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.