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Temporally precise single-cell-resolution optogenetics

Nature Neuroscience volume 20, pages 1796–1806 (2017)Cite this article

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A Publisher Correction to this article was published on 16 March 2018

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Abstract

Optogenetic control of individual neurons with high temporal precision within intact mammalian brain circuitry would enable powerful explorations of how neural circuits operate. Two-photon computer-generated holography enables precise sculpting of light and could in principle enable simultaneous illumination of many neurons in a network, with the requisite temporal precision to simulate accurate neural codes. We designed a high-efficacy soma-targeted opsin, finding that fusing the N-terminal 150 residues of kainate receptor subunit 2 (KA2) to the recently discovered high-photocurrent channelrhodopsin CoChR restricted expression of this opsin primarily to the cell body of mammalian cortical neurons. In combination with two-photon holographic stimulation, we found that this somatic CoChR (soCoChR) enabled photostimulation of individual cells in mouse cortical brain slices with single-cell resolution and <1-ms temporal precision. We used soCoChR to perform connectivity mapping on intact cortical circuits.

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Fig. 1: Soma-targeted optogenetics using the high-performance channelrhodopsin CoChR.
Fig. 2: Zero-spike crosstalk single-cell optogenetic control of cultured neurons with 1P illumination.
Fig. 3: Photocurrents of neurites expressing soCoChR-GFP are significantly smaller than in neurites expressing CoChR-GFP, in neurons virally expressing these opsins in mouse cortical brain slices.
Fig. 4: 2P power necessary to enable millisecond control of cortical neuron activation in mouse brain slice.
Fig. 5: Soma-targeted CoChR enables single-cell control with 3D photoactivation in brain slices.
Fig. 6: 2P holographic stimulation of soCoChR enables mapping of functional connectivity in brain slices.

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  • 16 March 2018

    In the supplementary information originally posted online, Supplementary Tables 1–5 and the Supplementary Note were missing. The error has been corrected online.

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Acknowledgements

We thank M. Gajowa for participating in preliminary opsin screening, F. Simony and M. Gajowa for help with viral injections, C. Tourain for technical support in building the holographic system, V. de Sars for software development and J. Cécile for help with CHO cell culture and transfection. V.E. thanks the Agence Nationale de la Recherche (ANR-10-INBS-04-01, France-BioImaging Infrastructure network; ANR-14-CE13-0016, Holohub), the National Institutes of Health (NIH 1-U01-NS090501-01), the FRC and the Rotary Club through the program Espoir en Tete and the Getty Lab. O.A.S. thanks the Simons Foundation for the Social Brain Fellowship and the ISEF (International Sephardic Educational Foundation) for an ISEF postdoctoral fellowship. This research was also developed with funding from the Defense Advanced Research Projects Agency (DARPA), contract No. N66001-17-C-4015. The views, opinions and/or findings expressed are those of the author and should not be interpreted as representing the official views or policies of the Department of Defense or the US Government. V.E. and E.S.B. thank the Human Frontiers Science Program (Grant RGP0015/2016) for financial support. E.S.B. additionally acknowledges, for funding, John Doerr, the Open Philanthropy Project, the HHMI-Simons Faculty Scholars Program, NIH R44EB021054, the MIT Media Lab, NIH 1R24MH106075, NIH 2R01DA029639, NIH 1R01NS087950, NIH 1R01MH103910, NIH Director’s Pioneer Award 1DP1NS087724 and NIH 1R01GM104948.

