics arises through a tight coupling of excitatory (E) and 15 inhibitory (I) signals. Genetically encoded voltage indicators (GEVIs) can report both spikes and subthreshold dynamics in vivo, but voltage only reveals the combined effects of E and I synaptic inputs, not their separate contributions individually. Here we combine optical recording of membrane voltage with simultaneous optogenetic manipulation to probe E and I individually in barrel cortex Layer 1 (L1) neurons in awake mice. Our studies reveal how the L1 microcircuit 20

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Section: Numerical Model Of L1 Microcircuitsupporting

confidence: 89%

Section: Main Textmentioning

confidence: 99%

All-optical electrophysiology reveals excitation, inhibition, and neuromodulation in cortical layer 1

Fan

Kheifets

Böhm

et al. 2019

Preprint

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“…However, rhodopsin fluorescence is very dim, requiring intense illumination for imaging 8 . Fusions of fluorescent protein (FP) domains or other bright fluorophores to rhodopsin GEVIs were therefore made to facilitate imaging 11,13,14 . These fusions enable voltage-sensitive electrochromic fluorescence resonance energy transfer (eFRET) from a bright fluorophore to the retinal cofactor within the rhodopsin, which acts as a dark quencher.…”

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confidence: 99%

A general approach to engineer positive-going eFRET voltage indicators

Abdelfattah

Valenti

Wong

et al. 2019

Preprint

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We engineered electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with "positive-going" fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transformed the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further applied this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.Genetically encoded voltage indicators (GEVIs) allow visualization of fast action potentials and subthreshold dynamics in groups of genetically defined neurons with high spatiotemporal resolution 1,2 . Monitoring voltage signals in vivo in large populations of neurons will enable dissection of detailed mechanistic links between brain activity and animal behavior. Despite recent advances, no current GEVI has ideal properties for routine, robust in vivo imaging.

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“…In contrast, fluorescent GEVIs allow recording both sub-threshold and threshold events from a few tens of neurons [19] simultaneously. Recently, neural spike were recorded from a large population of neurons (~ 100) for several minutes using a fluorescent hybrid GEVI [20] but requires a molecular complexation with a synthetic dye. Given the spike duration and the firing rates, there is a technical challenge in achieving a good SNR (> 5 as per Rose criterion [21]) at a frame rate of ~1kHz even with the best performing GEVI.…”

Section: Introductionmentioning

confidence: 99%

An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

Srinivasan

Griffin

Joshi

et al. 2019

Preprint

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1.AbstractGenetically encoded reporters have greatly increased our understanding of biology, especially in neuroscience. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach we have engineered Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential. Unlike other luciferase-luciferin bioluminescent systems, AMBER encodes the genes to express both the luciferase and luciferin. AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When AMBER is co-expressed with the luciferin producing genes it reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods we show that AMBER modulates its enzymatic activity as a function of the membrane potential. AMBER shows several-fold increase in the luminescent (ΔL/L) signal upon switching from the off to on state when the cell is depolarized. In vivo expression of AMBER in C. elegans allowed detecting pharyngeal pumping action and mechanosensory neural activity from multiple worms simultaneously. AMBER reports neural activity of multiple animals at the same time and can be used in social behavior assays to elucidate the role of membrane potential underlying behavior.2.Significance StatementThere have been many exciting advances in the development of genetically encoded voltage indicators to monitor intracelluar voltage changes. Most sensors employ fluorescence, which requires external light, potentially causing photobleaching or overheating. Consequently, there has been interest in developing luminescence reporters. However, they require addition of an exogenous substrate to produce light intracellularly. Here, we engineered a genetically encoded bioluminescent voltage indicator, AMBER, which unlike other bioluminescent activity indicators, does not require addition of an exogenous substrate. AMBER allows a large differential signal, a high signal-to-noise ratio, and causes minimal metabolic demand on cells. We used AMBER to record voltage activity in freely-moving C. elegans, demonstrating that AMBER is a important new tool for monitoring neuronal activity during social behavior.

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Bright and photostable chemigenetic indicators for extended in vivo voltage imaging

Cited by 411 publications

References 62 publications

All-optical electrophysiology reveals excitation, inhibition, and neuromodulation in cortical layer 1

All-optical electrophysiology reveals excitation, inhibition, and neuromodulation in cortical layer 1

A general approach to engineer positive-going eFRET voltage indicators

An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

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