Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation

Abstract

Optogenetics with microbial opsin genes has enabled high-speed control of genetically specified cell populations in intact tissue. However, it remains a challenge to independently control subsets of cells within the genetically targeted population. Although spatially precise excitation of target molecules can be achieved using two-photon laser-scanning microscopy (TPLSM) hardware, the integration of two-photon excitation with optogenetics has thus far required specialized equipment or scanning and has not yet been widely adopted. Here we take a complementary approach, developing opsins with custom kinetic, expression and spectral properties uniquely suited to scan times typical of the raster approach that is ubiquitous in TPLSMlaboratories. We use a range of culture, slice and mammalian in vivo preparations to demonstrate the versatility of this toolbox, and we quantitatively map parameter space for fast excitation, inhibition and bistable control. Together these advances may help enable broad adoption of integrated optogenetic and TPLSMtechnologies across experimental fields and systems.

Figure 1

Two-photon control of spiking with C1V1 variants in culture. (a) C1V1 mutants (white bands: mutations). (b) C1V1 and ChR2_HR single-photon photocurrents (C1V1, 1,075 ± 74 pA (n = 7); C1V1_T, 1,474 ± 130 pA (n = 18); C1V1_T/T, 1,175 ± 58 pA (n = 18); ChR2_HR, 1,075 ± 74 pA (n = 12), *P = 0.02; see Online Methods for parameters; Mann-Whitney two-tailed test; error bars, s.e.m.). (c) C1V1_T-ts-EYFP–expressing hippocampal neuron. Purple square: typical ROI. (d) TPLSM photocurrents. Bars indicate scan times; typical ROIs were 15 × 15 μm to 20 × 20 μm. (e) τ_off values (C1V1, 105 ± 1.1 ms (n = 6); C1V1_T, 60 ± 1.9 ms (n = 6); C1V1_T/T, 40 ± 1.3 ms (n = 5)). (f) TPLSM photocurrents for varying NA (C1V1: 40×/0.8, 239 ± 21 pA (n = 17), 20×/0.5, 490 ± 44 pA (n = 6), P = 0.00007; C1V1_T: 40×/0.8, 244 ± 20 pA (n = 14), 20×/0.5, 582 ± 32 pA (n = 6), P = 0.00005; C1V1_T/T: 40×/0.8, 269 ± 46 pA (n = 11), 20×/0.5, 585 ± 80 pA (n = 7), P = 0.001; * denotes significance at P < 0.05 level). (g) C1V1_T TPLSM photocurrents elicited by different light wavelengths. (h) TPLSM action spectra (C1V1, n = 5; C1V1_T, n = 5; C1V1_T/T, n = 6). Values were normalized to the maximum photocurrent within the cell across wavelengths. (i) Laser intensity versus TPLSM photocurrent (C1V1, n = 8; C1V1_T, n = 8; C1V1_T/T, n = 13). Normalization as in h. (j) Left, 1,040-nm, TPLSM-evoked spiking. Right, spiking efficacy (10 Hz: C1V1 n = 6, 36.7% ± 6% versus C1V1_T and versus C1V1_T/T, both n = 6, 100% ± 0%, P = 0.002; 20 Hz: C1V1 n = 6, 18.3% ± 3.7% versus C1V1_T n = 6, 56.7 ± 10%, P = 0.001 and versus C1V1_T/T n = 6, 91 ± 4%, P = 0.01; * denotes significance at P < 0.05 level). Black squares: spiking of the same cells in response to direct current injection (400-pA, 10-ms current pulses provided at the indicated frequency). Resting membrane potential (R_m), −65 mV; 2P, two photon.

