Figure 4. 2P power necessary to enable millisecond control of cortical neuron activation in mouse brain slice.

(a) Rise time of soCoChR-GFP (red) and CoChR-GFP (blue)-mediated photocurrents, measured in mouse brain slice cortical neurons, as a function of 2P stimulation power (n = 4–12 photostimulation powers per cell, 6 cells from 6 mice for CoChR-GFP; n = 2–11 photostimulation powers per cell, for 5 cells from 5 mice for soCoChR-GFP). Data collected from setup 1 are plotted with circles, and correspond to the power scale on the bottom x-axis; data collected from setup 2 are plotted with asterisks, and correspond to the top x-axis. The length of the top x-axis was rescaled with respect to the bottom x-axis by a power conversion factor k = 5.3 (see Methods). Lines connect data acquired from the same neuron. Blue and red arrows indicate average threshold powers relative to setup 1 for CoChR-GFP and soCoChR-GFP expressing neurons respectively (for CoChR-GFP: 28 ± 10 µW/µm², n = 7 cells from 5 mice; for soCoChR-GFP: 83 ± 39 µW/µm, n = 4 cells from 4 mice). (b) The AP latency, defined as the time from the onset of 2P stimulation to the peak of the AP, plotted vs. 2P stimulation power in setup 1 units (n = 3–5 powers per cell, for 4 cells from 3 mice for CoChR-GFP, blue dots; n = 3–5 powers per cell, for 3 cells from 3 mice for soCoChR-GFP, red dots). Lines connect data acquired from the same neuron. Horizontal dashed line denotes 15 ms latency, and vertical dashed line denotes 70 µW/µm² stimulation power, for comparison to panel c. Setups 1 and 2 were used interchangeably; power values used on setup 2 were scaled to equivalent power values for setup 1 (see Methods). See (c) for description of the gray area. (c) AP temporal jitter (measured as standard deviation of the AP latency across a series of 5 photostimulations for a given cell) plotted as a function of AP latency (n = 3–5 powers per cell, for 4 cells from 3 mice for CoChR-GFP, blue dots; n = 3–5 powers per cell, for 3 cells from 3 mice for soCoChR-GFP, red dots). Lines connect data acquired from the same neuron. Setups 1 and 2 were used interchangeably. Jitter below 1 ms (horizontal dashed line) was ensured when the latency was kept below 15 ms (vertical dashed line), corresponding to a photostimulation power higher than approximately 70 µW/µm². The gray areas in both (b) and (c) mark the region at which such conditions were satisfied.