Independent optical excitation of distinct neural populations

Journal name:
Nature Methods
Volume:
11,
Pages:
338–346
Year published:
DOI:
doi:10.1038/nmeth.2836
Received
Accepted
Published online
Corrected online

Abstract

Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system–mediated behavioral interference when using the variant CsChrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.

At a glance

Figures

  1. Novel channelrhodopsin spectral classes discovered through algal transcriptome sequencing.
    Figure 1: Novel channelrhodopsin spectral classes discovered through algal transcriptome sequencing.

    (ac) Maximum photocurrents in cultured neurons transfected with the different opsin-GFP fusions in response to far-red (660-nm), green (530-nm) and blue (470-nm) light; blue and green photon fluxes were matched, with illumination conditions defined as follows: 1-s pulse at 10 mW/mm2 for red, 5-ms pulse at 3.66 mW/mm2 for green and 5-ms pulse at 4.23 mW/mm2 for blue. Individual cell photocurrents are plotted as gray circles and overlaid on the population bar graph. See Supplementary Figure 6 for additional individual cell data. Bottom, phylogeny tree of the channelrhodopsins tested, based on transmembrane helix alignments. The scale indicates the number of substitutions per site. (d) Representative voltage-clamp traces in cultured neurons as measured under the screening conditions in ac (the long red light pulse was used to ensure we did not miss any red-sensitive channelrhodopsins in our screen). (e) Channelrhodopsin action spectra (HEK293 cells; n = 6–8 cells; measured using equal photon fluxes, ~2.5 × 1021 photons/s/m2). (fh) Channelrhodopsin kinetic properties as measured in cultured neurons (see also Supplementary Figs. 7 and 8). Off-kinetics (f) were measured under the conditions in ac; on-kinetics (g) and recovery kinetics (h) were measured with a 1-s pulse at 5 mW/mm2. All opsins were illuminated near their respective peak wavelength, which was either blue or green for all opsins except Chrimson, which was characterized at 625 nm (n = 5–12 cells for all kinetic comparisons). τoff, monoexponential fit of photocurrent decay. Peak current recovery ratios in h were determined from three 1-s light pulses, with the first pulse response used as the baseline for peak current recovery ratio calculations for both the second (1 s in dark after first pulse) and third pulse response (30 s in dark after second pulse). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA with Dunnett's post hoc test, with ChR2 as the reference in c,f,g, and C1V1TT as the reference in a,b. Exact P values and n values are in Supplementary Table 2. Plotted data are mean ± s.e.m. Opsins and the species they derive from are defined in the Online Methods.

  2. Comparison of optical spiking in cultured neurons expressing different channelrhodopsins.
    Figure 2: Comparison of optical spiking in cultured neurons expressing different channelrhodopsins.

    (ac) Green light–driven spiking fidelity. All green light spiking protocols used a train of 40 pulses, 2-ms pulse width, at 530 nm and at the indicated power (n = 5–8 cells for each opsin). (a) Representative green light–driven spiking traces at the indicated frequencies at 5 mW/mm2. (b) Green light–driven spike probability over a range of frequencies. The dashed line is the electrical spiking control from Chronos-expressing neurons (this control comprised a train of 40 pulses at the indicated frequencies; each current-injection pulse was 5 ms long and was varied from 200 to 800 pA depending on each neuron's spike threshold). (c) Spike latencies (time between the light-pulse onset and the spike peak) calculated for 5-Hz trains at 5 mW/mm2. (df) Comparison of spiking driven by red light (625 nm). (d) Representative current-clamp traces of red light response and spike fidelity (n = 5–8 cells for each opsin; 5-ms pulses, 5 Hz, 5 mW/mm2). (e) Comparison of wild-type Chrimson and Chrimson K176R mutant (ChrimsonR) high-frequency red light spiking (n = 10 and 4 cells, respectively; 40-pulse train, 2-ms pulse width, 5 mW/mm2). (f) Representative off-kinetics traces for Chrimson and ChrimsonR. ***P < 0.001; P = 0.0007 for CsChR and P < 0.0001 for Chronos; ANOVA with Dunnett's post hoc test with C1V1TT as reference. Plotted data are mean ± s.e.m.

  3. CsChrimson evokes action potentials in larval Drosophila motor neurons and triggers stereotyped behavior in adult Drosophila.
    Figure 3: CsChrimson evokes action potentials in larval Drosophila motor neurons and triggers stereotyped behavior in adult Drosophila.

