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. 2023 Apr 17;14:2182. doi: 10.1038/s41467-023-37968-x

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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. 1 — a Galantamine (Gal, 10 μM), mecamylamine (MEC, 10 μM), and saline were microinjected into the right VP through a cannula. A representative image of substance P (SP, purple) antibody-stained coronal brain section among nine repeats. b‒d Mechanical paw withdrawal thresholds (PWT) on both hind paws before (baseline), 15–30 min and 24 h after injection of Gal (b), MEC (c), and saline (d) into the VP (n = 9 mice). b Contralateral: F_{(1.167, 9.338)} = 29.45, P < 0.001; t = 6.83, df = 8, P < 0 001, Gal vs baseline. Ipsilateral: F_{(1.626, 13.00)} = 19.56, P < 0.001; t = 5.99, df = 8, P < 0 001, Ga1 vs baseline. c Contralateral: F_{(1.895, 15.16)} = 0.87, P = 0.44. Ipsilateral: F_{(1.384, 11.07)} = 5.48, P = 0.02; t = 3.31, df = 8, P = 0.01, MEC vs recovery. d Contralateral: F_{(1.336, 10.69)} = 0.80, P = 0.48. Ipsilateral: F_{(1.620, 12.96)} = 0.88. P = 0.45. e Whole-cell patch-clamp recordings were performed from virally labeled VP ChAT neurons to record responses to acetylcholine (ACh). f Representative images among five sets of repeats showing eYFP-labeled VP ChAT (ChAT) neurons. g ACh-evoked MEC-sensitive currents in VP ChAT and non-ChAT neurons. h Amplitude of ACh-evoked inward currents in ChAT (n = 19) and non-ChAT (n = 20) neurons in the VP from five mice. t = 2.12, df = 37, P = 0.04. i Schematic diagram for chemogenetic activation of VP ChAT neurons by delivering clozapine-N-oxide (CNO, 10 μM) into the VP through a cannula. Representative image is from 11 sections (one from each mouse) stained with ChAT (green) and SP (purple) antibodies and DAPI (blue). j Mechanical PWTs were measured before and 45–60 min after CNO or CNO-MEC was injected into the VP. n = 11 mice. k Mechanical PWTs were measured before and 45–60 min after CNO or CNO-saline was injected into the VP. n = 11 mice. One-way repeated-measures ANOVAs with Bonferroni tests and Geisser–Greenhouse correction were used in (b‒d) and (j, k). Two-tailed t-test was used in (h). *P < 0.05; **P < 0.01; ns not significant.