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).