Figure 2

Two-photon control of spiking with C1V1 variants in acute brain slices. (a) Top, targeted neurons during TPLSM excitation (red box, ROI). Bottom, two-photon image of patched/Alexa 594–filled neuron expressing C1V1_T-EYFP; lines schematize TPLSM pattern. Throughout, ROIs in slice were ~10 × 10 μm to 15 × 15 μm. (b) TPLSM spike generation under 1,040-nm illumination in neurons expressing C1V1_T-ts-EYFP or C1V1_T/T-ts-EYFP (C1V1_T-ts-EYFP in hippocampus (n = 15) and prefrontal cortical (n = 51) neurons; C1V1_T/T-ts-EYFP in hippocampus (n = 7) and prefrontal cortical (n = 8) neurons). Resting membrane potential (R_m), −65 to −70 mV; black ticks, scan onset. (c) TPLSM activation of a representative C1V1_T-ts-EYFP–expressing cell from targeting different axial (z) positions. R_m, −65 mV. (d) Percent successful spikes versus axial position in C1V1_T-ts-EYFP–expressing cortical neurons. R_m, −65 mV; n = 4; error bars, s.e.m. (e) Percent successful spikes versus lateral distance from the center of a representative cortical cell expressing C1V1_T-ts-EYFP. Blue, X; green, Y; R_m, −65 mV; n = 7; error bars: s.e.m. (f) Top, p2A strategy: opsin separated from fluorophore (XFP). Bottom, single-photon (1P) photocurrent for different configurations of C1V1, fluorophore and p2A (C1V1_T: EYFP-p2A-opsin 1,383 ± 80 pA (n = 8), opsin-ts-EYFP 1,474 ± 130 pA (n = 12), opsin-p2A-EYFP 1,811 ± 125 pA (n = 18; P = 0.04 versus two comparison groups); C1V1_T/T: EYFP-p2A-opsin 1,245 ± 67 pA (n = 8), opsin-ts-EYFP 1,175 ± 58 pA (n = 9), opsin-p2A-EYFP 1,541 ± 135 pA (n = 17, P = 0.009 versus opsin-ts-EYFP)). P values, Mann-Whitney two-tailed test for non-Gaussian distribution. (g) Left, cortical neurons expressing C1V1_T-ts-EYFP. Right, cortical neurons expressing C1V1_T-p2A-EYFP. (h) Top, current clamp: TPLSM-stimulation of C1V1_T-p2A-EYFP (20 Hz; R_m, −65 mV). Bottom, voltage clamp.

Figure 3

Spatial resolution of C1V1-mediated two-photon excitation in acute brain slices. (a) Two-photon image of dual whole-cell recording from Alexa 594–filled pyramidal cells expressing C1V1_T-p2A-EYFP. (b) Current-clamp traces during TPLSM activation of cell 1 or cell 2. Resting membrane potential (R_m), −65 mV. (See Online Methods for stimulation parameters.) Typical ROI sizes were as in Figure 2a. (c) Spike probability for TPLSM stimulation (stim) of cell 1 or 2. R_m, −65 mV; similar results were obtained in n = 4 cell pairs (*P = 0.03) wherein no spikes were seen in adjacent cells despite 100% fidelity in driving the targeted cell at 1 Hz, regardless of which cell was targeted. (d) TPLSM-elicited photocurrents in voltage-clamp mode. Values were normalized to the maximum photocurrent observed between cell 1 and cell 2 during stimulation of either cell 1 or cell 2 (n = 4 cell pairs, *P = 0.03; error bars, s.e.m.; P values, Mann-Whitney two-tailed test for non-Gaussian distribution). (e) Spike jitter (s.d. of spike timing stimulated at 1 Hz) versus TPLSM photocurrent for pyramidal cells expressing C1V1_T-p2A-EYFP (n = 12 cells). Inset, representative current clamp traces showing jitter. R_m, −65 mV; error bars, s.d. of mean time to spike. (f) Plot of 1,040-nm TPLSM photocurrent versus EYFP fluorescence of pyramidal cells expressing C1V1_T-p2A-EYFP. Cells were selected here not for high expression but to cover a broad range of expression, to assess relationship between fluorescence and photocurrent; linear fit R² = 0.85. a.u., arbitrary units.

Figure 4

Two-photon optogenetic control of spike firing in vivo in adult mammals. (a) Schematic of the experimental setup for in vivo two-photon control of superficial layer 2 and 3 (2/3) somatosensory neurons transduced with C1V1_T-p2A-EYFP. (b) Left, two-photon image of layer 2/3 pyramidal neurons transduced with C1V1_T-p2A-EYFP in somatosensory cortex (150–250 μm below pia). Right, two-photon image of layer 1 pyramidal neurons transduced with C1V1_T-p2A-EYFP in somatosensory cortex (50–150 μm below pia). (c) Two-photon image of dendritic spines on pyramidal cells transduced with C1V1_T-p2A-EYFP in layer 2/3 of somatosensory cortex. (d) Upper left, in vivo two-photon image of layer 2/3 pyramidal cells transduced with C1V1_T-p2A-EYFP (imaged during loose patch). Lower left, trace showing precise spike-train control with 5-Hz 1,040-nm raster-scanning illumination; the amplitude and waveform of these evoked spikes recorded in cell-attached mode matched the spontaneous spikes in each cell. Upper right, axial resolution of two-photon optogenetic control of spiking in vivo. Blue triangles indicate pyramidal neurons and red boxes illustrate ROI positioning. Spiking of layer 2/3 cells as a function of raster scan position is shown (20×/0.5-NA objective; λ: 1,040 nm; dwell time per pixel, 3.2 μs, scan resolution, 0.6 μm per pixel; line scan speed, 0.19 μm μs⁻¹; laser intensity, 20 mW). Typical ROI sizes were as in Figure 2a. Lower right, lateral resolution of two-photon optogenetic control of spiking in vivo. Similar results were observed in n = 3 neurons in vivo, all 150–250 μm below pia in layer 2/3 in somatosensory cortex.

