Generation of a Genetically Encoded Voltage Indicator MARINA reporter human iPS cell line using Cas9 (VULSCi002-A-2)
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PROVIDER: PRJEB81248 | ENA |
REPOSITORIES: ENA
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Project description:Voltage-sensitive fluorescent proteins (VSFPs) are a family of genetically-encoded voltage indicators (GEVIs) reporting membrane voltage fluctuation from genetically-targeted cells in cell cultures to whole brains in awake mice as demonstrated earlier using 1-photon (1P) fluorescence excitation imaging. However, in-vivo 1P imaging captures optical signals only from superficial layers and does not optically resolve single neurons. Two-photon excitation (2P) imaging, on the other hand, has not yet been convincingly applied to GEVI experiments. Here we show that 2P imaging of VSFP Butterfly 1.2 expresssing pyramidal neurons in layer 2/3 reports optical membrane voltage in brain slices consistent with 1P imaging but with a 2-3 larger ΔR/R value. 2P imaging of mouse cortex in-vivo achieved cellular resolution throughout layer 2/3. In somatosensory cortex we recorded sensory responses to single whisker deflections in anesthetized mice at full frame video rate. Our results demonstrate the feasibility of GEVI-based functional 2P imaging in mouse cortex.
| S-EPMC3715760 | biostudies-other
Project description:Genetically encoded voltage indicators (GEVIs) allow for the cell-type-specific real-time imaging of neuronal membrane potential dynamics, which is essential to understanding neuronal information processing at both cellular and circuit levels. Among GEVIs, near-infrared-shifted GEVIs offer faster kinetics, better tissue penetration, and compatibility with optogenetic tools, enabling all-optical electrophysiology in complex biological contexts. In our previous work, we employed the directed molecular evolution of microbial rhodopsin Archaerhodopsin-3 (Arch-3) in mammalian cells to develop a voltage sensor called Archon1. Archon1 demonstrated excellent membrane localization, signal-to-noise ratio (SNR), sensitivity, kinetics, and photostability, and full compatibility with optogenetic tools. However, Archon1 suffers from low brightness and requires high illumination intensities, which leads to tissue heating and phototoxicity during prolonged imaging. In this study, we aim to improve the brightness of this voltage sensor. We performed random mutation on a bright Archon derivative and identified a novel variant, monArch, which exhibits satisfactory voltage sensitivity (4~5% ΔF/FAP) and a 9-fold increase in basal brightness compared with Archon1. However, it is hindered by suboptimal membrane localization and compromised voltage sensitivity. These challenges underscore the need for continued optimization to achieve an optimal balance of brightness, stability, and functionality in rhodopsin-based voltage sensors.
| S-EPMC11855178 | biostudies-literature
Project description:Genetically encoded voltage indicators (GEVIs) promise to reveal the membrane potential of genetically targeted neuronal populations through noninvasive, chronic imaging of large portions of cortical space. Here we test a promising GEVI in mouse cortex during wakefulness, a challenging condition due to large hemodynamic activity, and we introduce a straightforward projection method to separate a signal dominated by membrane voltage from a signal dominated by hemodynamic activity. We expressed VSFP-Butterfly 1.2 plasmid in layer 2/3 pyramidal cells of visual cortex through electroporation in utero. We then used wide-field imaging with two cameras to measure both fluorophores of the indicator in response to visual stimuli. By taking weighted sums and differences of the two measurements, we obtained clear separation of hemodynamic and voltage signals. The hemodynamic signal showed strong heartbeat oscillations, superimposed on slow dynamics similar to blood oxygen level-dependent (BOLD) or "intrinsic" signals. The voltage signal had fast dynamics similar to neural responses measured electrically, and showed an orderly retinotopic mapping. We compared this voltage signal with calcium signals imaged in transgenic mice that express a calcium indicator (GCaMP3) throughout cortex. The voltage signal from VSFP had similar signal-to-noise ratios as the calcium signal, it was more immune to vascular artifacts, and it integrated over larger regions of visual space, which was consistent with its reporting mostly subthreshold activity rather than the spiking activity revealed by calcium signals. These results demonstrate that GEVIs provide a powerful tool to study the dynamics of neural populations at mesoscopic spatial scales in the awake cortex.
| S-EPMC4287159 | biostudies-literature
Project description:An improved genetically encoded voltage indicator (GEVI) was achieved by altering the charge composition of the region linking the voltage-sensing domain of the GEVI to a pH-sensitive fluorescent protein. Negatively charged linker segments reduced the voltage-dependent optical signal while positively charged linkers increased the signal size. Arginine scanning mutagenesis of the linker region improved the signal size of the GEVI, Bongwoori, yielding fluorescent signals as high as 20% ΔF/F during the firing of action potentials. The speed of this new sensor was also capable of optically resolving action potentials firing at 65 Hz. This large signal size enabled individual pixels to become surrogate electrodes. Plotting the highest correlated pixels based only on fluorescence changes reproduced the image of the neuron exhibiting activity. Furthermore, the use of a pH-sensitive fluorescent protein facilitated the detection of the acidification of the neuron during the firing of action potentials.