    (ac) Intracellular recordings from m6 muscles in 3rd instar larvae expressing CsChrimson in motor neurons. Responses to 470-nm, 617-nm and 720-nm light pulses of indicated power and increasing duration are shown. Dashes in each subpanel indicate −50 mV. (d) Probability of light-evoked excitatory junction potentials (EJPs) after pulses of 1, 2, 4, 8 or 16 ms in response to 470-nm and 617-nm light and after pulses of 10, 20, 40, 80 or 160 ms in response to 720-nm light. n = 6 muscles in 3 animals for all larvae experiments. (e) Mean number of EJPs evoked in response to light pulses. (fh) Behavioral response of adult flies to light (n = 5 flies in each case). (f) Proboscis extension reflex (PER) of flies (pUAS-CsChrimson-mVenus in attP18/w;Gr64f-GAL4/+;Gr64f-GAL4/+, shown as Gr64f × CsChrimson) in response to 25 pulses of lights at 470 nm, 617 nm and 720 nm (see Online Methods for PER scoring). (g) PER of Gr64f × CsChrimson flies to pulsed light in darkness (D) or in a visual arena with flowing blue random dots (A). (h) Startle response of control flies (pUAS-CsChrimson-mVenus in attP18/+;+/+;+/+, shown as WTB × CsChrimson) to visual stimuli as in g. The startle score is the number of moving legs after stimulation. ***P < 0.001, **P < 0.01. Error bars, s.e.m.

  4. Characterization of channelrhodopsin blue light (470-nm) sensitivities for two-color excitation in cultured neurons.
    Figure 4: Characterization of channelrhodopsin blue light (470-nm) sensitivities for two-color excitation in cultured neurons.

    (a) Current-clamp traces of representative Chrimson-expressing neuron under pulsed vs. continuous illumination. (b) Chrimson blue light–induced cross-talk voltages vs. irradiances for individual cells under pulsed illumination (5 ms, 5 Hz, n = 5 cells). (c) Photocurrent vs. blue irradiances (5-ms pulses; n = 4 cells for Chrimson, n = 8–10 cells for others). Vertical dashed lines indicate half-maximal points up the curves for ChR2 and Chronos as fitted. (d) Turn-on kinetics (1-s pulse; n = 4–7 cells; see Supplementary Fig. 17b,c for raw traces). (eg) Comparison between ChR2 and Chronos spike probability over three logs of blue irradiance. All pulsed illuminations used 10 pulses, 5 Hz, 5 ms pulse width. (e) Representative spiking traces at the indicated irradiances. (f) Spike probability vs. blue light irradiance, plotted for individual Chronos- or ChR2-expressing neurons and minimum irradiance threshold for 100% spiking (MIT100) as a function of GFP fluorescence. AU, arbitrary units. (g) Neuron spike threshold and resting potentials (n = 16–23 cells). Error bars, s.e.m.

  5. Independent optical excitation of neural populations in mouse cortical slice using Chrimson and Chronos.
    Figure 5: Independent optical excitation of neural populations in mouse cortical slice using Chrimson and Chronos.

    (ae) Spike and cross-talk characterization in opsin-expressing cells. Experimental optical configurations are depicted in a,f,j. (b) Chrimson and Chronos action spectra emphasizing (vertical shaded bars) the blue (470-nm) and red (625-nm) wavelengths used in this figure. (ce) Current-clamp characterizations of Chrimson or Chronos expressing neurons in slice to determine optimal irradiance range for two-color excitation. Chrimson-GFP and Chronos-GFP were independently expressed in cortical layer 2/3 neurons in separate mice. 5-ms, 5-Hz light pulses were used; n = 7 cells from 3 animals for Chrimson; n = 11 cells from 4 animals for Chronos. (c,d) Spike probability vs. irradiance for red (c) and blue light (d). The blue vertical shaded bar represents the blue irradiance range where Chronos drove spikes at 100% probability and no cross-talk spike was ever observed for any Chrimson neurons. (e) Chrimson subthreshold cross-talk voltage in individual neurons vs. blue irradiances; compare to Figure 4b. (fi) Postsynaptic currents (PSCs) in non-opsin-expressing neurons downstream of Chrimson and Chronos expressing neurons in brain slice with both opsins introduced into separate neural populations. Stimulation parameters: 0.3 mW/mm2 for blue, 4 mW/mm2 for red, 5-ms pulses; 6 neurons from 3 animals. All synaptic transmission slice experiments were done using wide-field illumination (Supplementary Fig. 18). (g) Triple-plasmid electroporation scheme for mutually exclusive Chrimson and Chronos expression in different sets of layer 2/3 cortical pyramidal cells. (h) Histology of intermingled Chrimson- (red) and Chronos-expressing (blue) neurons in layer 2/3 (left, taken at 10× magnification) and their axons (right, taken at 60× magnification). Scale bars: 100 μm (left), 20 μm (right). (i) PSCs in response to optical Poisson stimulation with blue and red light; shown are raw voltage traces (gray) with average trace (black) from a single neuron experiencing blue (top), red (center) or both (bottom) light pulses. (PSC traces from neurons downstream of mutually exclusive Chrimson- and Chronos-expressing neurons in response to blue or red light are in Supplementary Fig. 18e.) (jm) PSCs in non-opsin-expressing neurons downstream of Chronos- or Chrimson-expressing neurons. Conditions are as in fi, except pulses were delivered at 0.2 Hz. n = 7 cells from 2 animals for Chronos; n = 12 cells from 4 animals for Chrimson. The black trace is the averaged response; gray traces are individual trials. (k) Chronos-driven PSCs under blue or red light, obtained from a representative neuron (left), with population data (right). (l) Chrimson-driven PSCs under blue or red light traces, obtained from a representative neuron (left), with population data (right). (m) Chrimson-driven PSC amplitudes (top) and the probability of observing a PSC at all (bottom) vs. blue irradiances.

