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All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.

Hochbaum DR, Zhao Y, Farhi SL, Klapoetke N, Werley CA, Kapoor V, Zou P, Kralj JM, Maclaurin D, Smedemark-Margulies N, Saulnier JL, Boulting GL, Straub C, Cho YK, Melkonian M, Wong GK, Harrison DJ, Murthy VN, Sabatini BL, Boyden ES, Campbell RE, Cohen AE - Nat. Methods (2014)

Bottom Line: We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent.Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons.In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios.

View Article: PubMed Central - PubMed

Affiliation: 1] Applied Physics Program, School of Engineering and Applied Science, Harvard University, Cambridge, Massachusetts, USA. [2].

ABSTRACT
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 QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP 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 rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

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Optopatch2 in organotypic brain sliceA) Left and middle: eGFP fluorescence, indicating CheRiff distribution. Right: QuasAr2 fluorescence. Scale bars from left to right: 50 µm, 20 µm, 20 µm. B) Single-trial optical recordings of APs initiated by pulses of blue illumination (10 ms, 7.5 mW/cm2). Signal represents whole-soma fluorescence without photobleaching correction or background subtraction. C) Bursts of APs triggered by steps of blue illumination (500 ms, 1–10 mW/cm2). Inhibitory potentials (arrows) were sometimes observed during the stimulation intervals, but not during rest intervals, suggesting inhibitory feedback arising from optically induced network activity. For B) and C), red illumination was 1,200 W/cm2 nominal incident intensity, not corrected for light scatter. Fluorescence collected at a frame rate of 1 kHz on an EMCCD camera (Methods).
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Figure 5: Optopatch2 in organotypic brain sliceA) Left and middle: eGFP fluorescence, indicating CheRiff distribution. Right: QuasAr2 fluorescence. Scale bars from left to right: 50 µm, 20 µm, 20 µm. B) Single-trial optical recordings of APs initiated by pulses of blue illumination (10 ms, 7.5 mW/cm2). Signal represents whole-soma fluorescence without photobleaching correction or background subtraction. C) Bursts of APs triggered by steps of blue illumination (500 ms, 1–10 mW/cm2). Inhibitory potentials (arrows) were sometimes observed during the stimulation intervals, but not during rest intervals, suggesting inhibitory feedback arising from optically induced network activity. For B) and C), red illumination was 1,200 W/cm2 nominal incident intensity, not corrected for light scatter. Fluorescence collected at a frame rate of 1 kHz on an EMCCD camera (Methods).

Mentions: We expressed Optopatch2 in organotypic brain slice using biolistic gene delivery. Neurons that had taken up the gene were clearly visible via fluorescence of eGFP (indicating CheRiff) and QuasAr2 under wide-field epifluorescence imaging (20× N.A. 1.0 water immersion objective) (Fig. 5a). Upon illumination with pulses of blue light (10 ms, repeated at 5 Hz, 7.5 mW/cm2), the fluorescence under red excitation (1,200 W/cm2 nominal incident intensity, not corrected for light scatter) showed clearly resolved single-trial APs in the soma (Fig. 5b) and in dendrites (Supplementary Fig. 22). These traces represent raw fluorescence, without background subtraction or correction for photobleaching.


All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.

Hochbaum DR, Zhao Y, Farhi SL, Klapoetke N, Werley CA, Kapoor V, Zou P, Kralj JM, Maclaurin D, Smedemark-Margulies N, Saulnier JL, Boulting GL, Straub C, Cho YK, Melkonian M, Wong GK, Harrison DJ, Murthy VN, Sabatini BL, Boyden ES, Campbell RE, Cohen AE - Nat. Methods (2014)

Optopatch2 in organotypic brain sliceA) Left and middle: eGFP fluorescence, indicating CheRiff distribution. Right: QuasAr2 fluorescence. Scale bars from left to right: 50 µm, 20 µm, 20 µm. B) Single-trial optical recordings of APs initiated by pulses of blue illumination (10 ms, 7.5 mW/cm2). Signal represents whole-soma fluorescence without photobleaching correction or background subtraction. C) Bursts of APs triggered by steps of blue illumination (500 ms, 1–10 mW/cm2). Inhibitory potentials (arrows) were sometimes observed during the stimulation intervals, but not during rest intervals, suggesting inhibitory feedback arising from optically induced network activity. For B) and C), red illumination was 1,200 W/cm2 nominal incident intensity, not corrected for light scatter. Fluorescence collected at a frame rate of 1 kHz on an EMCCD camera (Methods).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4117813&req=5

Figure 5: Optopatch2 in organotypic brain sliceA) Left and middle: eGFP fluorescence, indicating CheRiff distribution. Right: QuasAr2 fluorescence. Scale bars from left to right: 50 µm, 20 µm, 20 µm. B) Single-trial optical recordings of APs initiated by pulses of blue illumination (10 ms, 7.5 mW/cm2). Signal represents whole-soma fluorescence without photobleaching correction or background subtraction. C) Bursts of APs triggered by steps of blue illumination (500 ms, 1–10 mW/cm2). Inhibitory potentials (arrows) were sometimes observed during the stimulation intervals, but not during rest intervals, suggesting inhibitory feedback arising from optically induced network activity. For B) and C), red illumination was 1,200 W/cm2 nominal incident intensity, not corrected for light scatter. Fluorescence collected at a frame rate of 1 kHz on an EMCCD camera (Methods).
Mentions: We expressed Optopatch2 in organotypic brain slice using biolistic gene delivery. Neurons that had taken up the gene were clearly visible via fluorescence of eGFP (indicating CheRiff) and QuasAr2 under wide-field epifluorescence imaging (20× N.A. 1.0 water immersion objective) (Fig. 5a). Upon illumination with pulses of blue light (10 ms, repeated at 5 Hz, 7.5 mW/cm2), the fluorescence under red excitation (1,200 W/cm2 nominal incident intensity, not corrected for light scatter) showed clearly resolved single-trial APs in the soma (Fig. 5b) and in dendrites (Supplementary Fig. 22). These traces represent raw fluorescence, without background subtraction or correction for photobleaching.

Bottom Line: We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent.Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons.In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios.

View Article: PubMed Central - PubMed

Affiliation: 1] Applied Physics Program, School of Engineering and Applied Science, Harvard University, Cambridge, Massachusetts, USA. [2].

ABSTRACT
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 QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP 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 rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

Show MeSH
Related in: MedlinePlus