Supplementary Figure 11: Spatial confinement of optical manipulation of the DRN

For 9 time points (3 seconds), the sites were targeted iteratively, illuminating each site for 4 ms at a time and jumping between sites within 1 ms.a, Overlay of an anatomical reference (gray, 5th percentile F0 baseline of 159 time points, including 100 time points before manipulation and 50 time points after manipulation) and maximum-projection of ∆F/F over these 9 time points (red).b, Selected sites to quantify ∆F/F activity traces. Two manipulation sites were selected (blue). For each of these, three sites at increasing distance from the manipulation site were selected as well, in 5 µm increments (orange, green and red, respectively).c, For all sites, ∆F/F traces were extracted from pixels in a 2 µm radius (5th percentile constant baseline). Time points with manipulations are indicated with gray background. Since the manipulations occurred concurrently with imaging, the responses are not always recorded directly. For most of the sites, there are only 2 time points where the detection piezo position is exactly pointed at the targeted z-slice while the respective site is activated, resulting in two primary peaks in the respective ∆F/F traces. The experiment shown in this figure was repeated independently three times with similar results. Scale bar, 25 µm.