Videos

  1. Experimental setup with a visual arena
    Video 1: Experimental setup with a visual arena
    The fly was tethered and centered in the visual arena. In this movie, a flowing random dot pattern is shown. The visual arena was removed from the setup in other conditions. Fly behavior was recorded using a camera with 850 nm IR illuminator.
  2. PER of a Gr64f X CsChrimson fly to 720 nm light in darkness
    Video 2: PER of a Gr64f X CsChrimson fly to 720 nm light in darkness
    A fly with CsChrimson expression in sugar receptors shows PER to deep red light stimulation.
  3. Startle response to 720 nm light in darkness
    Video 3: Startle response to 720 nm light in darkness
    A control fly without CsChrimson expression shows clear startle response to deep red light.
  4. PER of a Gr64f X CsChrimson fly to 720 nm light in a blue random dot arena
    Video 4: PER of a Gr64f X CsChrimson fly to 720 nm light in a blue random dot arena
    PER of a fly with CsChrimson expression in sugar receptors is not affected by visual distractors.
  5. Inhibited startle response to 720 nm light in a blue random dot arena
    Video 5: Inhibited startle response to 720 nm light in a blue random dot arena
    The startle response of a control fly without CsChrimson expression is effectively inhibited.
  6. Optogenetics in freely behaving intact flies
    Video 6: Optogenetics in freely behaving intact flies
    Top: Light-induced CO2 avoidance behavior (VT031497-Gal4 x UAS-CsChrimson in attP18). Bottom: A control group (WTB x UAS-CsChrimson in attP18). Circles show raw video images with false color red background indicating the illuminated quadrants. The effect of light is quantified (see Methods) and plotted as a single blue line corresponding to the presented examples and a plot representing the mean of all 9 sessions (±SEM error bars). Plots will be in red region if more than 50% of flies are in illuminated quadrants. Replay speed: 4x.

Accession codes

Primary accessions

NCBI Reference Sequence

Change history

Corrected online 28 August 2014
In the version of this article initially published, the Drosophila transgenic strains were incorrectly reported as generated using Chrimson (Fig. 3, Supplementary Figs. 14–16 and Supplementary Videos 2–6). Owing to a miscommunication, the Drosophila strains were actually generated with CsChrimson, a CsChR-Chrimson chimera replacing the Chrimson N terminus with the CsChR N terminus. CsChrimson has the same spectral and kinetic properties as Chrimson; these data have been added to the paper in the form of an addendum, and incorrectly listed strains have been fixed in the main text and supplementary materials. The error has been corrected in the HTML and PDF versions of the article.

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Author information

Affiliations

  1. The MIT Media Laboratory, Synthetic Neurobiology Group, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.

    • Nathan C Klapoetke,
    • Yong Ku Cho,
    • Tania K Morimoto,
    • Amy S Chuong &
    • Edward S Boyden
  2. Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA.

    • Nathan C Klapoetke,
    • Yong Ku Cho,
    • Tania K Morimoto,
    • Amy S Chuong &
    • Edward S Boyden
  3. MIT Center for Neurobiological Engineering, MIT, Cambridge, Massachusetts, USA.

    • Nathan C Klapoetke,
    • Yong Ku Cho,
    • Tania K Morimoto,
    • Amy S Chuong &
    • Edward S Boyden
  4. Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA.

