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. 2023 May 22;14:2939. doi: 10.1038/s41467-023-38583-6

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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. 6 — a Left: experimental strategy for silencing RTN^Phox2b/Atoh1 neurons bilaterally using the virally-driven inhibitory DREADD receptor hM4Di in Atoh1^FRTCre;Phox2b^Flpo adult mice. Right: transverse sections showing bilaterally transfected neurons in the pF area around the 7N motoneurons identified by Choline Acetyl Transferase (ChAT) expression. Scale bar, 250 µm. Representative of n = 4 mice. b Number and distribution of transfected (mCherry⁺) neurons across pF subregions as defined in Fig. 4f. Bars are means ± SD across n mice and gray circles are mean values of individual mice. See methods for details. From left to right: frequency: p = 0,2532; p > 0,9999; Ti: p = 0,1975; p = 0,6264; Te: p = 0,6042; p = 0,998; amplitude: p = 0,1716; p = 0,1975 (Wilcoxon matched-pairs tests). c Raw (DiaEMG) and integrated (Int.) recordings of diaphragm activity of one representative animal at rest before (CTL), during (CNO), and after (REC) administration of CNO at 10 mg/kg. d Quantification of respiratory parameters before, during and after CNO administration: frequency, inspiratory (Ti) and expiratory (Te) times, and normalized amplitude. Note that silencing RTN^Phox2b/Atoh1 neurons does not alter basal respiratory parameters. From left to right: frequency: p = 0,2532; p > 0,9999; Ti: p = 0,1975; p = 0,6264; Te: p = 0,6042; p = 0,998; amplitude: p = 0,1716; p = 0,1975 (Wilcoxon matched-pairs tests). e, f Similar representations as in c, d in mice running on a treadmill at 40 cm/s. Note the significantly lower diaphragm frequency and increased expiratory time (Te) when RTN^Phox2b/Atoh1 neurons are silenced. From left to right: frequency: p = 0,0069; p = 0,1025; Ti: p = 0,5311; p = 0,3134; Te: p = 0,0089; p = 0,0987; amplitude: p = 0,0146, p = 0,4805 (paired t tests). For all graphs, gray open circles are means of individual mice, red circles are means ± SD across n mice, * denotes p < 0,05, ** denotes p < 0,01, *** denotes p < 0,001, and ns denotes p > 0,05. Source data are provided as a Source data file. See also Fig. S11.