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. 2023 Mar 23;26(4):682–695. doi: 10.1038/s41593-023-01281-z

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© The Author(s) 2023

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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Fig. 4 — a,g, Top left: two-photon image of axons from an SS27485-Gal4 (a) or an SS36112-Gal4 (g) animal expressing OpGCaMP6f (cyan) and tdTomato (red). ROIs are numbered. Scale bars, 5 μm. Bottom: behavioral epochs are color-coded. Representative ΔF/F time series from two ROIs (green) overlaid with a prediction (black) obtained by convolving resting epochs (a) or puff stimuli (g) with Ca²⁺ indicator response functions. Explained variances are indicated (R²). b,h, Mean (solid line) and 95% confidence interval (gray shading) of ΔF/F traces for rest ANs (b) or puff-responsive ANs (h) during epochs of forward walking (left), resting (middle) or CO₂ puffs (right). 0 s indicates the start of each epoch. Data more than 0.7 s after onset (yellow region) are compared with an Otsu thresholded baseline (one-way ANOVA and two-sided Tukey post hoc comparison, ***P < 0.001, **P < 0.01, *P < 0.05, NS, not significant). c,i, Standard deviation projection image of an SS27485-Gal4 (c) or an SS36112-Gal4 (i) nervous system expressing smFP and stained for GFP (green) and Nc82 (blue). Cell bodies are indicated (white asterisk). Scale bars, 40 μm. d,j, Projection as in c and i but for one MCFO-expressing, traced neuron (black asterisk). The brain’s AVLP (cyan) and the VNC’s leg neuromeres (yellow) are color-coded. Scale bars, 40 μm. e,f,k,l, Higher magnification projections of brains (top) and VNCs (bottom) from SS27485-Gal4 (e,f) or SS36112-Gal4 (k,l) animals expressing the stochastic label MCFO (e,k) or the synaptic marker, syt:GFP (green) and tdTomato (red) (f,l). Insets magnify dashed boxes. Indicated are cell bodies (asterisks), bouton-like structures (white arrowheads) and VNC leg neuromeres (T1, T2 and T3). Scale bars for brain images and insets are 5 μm (e) or 10 μm (k) and 2 μm for insets. Scale bars for VNC images and insets are 20 μm and 10 μm, respectively.

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