Author information

Author notes

  1. Or A. Shemesh, Dimitrii Tanese and Valeria Zampini contributed equally to this work.

Authors and Affiliations

  1. Media Laboratory, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA

    Or A. Shemesh, Changyang Linghu, Kiryl Piatkevich & Edward S. Boyden

  2. Department of Biological Engineering, MIT, Cambridge, MA, USA

    Or A. Shemesh, Changyang Linghu, Kiryl Piatkevich & Edward S. Boyden

  3. Center for Neurobiological Engineering, MIT, Cambridge, MA, USA

    Or A. Shemesh, Changyang Linghu, Kiryl Piatkevich & Edward S. Boyden

  4. Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA

    Or A. Shemesh, Changyang Linghu, Kiryl Piatkevich & Edward S. Boyden

  5. McGovern Institute for Brain Research, MIT, Cambridge, MA, USA

    Or A. Shemesh, Changyang Linghu, Kiryl Piatkevich & Edward S. Boyden

  6. Neurophotonics Laboratory, Wave Front Engineering Microscopy Group, CNRS UMR8250, Université Paris Descartes, Paris, France

    Dimitrii Tanese, Valeria Zampini, Emiliano Ronzitti, Eirini Papagiakoumou & Valentina Emiliani

  7. Institut de la Vision, UM 80, UPMC, Paris, France

    Valeria Zampini & Emiliano Ronzitti

  8. Institut national de la santé et de la recherche médicale (Inserm), Paris, France

    Eirini Papagiakoumou

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Contributions

O.A.S. designed, screened and tested soma-targeted opsins. O.A.S., C.L. and K.P. performed and analyzed 1P experiments in cultured cells. V.Z. and D.T. performed and analyzed 2P experiments in cultured cells and brain slices. V.Z. performed and optimized virus injection and implemented electrophysiological recording on the 2P rigs. E.R. and E.P. designed and built up setup 1. D.T. designed and built up setup 2, optimized multicell stimulation and designed calibration procedures. O.A.S., E.S.B., D.T., V.Z. and V.E. interpreted data, designed experiments, and wrote the paper with contributions from all authors. E.S.B. and V.E. conceived and supervised the project.

Corresponding authors

Correspondence to Edward S. Boyden or Valentina Emiliani.

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Competing interests

O.A.S., E.S.B. and C.L. are inventors on pending patents covering the described work.

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Integrated supplementary information

Supplementary Figure 1 An N-terminal domain of kainate receptor subunit 2 is retained in the soma

(a) A hippocampal neuron expressing GFP. (b) A hippocampal neuron expressing KA2-GFP. (c) A hippocampal neuron expressing KA2(1-150)-GFP. Scale bars for a-c: 50 µm. (d, e, f) Box-and-whiskers plots of GFP brightness versus position along a neurite, normalized to GFP brightness at the soma for GFP (n = 5 neurites from 3 cells), KA2-GFP (n = 5 neurites from 3 cells) and KA2(1-150)-GFP (n = 5 neurites from 3 cells) respectively. Red line denotes the median. Top and bottom edges of the box indicate the 75th and 25th percentiles, respectively. Top and bottom whiskers indicate the highest and lowest values respectively. See Supplementary Table 2 for Bonferroni-corrected Kolmogorov-Smirnov tests for Figure S1.

Supplementary Figure 2 CoChR-NaV1.2(II–III)-GFP expression and photostimulation in cultured hippocampal neurons

(a-d) Images are presented for cultured hippocampal neurons expressing CoChR-NaV1.2(II-III)-GFP, along with the countermarker mCherry. (a) A hippocampal neuron in culture expressing CoChR-NaV1.2(II-III)-GFP and mCherry, seen in the GFP channel (scale bar: 100 µm). (b) Zoomed-in image from the yellow rectangle of a. (c) The neuron of b, seen in the mCherry channel (magenta), along with other nearby neurons. (d) Merge of b and c (scale bar for b-d: 20 µm). (e) Whole cell current clamp recording of a cultured hippocampal neuron expressing CoChR-NaV1.2(II-III)-GFP, under current injection (10 ms duration; gray rectangle) and optical stimulation (480nm, 34.84mW/mm2, 1 ms duration; blue rectangle). Rectangles not to scale. (f) Box-and-whiskers plot of GFP brightness versus position along a neurite, normalized to GFP brightness at the soma, extracted from neurites of cultured hippocampal neurons expressing CoChR-NaV1.2(II-III)-GFP (n = 5 neurites taken from 5 cells from 4 cultures). Red line denotes the median. Top and bottom edges of the box indicate the 75th and 25th percentiles, respectively. Top and bottom whiskers indicate the highest and lowest values respectively.

