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. 2020 May 19;11:2501. doi: 10.1038/s41467-020-16198-5

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© The Author(s) 2020

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–c Withdrawal threshold of hind paw in response to von Frey hair stimulation (a), distance in center (b, left) and total distance (b, right) in an OF test, time in open arms (c, left) and total entries in arms (c, right) in an EPM test at day 3 after CFA (10 μl) or NS (10 μl) injection. n = 14 for NS and n = 13 for CFA. d–f Immunofluorescent images (d) and statistical data showing c-Fos⁺ cells (e) and nNOS-expressing neurons co-labeled with c-Fos (f) in the vmPFC 4 h after CFA or NS injection. n = 6. g Scheme indicating the vmPFC area where AAV-hSyn-DIO-hM3Dq-eGFP (0.4 μl) and CNO (0.5 mM, 1 μl) or NS (1 μl) was microinjected into nNOS-Cre mice. h Coronal section showing the expression of hM3Dq-eGFP in the vmPFC of nNOS-Cre mice. Similar results were observed in 9 mice out of 10 mice. i Immunofluorescent images indicating the expression of hM3Dq-eGFP in vmPFC nNOS neurons. Arrows marked type I and arrowheads marked type II nNOS-expressing neurons. This experiment was conducted in triplicate with 5 mice and similar results were observed. j Bath application of CNO (5 μM) elicited action potential firing in neurons expressing hM3Dq-eGFP in brain slices of nNOS-Cre mice. k–m Distance in center (k, left) and total distance (k, right) in an OF test, time in open arms (l, left) and total entries in arms (l, right) in an EPM test, and withdrawal threshold of hind paw in a von Frey test (m) 21 days after AAV-hSyn-DIO-hM3Dq-eGFP microinjection. n = 15. n–p Latency to feed (n, left) and total feed (n, right) in NSF, time in side with light (o, left) and total distance (o, right) in LDB and withdrawal latency to thermal stimulation (p) 21 days after AAV-hSyn-DIO-hM3Dq-eGFP microinjection. n = 14. Data are the mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 (unpaired two-tailed Student’s t-test). Scale bars, 50 μm in (d) and (i), 1 mm in (h). Source data are provided as a Source data file. Exact p values and additional statistical information can be found in Source data.