Figure 5

Bistable two-photon optogenetic control in culture and slice with novel ChR variants. (a) Earlier SFOs (1P SFO) and novel multiple-mutant opsins (2P SFO). (b–j) Experiments in cultured neurons. (b) Single-photon photocurrents. ChR2_A: 161 ± 33 pA, n = 7; ChR2_AR: 532 ± 87 pA, n = 7, P = 0.0003 versus ChR2_A; ChR2_T: 185 ± 45 pA, n = 4; ChR2_TR: 645 ± 98 pA, n = 7, P = 0.0002 versus ChR2_T. (c) TPLSM-stimulated 2PSFO (ChR2_TR τ_off = 3.2 ± 0.05 s (n = 8), ChR2_AR τ_off = 53.3 ± 2.8 s (n = 13)). (d) TPLSM photocurrents. ChR2_AR: 125 ± 7 pA (n = 15), ChR2_TR: 109 ± 10 pA (n = 8). Error bars, s.e.m. (e) Action spectra. Values were normalized to the maximum photocurrent within the cell across wavelengths; ChR2_AR (n = 5), ChR2_TR (n = 4). ROIs were 15 × 15 to 20 × 20 μm. (f) Laser intensity versus 940-nm, TPLSM-photocurrent (ChR2_TR (n = 3), ChR2_AR (n = 3)); normalization as in e. (g) ChR2_AR TPLSM photocurrents for multiple–axial-plane stimulation. Inset, photocurrents from multiple- or single-plane stimulation (multiplane: 366 ± 15 pA (n = 2), single plane: 125 ± 15 pA (n = 15, *P = 0.01 versus multiplane)). P values, Mann-Whitney two-tailed test for non-Gaussian distribution. (h) TPLSM photocurrent versus axial position (n = 4). (i) Single-photon deactivation of TPLSM-elicited ChR2_AR photocurrents. Gray bar, timing of deactivating pulses. (j) TPLSM deactivation of 477-nm-elicited ChR2_AR photocurrents. (k) Current clamp (bottom, CC) and voltage clamp (top, VC) of ChR2_AR TPLSM-stimulation in cortical slice. TPLSM scan was applied across soma (n = 3). (l) Left, single hippocampal neuron in acute slice with (top) or without (bottom) TPLSM activation of ChR2_AR, stimulated with simulated excitatory postsynaptic current (sEPSC) train (black). Right, separate cell with sequence of conditions reversed (n = 4).

Figure 6

Two-photon optogenetic inhibition. (a) Version 3.0 of opsins for enhancing photocurrent magnitude. (b) Two-photon image of cultured hippocampal neuron transduced with eArch3.0-EYFP; red square: ROI borders. (c) Voltage-clamp traces showing outward currents for 1,040 nm, TPLSM-scanned eArch3.0-expressing neurons at different scan resolutions (blue: n = 7, photocurrent:176.1 ± 20.6 pA; yellow: n = 7 cells, photocurrent 278.7 ± 41.6 pA). (d) Action spectrum of eArch3.0 (n = 3; error bars: s.e.m.). Values were normalized to the maximum photocurrent within the cell across all wavelengths. (e) Laser intensity versus 1,040-nm, TPLSM photocurrent (n = 6). Normalization as in d. (f) TPLSM-evoked eArch3.0 photocurrents elicited with different NA objectives (40×/0.8 NA: n = 7; 20×/0.5 NA; n = 6, *P = 0.03 versus 40×/0.8 NA). P values, Mann-Whitney two-tailed test for non-Gaussian distribution. (g) Trace of continuous TPLSM scan of eArch3.0-expressing neuron. Trace within dashed box at left expanded at right. (h) Trace of TPLSM-mediated spike inhibition in a cultured neuron expressing eArch3.0; spiking evoked by 300-pA current injection. R_m, −65 mV. (i) Two-photon image of pyramidal prefrontal neurons expressing eArch3.0. (j) TPLSM activation of eArch3.0 showing spike-inhibition dependence on TPLSM axial (z) position targeting. R_m, −65 mV. (k) Axial position versus successful spikes. R_m, −65 mV; n = 4. Values were normalized to the maximum number of spikes within the cell across all axial positions. (l) TPLSM inhibition over long timescales with eArch3.0. Top, traces showing key temporal windows. Bottom, spike rate over time. R_m, −65 mV; n = 5. TPLSM scan initiated at 15 s and terminated at 75 s.