Supplementary Figure 3 CoChR-GFP-Kv2.1-motif expression is somatodendritic

(Ai, Aii) Hippocampal neurons expressing CoChR-GFP-Kv2.1motif. (B, C) Box-and-whisker plots of brightness versus position along a neurite, normalized to brightness at the soma for CoChR-GFP-Kv2.1motif (B, n = 5 neurites from 5 cells from 2 cultures) and CoChR-GFP (C, n = 7 neurites from 5 cells from 2 cultures). Top and bottom edges of the box indicate the 75th and 25th percentiles, respectively. Red line denotes the median. Top and bottom whiskers indicate the highest and lowest values respectively. n.s., not significant, comparing B vs. C; see Supplementary Table 3 for Bonferroni-corrected Kolmogorov-Smirnov tests for Figure S3. Scale bar: 20µm.

Supplementary Figure 4 In vitro single photon characterization of CoChR and soCoChR

(a) Action spectra for CoChR (n = 9 cells) and soCoChR (n = 10 cells) measured in cultured neurons. Plotted data are mean ± s.e.m. (b) Blue light driven spike probability as a function of irradiance for CoChR (magenta) and soCoChR (black) expressing cells. Plotted data are mean ± s.e.m (n = 6 cells for each opsin). All blue light spiking protocols used 2 ms width light pulses at 480 nm. (c) Blue light driven photocurrents as a function of irradiance for CoChR (magenta) and soCoChR (black) expressing cells. Plotted data are mean ± s.e.m (n = 7 cells for each opsin). All blue light protocols used 2 ms pulse width at 480 nm. (d) Blue light driven spike fidelity for CoChR (magenta) and soCoChR (black) expressing cells. All blue light spiking protocols used a train of 40 pulses, 2 ms pulse width, at 480 nm, at 5 mW/mm2. Plotted data are mean ± s.e.m (n = 7 – 10 cells for each opsin). See Supplementary Table 3 for full statistics for Figure S4.

Supplementary Figure 5 Membrane properties of neurons expressing somatic vs. untargeted molecules

Cultured hippocampal neurons expressing CoChR-GFP (n = 10 cells from 3 cultures), KA2(1-150)-GFP (n = 10 cells from 3 cultures), and soCoChR-GFP (n = 10 cells from 3 cultures) were patched 14 days following AAV transduction. (a) Resting potential. We performed a Kruskal-Wallis Test (not significant, P = 0.7549, chi-square = 0.5624). (b) Holding current. Kruskal-Wallis Test, not significant, P = 0.9875, chi-square = 0.0252. (c) Membrane capacitance. Kruskal-Wallis Test, not significant, P = 0.9817, chi-square = 0.0369. (d) Membrane resistance. Kruskal-Wallis Test, not significant, P = 0.9205, chi-square = 0.1656. For all panels, red line denotes the median. Top and bottom edges of the box indicate the 75th and 25th percentiles, respectively. Top and bottom whiskers indicate the highest and lowest values.

Supplementary Figure 6 Detailed schematics of the holographic microscopes, for both setup 1 and setup 2

PC, Polarizer Cube; L/2, Half Wave Plate; M, Mirror; L, Lens; SLM, Spatial Light Modulator; CL, Cylindrical Lens; D, Dichroic; GM, Galvanometric Mirrors; F, Filter; OBJ, Objective; C, Condenser; PoC, Pockels Cell; CCD, charge-coupled device; sCMOS, scientific complementary metal-oxide semiconductor camera. See detailed description in Methods section.

Supplementary Figure 7 2P action spectrum and normalized excitation cross section of CoChR

(a) Normalized peak current of CoChR as a function of wavelength, measured in cultured CHO cells. Photostimulation was performed using a 2P Gaussian spot (FWHM = 38 µm) at different wavelengths, at constant photon flux (2.4 ± 0.3 1026 photons/s·m²) over the investigated wavelength range. Plotted data are mean ± s.e.m., with raw data in open circles, n = 5 cells. (b) Normalized 2P excitation cross section, extracted from the photocurrent kinetics (see Methods). Plotted data are mean ± s.e.m., with raw data in open circles, n = 5 cells.

