Two-Photon Holographic Stimulation of ReaChR

Abstract

Optogenetics provides a unique approach to remotely manipulate brain activity with light. Reaching the degree of spatiotemporal control necessary to dissect the role of individual cells in neuronal networks, some of which reside deep in the brain, requires joint progress in opsin engineering and light sculpting methods. Here we investigate for the first time two-photon stimulation of the red-shifted opsin ReaChR. We use two-photon (2P) holographic illumination to control the activation of individually chosen neurons expressing ReaChR in acute brain slices. We demonstrated reliable action potential generation in ReaChR-expressing neurons and studied holographic 2P-evoked spiking performances depending on illumination power and pulse width using an amplified laser and a standard femtosecond Ti:Sapphire oscillator laser. These findings provide detailed knowledge of ReaChR's behavior under 2P illumination paving the way for achieving in depth remote control of multiple cells with high spatiotemporal resolution deep within scattering tissue.

Keywords: 2-photon excitation; action-potential generation; computer generated holography; cortex; neuroscience; opsin; optogenetics.

Figure 1

Characterization of currents evoked by 2P holographic stimulation and two-photon excitation spectrum. (A) Simplified scheme of the 2P holographic stimulation microscope. The output beam from an Ytterbium-doped photonic crystal fiber amplifier Laser System was attenuated by rotating a half-waveplate combined to a polarizer cube. The beam was relayed and expanded to cover the surface of an LCoS-SLM (Hamamatsu X10468-07). Zero-order excitation was suppressed by aberrating it with a cylindrical lens. Then the SLM was imaged through a telescope (L1, L2 lenses) at the back focal plane of an IR antireflection coated water-immersion objective (Nikon CFI APO 40X WI NIR NA 0.80) mounted on an upright microscope (Scientifica). (B) CHO cells were voltage clamped at −40 mV and the current response to 2P holographic stimulation (1000 ms, red bar, λ = 1030 nm) with patterns matching the cell shape was recorded. Evoked response for increasing Laser Power Densities (LPDs) in a single cell. Average of n = 3 repetitions. (C) The maximum current (I_max) reached by 2P holographic stimulation at 1030 nm of 4 CHO cells was normalized to the saturation current (I_sat) and plotted vs. LPD. Average of n = 3 repetitions for each cell and LPD. Data recorded above a LPD of 0.2 mW/μm² was not shown as all cells had reached the saturation current. (D) Rise time (T_R, gray dots) and decay time (T_D, black diamonds) for 2P holographic stimulation of a single CHO cell. Average of n = 3 repetitions. T_D and T_R were fit, respectively, by a linear function (black line) and following Equation 4 (in Methods see Section Currents evoked by photo-stimulation of opsins) (gray line). (E) CHO cells were voltage clamped at −40 mV and the current response to 2P Generalized Phase Contrast (GPC) stimulation (400 ms, red bar) was recorded at a wavelength range from 720 to 1030 nm, while keeping the photon flux constant (2.7 × 10²⁶ photons/s/m²). Average of n = 3 repetitions for each wavelength. (F) The maximum current reached by 2P GPC stimulation was normalized to the current generated with stimulation at 1000 nm and plotted vs. excitation wavelengths. A constant photon flux of 2.7 × 10²⁶ photons/s/m² was used. The current generated between 975 and 1030 nm (^*) was significantly larger than currents generated at other wavelengths (p = 0.05, Wilcoxon paired T-test; n = 6 cells). (G) Decay time (T_D, black diamonds) and rise time (T_R, gray dots) for a constant photon flux of 2.7 × 10²⁶ photons/s/m² for 2P holographic stimulation of CHO cells. Average of n = 6 cells. T_R between 975 nm and 1000 nm (^*) was significantly larger than currents generated at other wavelengths (p = 0.05, Wilcoxon paired T-test; n = 6 cells). (H) The 2P cross-section, normalized to its value for excitation at 1000 nm, was plotted vs. excitation wavelengths. The 2P cross-section at 975 and 1000 nm (^**) was very significantly larger than the cross-section at other wavelengths (p = 0.01, Wilcoxon paired T-test; n = 11 cells).

Figure 2

Conditions to evoke an action potential in neurons of mouse visual cortex Layer 2/3 with a fiber amplified Laser System at 1030 nm. (A) Maximum intensity projection of a z-stack of confocal images of a fixed sagittal brain slice from mouse injected with AAV1 CamKII-ReaChR-p2A-YFP, 2 weeks after injection. Scale bar: 50 μm. (B) 2P microscopy images of visual cortex layer 2/3 from an anesthetized mouse (1% isoflurane) injected with AAV1 Ef1α-ReaChR-p2A-dTomato, 7 weeks after injection. Scale bar: 50 μm. Selected images from a stack of 101 images (100 to 200 μm under the surface of the brain). (C) Cumulative distribution of the saturation current (in Methods see Section Voltage clamp measurements of currents evoked by photo-stimulation of opsins) generated by 2P holographic stimulation in L2/3 cells when cells were voltage-clamped at −70 mV. (D) Voltage response of a L2/3 pyramidal cell to a series of 10-ms long 2P holographic spots (15 μm diameter, over the cell soma) of constant Laser Power Density (LPD) (red bar). Steady state current injection was used to keep the cell voltage at −70 mV in resting conditions. The LPD was increased from 0 to 0.014 mW/μm² within the series until an action potential (AP) was evoked. Inset: overlay of infrared (IR) slice image (grayscale), fluorescence (green) and 2P stimulation mask (red circled area) of V1 layer 2/3 in an acute sagittal brain slice from mouse injected with AAV1 CamKII-ReaChR-p2A-YFP, 5 weeks after injection. Scale bar: 30 μm.