    • Nathan C Klapoetke,
    • Yasunobu Murata,
    • Amanda Birdsey-Benson,
    • Yong Ku Cho,
    • Tania K Morimoto,
    • Amy S Chuong,
    • Martha Constantine-Paton &
    • Edward S Boyden
  5. MIT McGovern Institute for Brain Research, MIT, Cambridge, Massachusetts, USA.

    • Nathan C Klapoetke,
    • Yasunobu Murata,
    • Amanda Birdsey-Benson,
    • Yong Ku Cho,
    • Tania K Morimoto,
    • Amy S Chuong,
    • Martha Constantine-Paton &
    • Edward S Boyden
  6. Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.

    • Sung Soo Kim,
    • Stefan R Pulver &
    • Vivek Jayaraman
  7. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.

    • Eric J Carpenter &
    • Gane Ka-Shu Wong
  8. Beijing Genomics Institute-Shenzhen, Shenzhen, China.

    • Zhijian Tian,
    • Jun Wang,
    • Yinlong Xie,
    • Zhixiang Yan,
    • Yong Zhang &
    • Gane Ka-Shu Wong
  9. Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Brian Y Chow
  10. Institute of Botany, Cologne Biocenter, University of Cologne, Cologne, Germany.

    • Barbara Surek &
    • Michael Melkonian
  11. Department of Medicine, University of Alberta, Edmonton, Alberta, Canada.

    • Gane Ka-Shu Wong

Contributions

N.C.K., E.S.B., M.C.-P. and V.J. contributed to the study design and data analysis. G.K.-S.W. and B.Y.C. oversaw transcriptomic sequencing. E.S.B. and M.C.-P. supervised mammalian opto/electrophysiological parts of the project. N.C.K. coordinated all experiments and data analysis. N.C.K., Y.K.C., A.S.C. and T.K.M. conducted and analyzed all in vitro electrophysiology. M.M., B.S., N.C.K., T.K.M., E.J.C., Z.T., J.W., Y.X., Z.Y. and Y.Z. conducted algal RNA experiments or transcriptome sequencing and analysis. N.C.K., Y.M. and A.B.-B. performed and analyzed all slice electrophysiology. V.J. prepared CsChrimson for injection into Drosophila. S.S.K. and V.J. designed adult fly behavior experiments. S.S.K. performed all fly behavior experiments and data analysis. S.R.P. designed, performed and analyzed all larval Drosophila experiments. Correspondence should be addressed to V.J. (vivek@janelia.hhmi.org) for CsChrimson flies. All authors contributed to the discussions and writing of the manuscript.

Competing financial interests

B.Y.C., E.S.B., G.K.-S.W., N.C.K. and Y.K.C. are inventors on pending patents covering the described work. E.S.B. is an equity holder in Eos Neuroscience.

Corresponding authors

Correspondence to:

Author details

Supplementary information

Video

  1. Video 1: Experimental setup with a visual arena (3.42 MB, Download)
    The fly was tethered and centered in the visual arena. In this movie, a flowing random dot pattern is shown. The visual arena was removed from the setup in other conditions. Fly behavior was recorded using a camera with 850 nm IR illuminator.
  2. Video 2: PER of a Gr64f X CsChrimson fly to 720 nm light in darkness (2.46 MB, Download)
    A fly with CsChrimson expression in sugar receptors shows PER to deep red light stimulation.
  3. Video 3: Startle response to 720 nm light in darkness (3.19 MB, Download)
    A control fly without CsChrimson expression shows clear startle response to deep red light.
  4. Video 4: PER of a Gr64f X CsChrimson fly to 720 nm light in a blue random dot arena (2.27 MB, Download)
    PER of a fly with CsChrimson expression in sugar receptors is not affected by visual distractors.
  5. Video 5: Inhibited startle response to 720 nm light in a blue random dot arena (1.92 MB, Download)
    The startle response of a control fly without CsChrimson expression is effectively inhibited.
  6. Video 6: Optogenetics in freely behaving intact flies (5.96 MB, Download)
    Top: Light-induced CO2 avoidance behavior (VT031497-Gal4 x UAS-CsChrimson in attP18). Bottom: A control group (WTB x UAS-CsChrimson in attP18). Circles show raw video images with false color red background indicating the illuminated quadrants. The effect of light is quantified (see Methods) and plotted as a single blue line corresponding to the presented examples and a plot representing the mean of all 9 sessions (±SEM error bars). Plots will be in red region if more than 50% of flies are in illuminated quadrants. Replay speed: 4x.

PDF files

  1. Supplementary Text and Figures (8,030KB)

    Supplementary Figures 1–22 and Supplementary Tables 1–4

Additional data