Supplementary Figure 8 Axial resolution of 2P holographic spots

Top (a) and lateral (b) view of the fluorescence emission generated by a 10 µm diameter holographic spot on a thin fluorescent layer. The fluorescence is collected by a second bottom objective and relayed to a CCD camera; the z-stack is acquired by scanning the vertical position of the upper objective. Scale bar, 10 µm. Measured performed on setup 1 (λ = 1030 nm). (c) Normalized axial intensity profile from the z-stack of panel b. (d) Simulated axial dependence of the photocurrent’s peak at different excitation powers using the excitation spot of panels a,b. The black curve represents a Gaussian fit of the spot’s axial profile shown in panel c (FWHM = 17.5 µm). Colored curves represent the simulated axial dependence of the photocurrent for different excitation powers, taking into account the experimental photocurrent dependence on the excitation power shown in the inset (see Methods for more details on the model used) Inset: Black dots represent measured normalized photocurrent from a CoChR expressing CHO cell (λ = 1030 nm, setup 1), fitted by an exponential (red curve). (e) Experimental (violet dots; n = 2 CoChR expressing cells) and simulated (red line) photocurrent’s peak axial profile for the 10 µm diameter holographic spot of panels a,b using an excitation power of 80 µW/µm2. The blue curve is a Gaussian fit of the experimental data (FWHM = 43 µm). The black curve represents the Gaussian fit of the spot’s axial profile shown in panel c. (f) Lateral (x,z) view of the fluorescence emission generated by a 10 µm diameter holographic temporally focused spot on a thin fluorescent layer. Scale bar, 10 µm. Measurement performed on setup 2, modified by adding a diffraction grating for temporal focusing. (g) Experimental (violet dots) axial dependence of the photocurrent’s peak and Gaussian fit (blue line) generated by using the temporally focused spot shown in (f) (n = 1 CoChR expressing cell; n = 1 soCoChR expressing cell). The black curve represents the optical axial resolution of the temporally focused spot of panel f.

Supplementary Figure 9 Fluorescence plots along neurites, for cells expressing CoChR-GFP or soCoChR-GFP in slices

(a-b) Box-and-whiskers plots of GFP intensity versus position along a neurite, normalized to GFP intensity at the soma, extracted from 2P images (setup 1) of neurites of (a) CoChR-GFP (n = 9 neurites taken from 4 cells from 3 slices from 3 mice) and (b) CoChR-KA2(1-150)-GFP expressing cells (soCoChR-GFP; n = 9 neurites taken from 6 cells from 2 slices from 2 mice) in acute slices. Red line denotes the median. Top and bottom edges of the box indicate the 75th and 25th percentiles, respectively. Top and bottom whiskers indicate the highest and lowest values, respectively. For CoChR the GFP fluorescence decays slower than the corresponding current (Fig. 3e); this is probably due to a contribution to the fluorescence from the dense distribution of out of focus processes from neighbouring cells.

Supplementary Figure 10 Absolute currents at different distances from the soma for CoChR- and soCoChR-expressing cells in slices

A bar plot showing the peak currents induced by illumination of neurites at different distances from the soma (corresponding to experiments reported in Fig. 3e), for CoChR-GFP (blue bars) and soCoChR-GFP (red bars) -expressing neurons. Bars represent mean ± s.e.m.; dots denote values for single neurites.

