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. 2021 Nov 2;12:6307. doi: 10.1038/s41467-021-26275-y

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© The Author(s) 2021

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. 1 — a Rosa^FTLG allele used for intersectional transgenic labeling of boutons from vGlut2/Phox2b interneurons (left) and schematic of the results (right). b Coronal sections through the hindbrain of a Phox2b::Flpo; vGlut2::Cre;Rosa^FTLG mouse at P4, showing synaptic boutons (black) from vGlut2/Phox2b interneurons in relation to motor nuclei (ChAT⁺, blue) at low (left), and higher (middle) magnifications, and close-ups of boutons (green) on motoneurons (right), which are either Phox2b⁺ (purple) or Phox2b⁻ (blue). c (left) Strategy for mono-synaptically restricted transsynaptic labeling of premotor neurons from the posterior digastric muscle (PD) in a Phox2b::Cre;Rosa^nlsLacZ mouse, with G-deleted rabies virus (RV) encoding mCherry and complemented by a G-encoding helper HSV virus (HSV-YFP-G), and summary of the results. (right panel) The only seed neurons are Acc7 motoneurons, double-labeled by the HSV-G and RV-mCherry viruses. d, e Coronal sections through the hindbrain at P8 showing labeled premotor neurons (black on the left panels) in the IRt (d) and Peri5 (e), which for the most part (72.7% ± 3.5 SEM, n = 4 animals) express Phox2b (right panels). AD anterior digastric, IRt intermediate reticular formation, nTS nucleus of the solitary tract, PD posterior digastric, Peri5 peritrigeminal area, RF reticular formation, RTN retrotrapezoid nucleus. Scale bars, b 1 mm for the left column, c 250 μm, d, e 500 μm.