Fig. 3.

Activation of the ACC→VTA excitatory projection by chronic itch. A Schematic showing the timeline of the immunostaining experiments. B Scheme for retrograde labelling of VTA-projecting ACC neurons. C Representative images of c-Fos co-staining with mCherry in the ACC for both DW- and DCP-treated groups. Arrows indicate c-Fos⁺/mCherry⁺ neurons. Scale bars, 200 μm (left) and 50 μm (right). D, E Numbers of mCherry⁺ neurons in total (D) and different sections (E) of the ACC. n = 5 or 4 mice per group. F, G Numbers of c-Fos⁺/mCherry⁺ neurons in total (F) and different sections (G) of the ACC. n = 5 or 4 mice; *P <0.05, **P <0.01; unpaired Student’s t test for (F); two-way ANOVA followed by Bonferroni post hoc analysis for (G). H Percentage of c-Fos⁺/ mCherry⁺ neurons in mCherry⁺ neurons for different bregma planes of the ACC. n = 5 or 4 mice; ***P <0.001; two-way ANOVA followed by Bonferroni post hoc analysis. I Scheme for retrograde labelling of VTA-projecting ACC neurons in Vgat-Cre::H2B-GFP mice. J Representative images of GFP co-staining with mCherry in the ACC. Arrows indicate GFP⁺/mCherry⁺ neurons. Scale bars, 200 μm (left) and 50 μm (right). K Summary of the numbers of mCherry⁺ neurons in different sections of the ACC. n = 5 mice per group. L Numbers of GFP⁺/mCherry⁺ neurons in different parts of the ACC. n = 5 mice per group. M Percentage of GFP⁺/mCherry⁺ neurons in mCherry⁺ neurons for different sections of the ACC. Few co-labeled neurons are detected. n = 5 mice per group. Data are presented as mean ± SEM.