Temporally precise single-cell-resolution optogenetics

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.

Figure 1. Soma-targeted optogenetics using the high-performance channelrhodopsin CoChR

(a, b) Concept of soma-targeted optogenetics. Untargeted opsins (a) express over the entire neural membrane. One can aim light at a given neural soma, but each cell body is surrounded by opsin-bearing neurites from other cells, resulting in artifactual activation of those cells. Restricting opsin expression to the cell body would prevent such side effects, enabling single cell optogenetic stimulation (b). (c–j) Images are presented for cultured hippocampal neurons expressing wild-type vs. selectively trafficked CoChR fused to GFP, along with the cytosolic countermarker mCherry. (c) A hippocampal neuron in culture expressing CoChR-GFP and mCherry, seen in the GFP channel (scale bar: 100 µm). (d) Zoomed-in image from the yellow rectangle of c. (e) The neuron of c, seen in the mCherry channel (magenta), along with surrounding neurons. (f) Merge of d and e (scale bar for d–f: 20 µm). (g–j) As in c–f, but for a neuron expressing CoChR-KA2(1–150)-GFP (soma-targeted CoChR-GFP, abbreviated soCoChR-GFP). (k) Whole cell current clamp recording of a cultured hippocampal neuron expressing CoChR-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. (l) As in k, but for a neuron expressing CoChR-KA2(1–150)-GFP (soCoChR-GFP). (m) 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-GFP (n = 7 neurites taken from 5 cells from 2 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. (n) As in m, but for neurons expressing CoChR-KA2(1–150)-GFP (soCoChR-GFP; n = 9 neurites taken from 7 cells from 3 cultures). **P < 0.01 and ***P < 0.001, Bonferroni-corrected Kolmogorov-Smirnov test of brightness between wild-type and soma-targeted; see Supplementary Table 3 for full statistics for Figure 1). (o) Representative photocurrents for cultured hippocampal neurons expressing CoChR-GFP (magenta) and CoChR-KA2(1–150)-GFP (soCoChR-GFP; black), under voltage clamp conditions, using 5 ms light pulses (480nm, 34.84 mW/mm²; light blue rectangle). (p) Box-and-whiskers plot showing peak current amplitudes (pA) for CoChR-GFP and CoChR-KA2(1–150)-GFP (soCoChR-GFP) in cultured hippocampal neurons illuminated with blue light (480 nm, 34.84 mW/mW², 5 ms; n = 13 neurons from 3 cultures and 13 neurons from 3 cultures for CoChR-GFP and CoChR-KA2(1–150)-GFP 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. Not significant; Wilcoxon rank sum test; see Supplementary Table 3 for full statistics. (q) Box-and-whiskers plot showing photocurrent decay time (τoff, in ms) for CoChR-GFP and CoChR-KA2(1–150)-GFP (soCoChR-GFP) in cultured hippocampal neurons illuminated with blue light (480 nm, 34.84 mW/mW², 5 ms; n = 13 neurons from 3 cultures and 13 neurons from 3 cultures for CoChR-GFP and CoChR-KA2(1–150)-GFP 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. ***P < 0.0001; Wilcoxon rank sum test; see Supplementary Table 3 for full statistics.

Figure 2. Zero-spike crosstalk single cell optogenetic control of cultured neurons with one-photon illumination

