Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells

Park, Jeehae; Werley, Christopher A.; Venkatachalam, Veena; Kralj, Joel M.; Dib‐Hajj, Sulayman D.; Waxman, Stephen G.; Cohen, Adam E.

doi:10.1371/journal.pone.0085221

Cited by 80 publications

(90 citation statements)

References 24 publications

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“…Expression of Kir2.1 lowered the resting potential to approximately −60 mV, close to the resting potential of neurons 55, 56 . We reasoned that this effect would center the ITV close to the physiologically relevant range.…”

Section: Methodsmentioning

confidence: 69%

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

et al. 2014

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All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and 2, which show improved brightness and voltage sensitivity, microsecond response times, and produce no photocurrent. We engineered a novel channelrhodopsin actuator, CheRiff, which shows improved light sensitivity and kinetics, and spectral orthogonality to the QuasArs. A co-expression vector, Optopatch, enabled crosstalk-free genetically targeted all-optical electrophysiology. In cultured neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials in dendritic spines, synaptic transmission, sub-cellular microsecond-timescale details of action potential propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In brain slice, Optopatch induced and reported action potentials and subthreshold events, with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without use of conventional electrodes.

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Section: Methodsmentioning

confidence: 69%

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

et al. 2014

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“…Site-saturation libraries were generated using a modified version of the QuikChange method (Stratagene), as previously described (15). For the single-site-saturation libraries, a mix of three mutagenic primers containing the triplet sequences NDT, VHG, and TGG were used in ratio of 12:9:1 for mutagenic PCR amplification (Table S1, primers [7][8][9][10][11][12][13][14][15][16][17][18][19][20]. For the double-site-saturation library, a mixture of 9 mutagenic primers (and a reverse primer) was used for PCR amplification (Table S1, primers [21][22][23][24][25][26][27][28][29][30].…”

Section: Methodsmentioning

confidence: 99%

“…When protonpumping activity is abolished by mutating residue D95, Arch fluorescence remains sensitive to transmembrane voltage (3). D95E is the only reported mutation at this residue to retain wild type-like kinetics of voltage-induced fluorescent changes (14). Currently, Arch and its variants have extremely low quantum efficiencies, which greatly limits their utility as voltage sensors and more generally as optical markers.…”

mentioning

confidence: 99%

Directed evolution of a far-red fluorescent rhodopsin

McIsaac¹,

Engqvist²,

Wannier

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

115

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Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with livecell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK a of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼620 nm/730 nm).optogenetics | opsins | bioelectricity | voltage sensor | near-infrared

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“…We need high-throughput, multi-parameter screens. Mammalian cell lines capable of mimicking action potential transients provide a possible substrate for such a screen [21]. Technologies for in situ identification of novel mutants or scaffolds within a mixed population would further increase screening throughput.…”

Section: Genetically Encoded Optical Sensors Of Brain Functionmentioning

confidence: 99%

Roadmap on neurophotonics

Cho

Zheng

Augustine

et al. 2016

J. Opt.

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Mechanistic understanding of how the brain gives rise to complex behavioral and cognitive functions is one of science’s grand challenges. The technical challenges that we face as we attempt to gain a systems-level understanding of the brain are manifold. The brain’s structural complexity requires us to push the limit of imaging resolution and depth, while being able to cover large areas, resulting in enormous data acquisition and processing needs. Furthermore, it is necessary to detect functional activities and ‘map’ them onto the structural features. The functional activity occurs at multiple levels, using electrical and chemical signals. Certain electrical signals are only decipherable with sub-millisecond timescale resolution, while other modes of signals occur in minutes to hours. For these reasons, there is a wide consensus that new tools are necessary to undertake this daunting task. Optical techniques, due to their versatile and scalable nature, have great potentials to answer these challenges. Optical microscopy can now image beyond the diffraction limit, record multiple types of brain activity, and trace structural features across large areas of tissue. Genetically encoded molecular tools opened doors to controlling and detecting neural activity using light in specific cell types within the intact brain. Novel sample preparation methods that reduce light scattering have been developed, allowing whole brain imaging in rodent models. Adaptive optical methods have the potential to resolve images from deep brain regions. In this roadmap article, we showcase a few major advances in this area, survey the current challenges, and identify potential future needs that may be used as a guideline for the next steps to be taken.

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Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells

Cited by 80 publications

References 24 publications

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

Directed evolution of a far-red fluorescent rhodopsin

Roadmap on neurophotonics

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