Figure 3

AP threshold with standard femtosecond Ti:Sapphire oscillator and spatial specificity of 2P holographic stimulation of ReaChR. (A) Widefield fluorescence image of L2/3 ReaChR-expressing neuron filled with 20 μM Alexa 594. Scale bar: 15 μm. (B) Voltage response (single recording) to a 10 ms long (red line) 2P holographic stimulation with a standard femtosecond Ti:Sapphire oscillator at 950 nm using a circular spot on the cell soma (1 in panel A, 15 μm diameter) for increasing LPDs. (C) Average voltage response (n = 3 repetitions) to a 10 ms long (red line) 2P holographic stimulation with a standard femtosecond Ti:Sapphire oscillator at 950 nm on numbered circular spots (15 μm diameter) in panel A. LPD: 0.2 mW/μm². (D) Distribution of average voltage response (n = 3 repetitions, 25 spots, 3 cells) to a 10 ms long (red line) 2P holographic stimulation with a standard femtosecond Ti:Sapphire oscillator at 950 nm on cell processes. Distance from the soma from 15 to 100 μm, as shown in panel A. LPD corresponding to the one used to reach the AP threshold for 2P stimulation on the cell soma for each cell (0.15 to 0.25 mW/μm²).

Figure 4

Optimization of parameters to evoke an action potential in neurons of mouse visual cortex Layer 2/3 with a fiber amplified Laser System at 1030 nm. (A) Laser power density (LPD) necessary to evoke an AP with 1 to 10 ms long 2P holographic spots (15 μm diameter, over the cell soma) (n = 16 cells, average of 3 repetitions for each). Individual cells (gray), average (orange bars) and s.d. (error bars). (^**) (p = 0.01, Wilcoxon paired T-test). (B) Latency between the AP evoked by 1 to 10 ms long 2P holographic spots (15 μm diameter, over the cell soma) at the LPD for AP threshold and the onset of the 2P stimulation (n = 16 cells, average of 3 repetitions for each). Individual cells (gray), average (orange bars) and s.d. (error bars). The latency did not vary significantly with the pulse length at AP threshold. (C) Same as (B) with AP jitter. The jitter did not vary significantly with the pulse length at AP threshold. (D) Voltage response of a L2/3 pyramidal cell infected with AAV1 CamKII-ReaChR-p2A-YFP to a series of 10-ms long 2P holographic spots (15 μm diameter, over the cell soma) of constant Laser Power Density (LPD) (red bar). Steady state current injection was used to keep the cell voltage at −70 mV in resting conditions. Three repetitions for each LPD. (E) Latency between the AP evoked by 10 ms long 2P stimulation and the onset of 2P stimulation (n = 7 pyramidal cells, average of 3 repetitions for each), at LPDs varying from the LPD necessary to reach the AP threshold to 5 times this value. Example of recording on panel D. Individual cells (gray), average (orange bars) and s.d. (error bars). The latency significantly (^*) (p = 0.05, Wilcoxon paired T-test) or very significantly (^**) (p = 0.01, Wilcoxon paired T-test) decreased when the LPD was increased. (F) Same as (E) with AP jitter. The jitter very significantly decreased (^**) (p = 0.01, Wilcoxon paired T-test) when LPD was increased in comparison to the jitter at LPD used to reach the AP threshold.

Figure 5

Conditions to evoke a train of action potentials in neurons of mouse visual cortex Layer 2/3 with a fiber amplified Laser System at 1030 nm. (A) Trains of 2P holographic stimulation pulses were used to evoke trains of APs in single L2/3 cells: example of a pyramidal cell. Trains of 10 pulses at frequencies ranging from 5 to 40 Hz and LPDs at AP threshold were used. (B) Same as (A) for a fast spiking cell. (C) Plot of the maximum AP frequency vs. the photo-stimulation light pulses frequency, for pyramidal cells and LTS cells (red triangles, average of n = 19 cells) and FS cells (green diamonds, average of n = 4 cells). Black line corresponds to an AP frequency equal to the light pulse frequency. (D) Maximum AP frequency reached for trains of a given number of light pulses for pyramidal cells and LTS cells (red triangles, average of n = 19 cells) and FS cells (green diamonds, average of n = 4 cells).