(a,b) Schematic of the experiment. Cultured hippocampal neurons two weeks after transduction with AAV8-Syn-CoChR-GFP (untargeted CoChR, a) or AAV8-Syn-soCoChR-GFP (somatic CoChR, b). One cell in the field of view was patched (current clamp). The patched cell along with 9 neighboring cells were photo-stimulated using a digital micromirror device (DMD) consecutively. (c) Cultured hippocampal neurons expressing CoChR-GFP; regions of stimulation are highlighted by magenta circles. Numbers denote the order of stimulation. Scale bar: 20 µm. (d) As in c, but for soCoChR-GFP. (e) Representative current-clamp recording for a cultured hippocampal neuron expressing CoChR-GFP. Cells were photostimulated sequentially using the DMD (470 nm, 40.7 mW/mm², 1 ms light pulse). The order of the photo-stimulation, as indicated by the numbers in c, is given on the x-axis. The patched cell is numbered 1. The distance of cells 2–9 from the patched cell is given below each cell number, in microns. (f) As in e, but for soCoChR-GFP. (g) Box-and-whiskers plot showing the percentage of cultured hippocampal neurons which fired an action potential (AP) upon direct photostimulation, using light pulses as above (n = 5 cells from 4 cultures for CoChR-GFP; n = 5 cells from 5 cultures for soCoChR-GFP). Red line denotes the median. (h) Box-and-whiskers plot showing, averaged across all patched neurons, the percentage of photostimulated neighboring cells that yielded an AP in each patched cell (n = 5 cells from 4 cultures for CoChR-GFP; n = 5 cells from 5 cultures for soCoChR-GFP). 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. *P=0.0476, Wilcoxon rank sum test; Supplementary Table 3 for full statistics. (i) Box-and-whiskers plot showing, averaged across all patched neurons, the percentage of photostimulated neighboring cells that yielded a depolarization in each patched cell (n = 5 cells, in 4 cultures for CoChR-GFP; n = 5 cells, in 5 cultures for soCoChR-GFP). Red line denotes the median. Top and bottom edges of the box indicate the 75th and 25th percentiles, respectively. **P=0.0079, Wilcoxon rank sum test; Supplementary Table 3 for full statistics.

Figure 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 slice

(a–b) Schematic representation of the two experimental setups used for holographic illumination. In setup 1 (a), holographic photostimulation was achieved using an amplified fiber laser and was coupled with a two-photon (2P) scanning imaging system. In setup 2 (b) holographic photostimulation was achieved using a conventional Ti:Sapphire pulsed laser and was coupled with a widefield epifluorescence imaging system. Detailed descriptions of the setups are reported in Methods section and Supplementary Figure 6. (c) Schematic of 2P holographic stimulation along one neurite, showing Alexa 594 fluorescence (obtained via 2P scanning at 780 nm) from dye injected into a patched CoChR-GFP expressing neuron. The Alexa 594 fluorescence was used to guide holographic spot placement (red circles) to different points along a neurite, at different distances from the soma. Red arrow indicates the order of photostimulation. The bright emission to the left of the cell represents the patch pipette filled with Alexa 594 (scale bars: 50 µm along all three axes, image acquired with setup 1). (d) Representative whole-cell currents recorded from a CoChR-GFP expressing neuron (left) and a soCoChR-GFP expressing neuron (right; some 50 Hz electrical noise is apparent in the traces on the right), when illuminated with a power density corresponding to the spiking threshold power density (18 µW/µm² and 101 µW/µm² for the CoChR-GFP and the soCoChR-GFP expressing cells respectively; λ = 1030 nm, using setup 1). (e) Bar plot of the integral of the elicited photocurrent, normalized to that obtained with the spot at the soma, as a function of distance from the soma, for CoChR-GFP (blue bars) and soCoChR-GFP (red bars) expressing neurons (spacing between spots ~10 µm, λ = 1030 nm or 920 nm for setup 1 or setup 2, respectively; data were pooled across both setups). Bars reports mean ± s.e.m. Dots denote values for single neurites. For each cell, the photostimulation was done at the power density threshold determined for that cell (average powers: 29±10 µW/µm² for CoChR-GFP expressing cells; 90±60 µW/µm² for soCoChR-GFP expressing cells; power values referred to setup 1, see Methods). The normalized current integral was significantly higher in CoChR-GFP relative to soCoChR-GFP expressing cells for distances of 30 µm or more from the soma. ** P<0.0017 for distances ≥ 30 µm; Kolmogorov-Smirnov (KS) test with Bonferroni correction (n=16 neurites from 8 CoChR-GFP cells from 7 mice; n=27 neurites from 16 soCoChR-GFP cells from 13 mice; see Supplementary Table 4 for full statistics for Fig. 3). Data obtained using both setup 1 and setup 2.

