Optical recording of action potentials in mammalian neurons using a microbial rhodopsin

Kralj, Joel M.; Douglass, Adam D.; Hochbaum, Daniel; Maclaurin, Dougal; Cohen, Adam E.

doi:10.1038/nmeth.1782

Cited by 430 publications

(502 citation statements)

References 35 publications

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“…[3][4][5][6][7][8] Nevertheless, though a useful correlation often exists between Ca 2+ dynamics and neuronal spiking across a range of spike frequencies, the slowkinetics and saturation of [Ca 2+ ]-related fluorescent signals constrain the utility of Ca 2+ imaging as a means of probing spiking dynamics in many neuron types. 4,9 Numerous voltage-sensitive indicators 1,[10][11][12][13][14][15][16][17][18] permit direct imaging of cellular membrane potentials. Organic voltage-sensitive dyes have allowed functional mapping studies in awake mammals 14 and studies of individual cells' dynamics in invertebrates 19 and mammalian brain slices.…”

Section: Introductionmentioning

confidence: 99%

“…11,15 These have been complemented by hybrid approaches that combine genetically encoded, membrane-targeted fluorescent proteins with synthetic voltage-sensing molecules. 16 Nevertheless, these probes have been limited by slow kinetics and limited dynamic range, 11 low quantum yields, 15,21 and possible interference with cellular capacitance. 16 These constraints have precluded concurrent detection of single action potentials across large populations of individual neurons in the live mammalian brain.…”

Section: Introductionmentioning

confidence: 99%

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Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Marshall

Schnitzer

2013

ACS Nano

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Biophysicists have long sought optical methods capable of reporting the electrophysiological dynamics of large-scale neural networks with millisecond-scale temporal resolution. Existing fluorescent sensors of cell membrane voltage can report action potentials in individual cultured neurons, but limitations in brightness and dynamic range of both synthetic organic and genetically encoded voltage sensors have prevented concurrent monitoring of spiking activity across large populations of individual neurons. Here we propose a novel, inorganic class of fluorescent voltage sensors: semiconductor nanoparticles, such as ultrabright quantum dots (qdots). Our calculations revealed that transmembrane electric fields characteristic of neuronal spiking (~10 mV/nm) modulate a qdot's electronic structure and can induce ~5% changes in its fluorescence intensity and ~1 nm shifts in its emission wavelength, depending on the qdot's size, composition, and dielectric environment. Moreover, tailored qdot sensors composed of two different materials can exhibit substantial (~30%) changes in fluorescence intensity during neuronal spiking. Using signal detection theory, we show that conventional qdots should be capable of reporting voltage dynamics with millisecond precision across several tens or more individual neurons over a range of optical and neurophysiological conditions. These results unveil promising avenues for imaging spiking dynamics in neural networks and merit in-depth experimental investigation. Toward addressing these challenges, we studied whether tools from nanotechnology, specifically, bright, multifunctional semiconductor quantum dots (qdots), 22,23 might be useful for imaging neuronal membrane potentials. Qdots are known to possess voltagesensitive optical properties, including a red-shifting of the qdot's fluorescence emission peak, spectral broadening, and a decrease in the intensity of fluorescence emission ( Figure 1C,D). [24][25][26][27] Qdots also are highly resistant to photobleaching and exhibit large quantum yields and absorption cross sections for one-(σ 1 ) and two-photon (σ 2 ) excitation. For example, CdSe qdots with radius R ~ 3 nm exhibit cross sections of σ 1 > 1 nm 2 and σ 2 > 3 × 10 4 GM, as compared to σ 1 ~ 10 −2 nm 2 and σ 2 ~ 3 × 10 2 GM for green fluorescent protein. 28,29 Marshall and Schnitzer Page 2 ACS Nano. Author manuscript; available in PMC 2017 December 15. Graphical Abstract HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptTo evaluate the spike detection capabilities of qdot indicators quantitatively, we applied a theoretical approach that incorporates the physical, biophysical, and statistical components of the problem. We calculated the effect of applied electric fields on the qdot's electronic structure and examined how this modulates a qdot's fluorescence intensity and emission spectra. We also studied nonspherical, nonhomogenous qdots, which we refer to more generally as semiconductor nanocrystals, and found that these nanoparticles can demonstrate muc...

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Marshall

Schnitzer

2013

ACS Nano

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“…Microbial opsins are integral membrane proteins with seven transmembrane helices and a conserved lysine residue that forms a covalent Schiff-base linkage to all-trans retinal. The apoprotein covalently bonded to a retinal molecule forms a colored holoprotein referred to as rhodopsin (2), variants of which have recently been reported to naturally fluoresce at wavelengths >680 nm in live cells (3,4). Rhodopsins thus offer novel substrates for engineering genetically encoded fluorescent markers in a desirable spectral range for livecell imaging.…”

mentioning

confidence: 99%

“…In the field of optogenetics, rhodopsins have been used as actuators of neuronal activity: When certain rhodopsin variants are expressed in neurons, light-driven ion flux can be exploited to activate (9) or inhibit (10) neuronal activity with fast kinetics in a selective and reversible fashion. The absorbance spectrum of rhodopsins can be sensitive to membrane voltage (11), and at least three rhodopsins have been reported to exhibit dim fluorescence that is sensitive to changes in membrane voltage (3,4,12), a property that has been harnessed to develop genetically encoded fluorescent sensors of neuronal activity (3,12,13). Although significant progress has been made in developing opsin-based tools that enable selective activation/inhibition of neurons, progress toward developing brighter opsin-based biological sensors has been limited.…”

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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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“…26,36 Sufficiently bright and side-effect-free GEVIs that respond to both depolarization and repolarization with effective time constants of 1 ms or lower remain to be presented. It should be emphasized, however, that a reasonable SNR at high temporal resolution (as required to resolve action potentials) can only be achieved if the indicator can deliver both large photon fluxes (i.e., is bright and photostable) and high sensitivity.…”

Section: ■ Outlookmentioning

confidence: 99%

Genetically Engineered Fluorescent Voltage Reporters

Mutoh

Akemann

Knöpfel

2012

ACS Chem. Neurosci.

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Fluorescent membrane voltage indicators that enable optical imaging of neuronal circuit operations in the living mammalian brain are powerful tools for biology and particularly neuroscience. Classical voltage-sensitive dyes, typically low molecular-weight organic compounds, have been in widespread use for decades but are limited by issues related to optical noise, the lack of generally applicable procedures that enable staining of specific cell populations, and difficulties in performing imaging experiments over days and weeks. Genetically encoded voltage indicators (GEVIs) represent a newer alternative that overcomes several of the limitations inherent to classical voltage-sensitive dyes. We critically review the fundamental concepts of this approach, the variety of available probes and their state of development.

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Optical recording of action potentials in mammalian neurons using a microbial rhodopsin

Cited by 430 publications

References 35 publications

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Directed evolution of a far-red fluorescent rhodopsin

Genetically Engineered Fluorescent Voltage Reporters

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