Figure 3. Photocurrents of neurites expressing soCoChR-GFP are significantly smaller than in neurites expressing CoChR-GFP, in neurons virally expressing these opsins in mouse cortical brain slice.

(a–b) Schematic representation of the two experimental setups used for holographic illumination. In setup 1 (a), holographic photostimulation was achieved using an amplified fiber laser and was coupled with a two-photon (2P) scanning imaging system. In setup 2 (b) holographic photostimulation was achieved using a conventional Ti:Sapphire pulsed laser and was coupled with a widefield epifluorescence imaging system. Detailed descriptions of the setups are reported in Methods section and Supplementary Figure 6. (c) Schematic of 2P holographic stimulation along one neurite, showing Alexa 594 fluorescence (obtained via 2P scanning at 780 nm) from dye injected into a patched CoChR-GFP expressing neuron. The Alexa 594 fluorescence was used to guide holographic spot placement (red circles) to different points along a neurite, at different distances from the soma. Red arrow indicates the order of photostimulation. The bright emission to the left of the cell represents the patch pipette filled with Alexa 594 (scale bars: 50 µm along all three axes, image acquired with setup 1). (d) Representative whole-cell currents recorded from a CoChR-GFP expressing neuron (left) and a soCoChR-GFP expressing neuron (right; some 50 Hz electrical noise is apparent in the traces on the right), when illuminated with a power density corresponding to the spiking threshold power density (18 µW/µm² and 101 µW/µm² for the CoChR-GFP and the soCoChR-GFP expressing cells respectively; λ = 1030 nm, using setup 1). (e) Bar plot of the integral of the elicited photocurrent, normalized to that obtained with the spot at the soma, as a function of distance from the soma, for CoChR-GFP (blue bars) and soCoChR-GFP (red bars) expressing neurons (spacing between spots ~10 µm, λ = 1030 nm or 920 nm for setup 1 or setup 2, respectively; data were pooled across both setups). Bars reports mean ± s.e.m. Dots denote values for single neurites. For each cell, the photostimulation was done at the power density threshold determined for that cell (average powers: 29±10 µW/µm² for CoChR-GFP expressing cells; 90±60 µW/µm² for soCoChR-GFP expressing cells; power values referred to setup 1, see Methods). The normalized current integral was significantly higher in CoChR-GFP relative to soCoChR-GFP expressing cells for distances of 30 µm or more from the soma. ** P<0.0017 for distances ≥ 30 µm; Kolmogorov-Smirnov (KS) test with Bonferroni correction (n=16 neurites from 8 CoChR-GFP cells from 7 mice; n=27 neurites from 16 soCoChR-GFP cells from 13 mice; see Supplementary Table 4 for full statistics for Fig. 3). Data obtained using both setup 1 and setup 2.