Figure 4. 2P power necessary to enable millisecond control of cortical neuron activation in mouse brain slice

(a) Rise time of soCoChR-GFP (red) and CoChR-GFP (blue)-mediated photocurrents, measured in mouse brain slice cortical neurons, as a function of 2P stimulation power (n = 4–12 photostimulation powers per cell, 6 cells from 6 mice for CoChR-GFP; n = 2–11 photostimulation powers per cell, for 5 cells from 5 mice for soCoChR-GFP). Data collected from setup 1 are plotted with circles, and correspond to the power scale on the bottom x-axis; data collected from setup 2 are plotted with asterisks, and correspond to the top x-axis. The length of the top x-axis was rescaled with respect to the bottom x-axis by a power conversion factor k = 5.3 (see Methods). Lines connect data acquired from the same neuron. Blue and red arrows indicate average threshold powers relative to setup 1 for CoChR-GFP and soCoChR-GFP expressing neurons respectively (for CoChR-GFP: 28 ± 10 µW/µm², n = 7 cells from 5 mice; for soCoChR-GFP: 83 ± 39 µW/µm, n = 4 cells from 4 mice). (b) The AP latency, defined as the time from the onset of 2P stimulation to the peak of the AP, plotted vs. 2P stimulation power in setup 1 units (n = 3–5 powers per cell, for 4 cells from 3 mice for CoChR-GFP, blue dots; n = 3–5 powers per cell, for 3 cells from 3 mice for soCoChR-GFP, red dots). Lines connect data acquired from the same neuron. Horizontal dashed line denotes 15 ms latency, and vertical dashed line denotes 70 µW/µm² stimulation power, for comparison to panel c. Setups 1 and 2 were used interchangeably; power values used on setup 2 were scaled to equivalent power values for setup 1 (see Methods). See (c) for description of the gray area. (c) AP temporal jitter (measured as standard deviation of the AP latency across a series of 5 photostimulations for a given cell) plotted as a function of AP latency (n = 3–5 powers per cell, for 4 cells from 3 mice for CoChR-GFP, blue dots; n = 3–5 powers per cell, for 3 cells from 3 mice for soCoChR-GFP, red dots). Lines connect data acquired from the same neuron. Setups 1 and 2 were used interchangeably. Jitter below 1 ms (horizontal dashed line) was ensured when the latency was kept below 15 ms (vertical dashed line), corresponding to a photostimulation power higher than approximately 70 µW/µm². The gray areas in both (b) and (c) mark the region at which such conditions were satisfied.

Figure 5. Soma-targeted CoChR enables single cell control with 3D photoactivation in brain slices

(a) Schematic of 3D holographic activation. One cell expressing CoChR-GFP or soCoChR-GFP was patched, then neighboring cells at different z-planes were illuminated with 10–14 µm diameter holographic spots. (b) Reconstruction of multi-spot 3D holographic pattern obtained by measuring induced fluorescence from a thin layer of rhodamine 6G (spin coated on a glass coverslip), imaged through a second microscope objective; spot diameter: 10 µm. Scale bars: 35 µm along all three axes. (c) 2P images (imaging λ = 920 nm) from a 3D z-stack used to draw 10–14 µm holographic spots (λ = 1030 nm, setup 1) on neighboring cells expressing CoChR-GFP (left, yellow circles numbered 1–7) or soCoChR-GFP (right, yellow circles numbered 1–7) and on the patched cell (yellow circle, numbered 8), inside a volume of approximately 200×200×70 µm (scale bars: 50 µm). (d) Whole-cell recording of a CoChR-GFP expressing cell (left) and a soCoChR-GFP expressing cell (right) while sequentially positioning the holographic spot on neighboring cells one at a time (represented in panel (c); λ = 1030 nm, setup 1, photostimulation power: 100 µW/µm² for CoChR-GFP and for soCoChR, 30 ms duration). The distance of cells 1–7 from the patched cell 8 is given below each cell number, in gray. (e) Bar plot showing the percentage of neighboring cells that, when stimulated, yielded an AP in a given patched cell (and averaged across all patched cells; n = 7 cells from 6 mice for CoChR-GFP; n = 7 cells from 7 mice for soCoChR-GFP. Values are mean ± s.e.m.). Dots denote values for single cells. λ = 1030 nm, setup 1, average photostimulation power: CoChR-GFP 142±24 µW/µm², soCoChR-GFP: 173±26 µW/µm², 30 ms duration. **P < 0.01, Wilcoxon rank sum test (P=0.004). (f) Whole-cell recording of a CoChR-GFP expressing cell (left) and a soCoChR-GFP cell (right; both cells presented in d) during simultaneous photostimulation of the neighboring cells represented in c, without a stimulation spot on the soma of the patched cell. Photostimulation power density for each spot was equal to the one used in d (λ = 1030 nm, setup 1, photostimulation power: 100 µW/µm² for CoChR-GFP and for soCoChR-GFP, 30 ms duration, red dash under traces). (g) Bar plot showing the percentage of simultaneous photostimulations of neighboring cells that yielded APs in a given patched cell (averaged across all patched cells). Grey bars indicate the generation of 1 AP; blue bars indicate the generation of more than one AP (n = 7 cells expressing CoChR-GFP from 6 mice; n = 7 cells expressing soCoChR-GFP from 7 mice). Values are mean ± s.e.m. Dots denote values for single cells. λ = 1030 nm, setup 1, average photostimulation power: 142±24 µW/µm² and 173±26 µW/µm² for CoChR-GFP expressing cells and for soCoChR-GFP expressing cells respectively; 30 ms duration. For generation of 1AP: *P < 0.05; χ²-test (χ² = 3.97, P = 0.046, df=1). For generation of more than one AP, χ² = 3.15, P = 0.08, df=1. Average distances between patched cells and neighboring cells are reported in Supplementary Figure S12 and Supplementary Table 4.