Supplementary Figure 11 Multi-spot holographic illumination and diffraction efficiency correction

(a) Max projection of the fluorescence generated by a sequence of 15 µm diameter holographic spots placed on a square grid. The generation of a variable size (from 0 to 15 µm) correction holographic spot (not shown) is applied to correct for the position-dependent diffraction efficiency (see Methods). Scale bar: 30 µm. (b) Vertical and horizontal intensity profiles corresponding to panel a with and without the correction of diffraction efficiency, averaged over the 5 columns and lines of the matrix, respectively. (c) Fluorescence generated by the simultaneous projection of 7 randomly placed holographic spots with equal intensity, corrected for diffraction efficiency. Scale bar: 30 µm. (d) Graded input pattern used to calculate the hologram generating the illumination pattern in panel c (see Methods). Scale bar: 30 µm. (e) Axial intensity profile of the fluorescence induced by a 10 µm holographic spot displaced over different axial positions relative to the microscope objective focal plane without diffraction efficiency correction (black curves) and with the correction (red curve) using an external correction spot (not shown). (f) Superposition of the axial intensity profiles of panel e (with correction of diffraction efficiency), after being centered at z = 0. The black curve represents the average profile with FWHM = 18.5 µm. (g) Axial intensity profile of the multispot holographic pattern reported in Figure 5b. The black curve represents the average profile with FWHM = 17.5 µm. (h) Lateral view of the 3D fluorescence pattern generated by 3 holographic spots (diameter 10 µm) of equal intensity at axial positions of z = -120 µm; 0; + 120µm. Scale bar: 30 µm. (i) Graded input pattern used to calculate the hologram generating the illumination pattern in panel h. As in panel d, different input intensities were assigned to each spot to compensate for the diffraction efficiency dependence on the vertical position and to obtain the same output power density on each spot. (j) Superposition of the axial intensity profiles of the spots of panel h. The black curve represents the average profile with FWHM = 18 µm.

Supplementary Figure 12 Average distances between the soma of the patched cell and the somata of neighboring photostimulated cells

This boxplot corresponds to experiments reported on Fig. 5e,g. Blue and red symbols represent average distances between the several photostimulated neighboring cells (n = 5 to 8 cells) and the patched cells (7 patched cells expressing CoChR-GFP from 6 mice, blue symbols; 7 patched cells expressing soCoChR-GFP from 7 mice, red symbols). Horizontal dashed lines represent the average distance over all experiments (n = 7 patched CoChR-GFP expressing cells, n = 7 patched soCoChR-GFP expressing cells). The height of the rectangles represents two standard deviations of the distribution of the average distances over different experiments. The differences in the distances in the two groups were not significant under a Wilcoxon rank sum test. Statistics are reported in Supplementary Table 4.

Supplementary Figure 13 Mapping of functional connectivity in brain slices

(a) Currents recorded in whole-cell configuration from a CoChR-GFP expressing cell (same patched cell shown in Fig. 6b) while sequentially positioning the holographic spot on neighboring cells (λ = 920 nm, setup 2, photostimulation power: 120 µW/µm²; three light pulses at 50 Hz of 10 ms duration). Currents corresponding to neighboring cells numbered 3, 5 and 18 (green numbers) are shown in Fig. 6. The distances between each neighboring cell and the patched cell are reported next to each curve (grey numbers). Each trace is the average of three trials. Currents were recorded before the perfusion of synaptic receptor blockers (I TOT ) and after the perfusion of synaptic receptor blockers (I ART ). The current component sensitive to application of synaptic receptor blockers was: PSCs = I TOT - I ART . Black traces: I TOT where the ratio PSCs/I ART  > 1. Gray traces: I TOT where the ratio PSCs/I ART  < 1. Red traces: I TOT where no PSCs were observed (I TOT  = I ART ). Blue traces: I ART . (b) Currents recorded in whole-cell configuration from a soCoChR-GFP expressing cell while sequentially positioning the holographic spot on neighboring cells (λ = 1030 nm, setup 1, photostimulation power: 120 µW/µm²; three light pulses at 50 Hz of 10 ms duration). Currents corresponding to neighboring cells numbered 6, 9 and 12 are shown in Fig. 6. The distances between each neighboring cell and the patched cell are reported next to each curve (grey numbers). Color code as in panel (a).

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Shemesh, O.A., Tanese, D., Zampini, V. et al. Temporally precise single-cell-resolution optogenetics. Nat Neurosci 20, 1796–1806 (2017). https://doi.org/10.1038/s41593-017-0018-8

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