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. 2022 Dec 17;39(5):793–807. doi: 10.1007/s12264-022-00996-6

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© The Author(s) 2022

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Contribution of ACC neurons to chronic itch-induced scratching behaviors. A Schematic showing the experimental timeline for establishing the chronic itch model. DCP, diphenylcyclopropenone; DW, distilled water. B Time course of DCP-induced scratching behavior. Numbers of scratching bouts within 60 min were counted. n = 5 mice per group; **P <0.01, ***P <0.001; two-way ANOVA followed by Bonferroni post hoc analysis. C Representative immunostaining images of c-Fos expression in the ACC. Scale bars, 200 μm (left) and 30 μm (right). D, E Numbers of c-Fos⁺ neurons in total (D) and different bregma planes (E) of the ACC in the DCP-treated group compared with the DW control group. n = 4 or 5 mice; ***P <0.001; unpaired Student’s t test for (D); two-way ANOVA followed by Bonferroni post hoc analysis for (E). F Schematic showing the experimental timeline for intrathecal ablation of itch-specific GRPR⁺ neurons. Sap, saporin. G Scratching behavior evoked by DCP is significantly blocked in mice treated with bombesin-sap compared with blank-sap. ***P <0.001; unpaired Student’s t test. H, I Representative image (H) and quantification (I) showing a dramatic reduction of DCP-induced c-Fos expression in the ACC when the ascending itch signal is blocked by bombesin-sap. n = 3 mice per group; *P <0.05; unpaired Student’s t test. Scale bar, 200 μm (left), 50 μm (right). J Timeline for chemogenetic inhibition of ACC neuronal activity and behavioral tests. K Schematic of the sites of viral injection into the ACC. L A representative image showing the expression of hM4Di-mCherry in the ACC. Scale bars, 200 μm (left) and 50 μm (right). M, N Whole-cell patch clamp recordings reveal that bath application of CNO (5 μmol/L) blocks action potential firing of ACC pyramidal neurons. M Representative trace. N Quantification data. n = 5 neurons. ***P <0.001; paired Student’s t test. O Quantitative analyses of the number of scratching bouts within 60 min in mice treated with saline or CNO to chemogenetically inactivate ACC neurons. Sal: saline. n = 6 mice per group; ***P <0.001; unpaired Student’s t test. P, Q Total distance travelled (P) and average velocity (Q) in the open field. No significant difference was detected between the two groups. n = 6 mice per group. Data are presented as the mean ± SEM.