Figure 6. 2P holographic stimulation of soCoChR enables mapping of functional connectivity in brain slices

(a) Schematic of the connectivity experiment: a 2P image (λ = 920 nm, setup 1) of the volume around a patched opsin positive cell (yellow spot), expressing CoChR-GFP (top) or soCoChR-GFP (bottom), is used to sequentially position a 10–14 µm holographic spot (red spots) on opsin expressing nearby cells and to calculate the corresponding phase masks. Scale bar: 40 µm. (b) Representative currents recorded in whole-cell configuration from a CoChR-GFP (top) or soCoChR-GFP (bottom) expressing cell while sequentially positioning the holographic spot on neighboring cells. Red bars represent the photostimulation periods for each cell (three light pulses at 50 Hz of 10 ms duration). Photostimulation power: 120 µW/µm² for CoChR-GFP; 150 µW/µm² for soCoChR-GFP; λ = 1030 nm, setup 1. Black traces: currents recorded before the perfusion of receptor blockers (average of three trials on each spot position). Blue traces: artifactual currents recorded after the perfusion of receptor blockers (average of three trials on each spot position). (c) Bar plots showing the peak amplitudes of currents sensitive to receptor blockers (PSCs; gray bars), and the peak amplitudes of artifactual currents recorded after the perfusion of receptor blockers (I_ART, blue bars) (n = 4 CoChR-GFP cells from 4 mice; n = 5 soCoChR-GFP cells from 5 mice). Currents were recorded in whole-cell configuration from cells expressing CoChR-GFP or soCoChR-GFP while sequentially positioning the holographic spot on neighboring cells one at a time (λ = 920 or 1030 nm, setup 1 or 2; photostimulation power (relative to setup 1): 130 ± 30 mW/µm² for CoChR-GFP, and 180 ± 20 mW/µm² for soCoChR-GFP; three light pulses at 50 Hz of 10 ms duration). For mean peak current comparison: *P < 0.05; Wilcoxon rank sum test (P=0.015). (d) Bar plots showing the ratio PSC / I_ART (n = 54 out of 78 photostimulated neighboring cells triggering PSCs in the patched cell, from 4 CoChR-GFP cells from 4 mice; n = 15 out of 109 photostimulated neighboring cells triggering PSCs in the patched cell from 5 soCoChR-GFP cells from 5 mice). Currents were recorded in whole-cell configuration from cells expressing CoChR-GFP or soCoChR-GFP while sequentially positioning the holographic spot on neighboring cells one at a time (λ = 920 or 1030 nm, setup 1 or 2; photostimulation power (relative to setup 1): 130 ± 30 mW/µm² for CoChR-GFP, and 180 ± 20 mW/µm² for soCoChR-GFP; three light pulses at 50 Hz of 10 ms duration). For comparison of ratio PSC / I_ART: **P < 0.01; Wilcoxon rank sum test (P = 0.003).