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.