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. 2018 Jul 9;8:10307. doi: 10.1038/s41598-018-28680-8

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Identification of Seta and Leta, backward-specific interneurons expressing 5-HT2A-GAL4. (a) A cartoon showing the design of GAL4 knock-in. The GAL4-coding sequence along with the 2A peptides was inserted by the CRISPR/Cas9 method to the target NM receptor gene locus, just downstream of the C-terminus of the NM coding sequence. dsRed flanked by loxP sites was used as a marker for successful insertion. (b) Expression of 5-HT2A-GAL4 visualized with mCD8-GFP. (c–c”) motor pattern mapping in 5-HT2A-GAL4- and RRa-GAL4-targeted cells. (c) 18 ROIs representing each hemisegment used for the analyses (visual cues). (c’) Dominant motor pattern mapping identified voxels specific to BW. Motor patterns are colored as in Fig. 3. Two ROIs (labeled as Seta and Leta) are selected for the ROI correlation mapping in (c”). (c”) ROI correlation mapping reveals the outline of Seta and Leta. Pearson’s r between each voxel and a ROI in Seta and Leta shown in (c’) was calculated. The mapping results for Seta (red) and Leta (green) and GCaMP signals (blue) are merged. Axons of Seta and Leta are revealed in yellow (red + blue) and green/blue color (green + blue) and are found to be present in the CI and DM tracts (Fas2 coordinate)³³, respectively (arrows). c’ and c” are z-stacked images. (d) Mean activity of Seta (top, red) and Leta (middle, green) (derived of three samples) aligned with motor activities during AT, BW, FW and PT (bottom, grayscale image). Colored bars represent the duration of each motor pattern. (AT:3.6 ± 0.2 s, BW:5.2 ± 0.2 s, FW:3.4 ± 0.4 s, PT:3.0 ± 0.1 s, QS:8.4 ± 0.9 s, mean ± standard deviation). (e) Maximum activity level (fluorescence changes, ΔF/F) of Seta and Leta during each motor patterns. While both Seta and Leta are active during BW, only Seta is active during AT and PT. ***p < 0.001, ANOVA with post-hoc Tukey HSD test, compared to QS. (f–f”) ROI correlation mapping in 5-HT2A-GAL4-targeted cells. Unlike in c, ROI mapping was applied only to 5-HT2A-GAL4-targeted cells. The morphology of Seta (f,f”) and Leta (f’,f”) can be seen more clearly. Scale bar 100 μm.