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. 2024 Jun 5;630(8017):686–694. doi: 10.1038/s41586-024-07523-9

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 1 — (a) Z-projected confocal images of the brain (top) and VNC (bottom) show the expression of UAS-CsChrimson.mVenus (green) in command-like DNs, membrane-bound tdTomato in the Dfd driver line (red), and neuropil (‘nc82’, blue). The location of command-like DN cell bodies is indicated (white arrowheads). Scalebars are 100 μm. (b) Z-projected confocal image of Dfd driver line expression of soma-targeted mCherry. Only brain neurons in the GNG are labeled. Scalebar is 100 μm. (c) Confocal image of the posterior GNG with Dfd driver line expression of soma-targeted mCherry and aDN2 expression of UAS-CsChrimson.mVenus (green). The two GNG-DNs in the aDN2 driver line are not targeted by the Dfd driver line. Scalebar is 20 μm. Immunohistochemistry and confocal imaging experiments in (a-c) were performed once due to the reliability of these methods. (d) Behavioral responses to optogenetic stimulation of the neck connective at different laser intensities for DNp09 (left; 4 flies, total 49 trials per condition), aDN2 (left-middle; 4 flies, total 60 trials per condition), MDN (right-middle; 4 flies, total 50 trials per condition), and no DN control (right; 3 flies, total 60 trials per condition) animals. Flies reliably (i) walk forward upon DNp09 stimulation for stimuli ≥ 21 μW, (ii) groom upon aDN2 stimulation only for the highest stimulation power (41.6 μW) but rest at 21 μW, and (iii) walk backward upon MDN stimulation for stimuli ≥ 10.5 μW. For all stimulation intensities, control flies walk more and rest less. Thus, we selected 21 μW as our default laser stimulation power and 41.6 μW for aDN2 stimulation specifically. (e) MDN stimulation with focused laser light elicits backward walking when illuminating the anterior dorsal thorax (left, as in Figs. 4 and 5), the neck (middle, as in Fig. 2) or the head (right). 3 flies, total 30 stimulation trials per condition. (f) Stimulation of a brain-specific neuron (‘Bolt protocerebral neurons’ or BPN) known to drive forward walking³ with focused laser light elicits forward walking when illuminating the head (right), but not the thorax (left). Laser light focused on the neck (middle) can only elicit weak forward walking at 41.6 μW. 4 flies, total 40 stimulation trials per condition. (g) Silencing GNG neurons (Dfd-LexA > LexAop-GtACR1) with focused 561 nm laser light elicits anterior grooming when illuminating the head (right), neck (middle), or thorax (left). 3 flies, total of 30 stimulation trials per condition. All velocity traces in (d,e,f) show mean ± 95% confidence interval of the mean across stimulation trials.