| S-EPMC5557843 | biostudies-literature
Project description:The hybrid voltage sensor (hVOS) combines membrane-targeted green fluorescent protein and the hydrophobic anion dipicrylamine (DPA) to provide a promising tool for optical recording of electrical activity from genetically defined populations of neurons. However, large fluorescence signals are obtained only at high DPA concentrations (>3 mum) that increase membrane capacitance to a level that suppresses neural activity. Here, we develop a quantitative model of the sensor to guide its optimization and achieved an approximate threefold increase in fractional fluorescence change at a lower DPA concentration of 2 mum. Using this optimized voltage reporter, we perform optical recordings of evoked activity in the Drosophila antennal lobe with millisecond temporal resolution but fail to detect action potentials, presumably because spike initiation and/or propagation are inhibited by the capacitive load added even at reduced DPA membrane densities. We evaluate strategies for potential further improvement of hVOS quantitatively and derive theoretical performance limits for optical voltage reporters in general.
| S-EPMC2714581 | biostudies-literature
Project description:Genetically encoded voltage indicators (GEVIs) can measure millisecond-scale subthreshold neural responses with cell type specificity. Here, we successfully expressed, for the first time, a GEVI in excitatory V1 neurons in macaque monkeys. We then used widefield fluorescent imaging to measure V1 dynamics in response to visual stimuli with diverse temporal waveforms and contrasts, and compared these responses to signals measured using a genetically encoded calcium indicator (GECI) and a synthetic voltage-sensitive dye (VSD). When compared to GECI, GEVI captures faster response dynamics, tracks higher temporal frequencies, and responds to lower contrasts. To quantitatively characterize these three signals, we developed a simple nonlinear model that predicts the response dynamics to stimuli with arbitrary temporal waveforms and contrasts. Our results are consistent with the hypothesis that GEVI signals reflect the dynamics of locally summed membrane potentials, thus opening the door for a new class of experiments in behaving primates.
| S-EPMC11838743 | biostudies-literature
Project description:We report development of the first genetically encoded bioluminescent indicator for membrane voltage called LOTUS-V. Since it is bioluminescent, imaging LOTUS-V does not require external light illumination. This allows bidirectional optogenetic control of cellular activity triggered by Channelrhodopsin2 and Halorhodopsin during voltage imaging. The other advantage of LOTUS-V is the robustness of a signal-to-background ratio (SBR) wherever it expressed, even in the specimens where autofluorescence from environment severely interferes fluorescence imaging. Through imaging of moving cardiomyocyte aggregates, we demonstrated the advantages of LOTUS-V in long-term imaging are attributable to the absence of phototoxicity, and photobleaching in bioluminescent imaging, combined with the ratiometric aspect of LOTUS-V design. Collectively LOTUS-V extends the scope of excitable cell control and simultaneous voltage phenotyping, which should enable applications in bioscience, medicine and pharmacology previously not possible.
| S-EPMC5322354 | biostudies-literature
Project description: Not available
| S-EPMC10618466 | biostudies-literature
Project description:Imaging voltage using fluorescent-based sensors could be an ideal technique to probe neural circuits with high spatiotemporal resolution. However, due to insufficient signal-to-noise ratio (SNR), imaging membrane potential in mammalian preparations is still challenging. In recent years, many genetically encoded voltage indicators (GEVIs) have been developed. To compare them and guide decisions on which GEVI to use, we have characterized side by side the performance of eight GEVIs that represent different families of molecular constructs. We tested GEVIs in vitro with 1-photon imaging and in vivo with 1-photon wide-field imaging and 2-photon imaging. We find that QuasAr2 exhibited the best performance in vitro, whereas only ArcLight-MT could be used to reliably detect electrical activity in vivo with 2-photon excitation. No single GEVI was ideal for every experiment. These results provide a guide for choosing optimal GEVIs for specific applications.
| S-EPMC7075032 | biostudies-literature
Project description:TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life. IS605-family TnpB homologs function in bacteria as programmable RNA-guided homing endonucleases driving transposon maintenance through DSB-stimulated homologous recombination. Here we uncover molecular mechanisms of transposition lifecycle of IS607-family elements that, remarkably, also encode group I introns. We discover molecular features for a candidate ‘IStron’ from Clostridium botulinum that allow the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts have evolved a sensitive equilibrium to balance competing and mutually exclusive activities that promote transposon maintenance while limiting adverse fitness costs on the host. Collectively, this work highlights molecular innovation in the multi-functional utility of transposon-encoded noncoding RNAs.
2024-05-06 | GSE261343 | GEO