Fig 2

Fig 2

(A) Schematic of circuit-breaking screen: 28 Gal4 enhancer trap lines were crossed to UAS:epNTR, and labeled neurons were ablated and tested for escape behavior. Right: heat maps representing maximum horizontal and sagittal projections of brain coverage (number of lines labeling a given voxel). (B) Histogram of the change in LLC probability following ablation (compared to MET-treated non-epNTR-expressing sibling controls) for each line screened. Magenta: Gal4 lines with a >50% reduction. A minimum of 7 larvae were used per condition per line. %LLC is the percentage of trials on which an LLC was performed as a fraction of trials with no SLC response. (C) LLC probability for lines highlighted in (B). LLC probability after ablation (magenta) and in MET-treated sibling controls (black). y252-Gal4 (n = 22 control, 31 ablated larvae), y293-Gal4 (n = 17, 17), y330-Gal4 (n = 8, 7). ***p < 0.001, **p < 0.01, *p < 0.05, t test. (D) Maximum horizontal projections for Gal4 lines with reduced LLC probability after ablation. Expression is color-coded for depth (μm below image top). (E) Expression overlap between y252-Gal4 and y293-Gal4 (magenta) and between all 3 lines (green). Boxed area enlarged in (E'). (F) Coronal (top) and dorsal (bottom) projections of confocal substacks through the R1 cluster in y293-Gal4; UAS:Kaede (green); vmat2:GFP^pku2 (magenta) larvae. Prior to imaging, Kaede was photoconverted to red. Arrows indicate LC and Ra labeled by vmat2:GFP. Both Kaede and GFP are expressed in the raphe (white). (G-H) LLC probability after laser ablation of R1 (G, n = 9) and R6 (H, n = 16) in y293-Gal4. *p < 0.05, t test. (I) Percent of LLCs made in a rightward direction after left R1 ablation (“Uni,” n = 14) and nonablated controls (n = 24). *p < 0.05, t test. Scale bars: 100 μm in (A, D, E); 25 μm in (F); 40 μm in (G-I). Underlying numerical data are included in S1 Data, and unprocessed image stacks in (F) are available at https://doi.org/10.5281/zenodo.3382102. a, anterior; d, dorsal; dpf, day post-fertilization; epNTR, engineered nitroreductase variant; GFP, green fluorescence protein; LC, locus coeruleus; LLC, long-latency C-start; m, medial; MET, metronidazole; R1, rhombomere 1; Ra, raphe; SLC, short-latency C-start.