FIGURE 5.

ZI PV neurons negatively regulate scratching behaviors during chronic itch. (A) Schematic showing the viral targeting of AAV-DIO-GCaMP6s-EYFP and AAV-DIO-EYFP into the ZI of PV-Cre mice with AD. (B) Histological verification of viral expression (green) and optical fiber implantation in the ZI in a representative mouse. (C) Representative GCaMP6s fluorescence trace (top) and behavioral trace (bottom) recorded simultaneously in ZI PV neurons in response to calcipotriol injection. (D) Heatmap illustrating GCaMP6s fluorescence aligned to the beginning of individual scratching trains in all the mice in response to calcipotriol stimuli. Each row represents Ca²⁺ signals corresponding to one scratching train. (E) Mean fluorescent signal in response to calcipotriol stimuli in all the mice recorded, with shaded areas indicating the SEM. The black and gray line represent the signals of PV ZI neurons in mice with AAV-GCaMP6s and AAV-EYFP injection, respectively. The red line represents statistically significant increase from the baseline (P < 0.05; multivariate permutation test). (F) Area under the curve showing fluorescence changes of ZI PV neurons in mice with AAV-GCaMP6s and AAV-EYFP injection in both pre-scratching and post-scratching periods under the condition of calcipotriol stimuli. Repeated ANOVA followed by simple effects analysis. n = 4 mice in EYFP group and 6 mice in GCaMP6s group, F(1,8) = 14.355, P < 0.01. (G) Schematic illustration of viral injection in mice with AD for chemogenetic modulation of ZI PV neurons. (H) Histological verification of viral expression within ZI in a representative mouse with AAV-DIO-hM3Dq-mCherry injection. Scale bar = 250 μm. (I) Chemogenetic activation of ZI PV neurons reduces scratching behaviors induced by calcipotriol injection, while the inhibition of them exerts no effect on scratching behaviors. One-way ANOVA with post hoc LSD test for multiple comparisons. F(2,6) = 49.27, P < 0.001. n = 7, 8, and 6 in EGFP, hM3Dq, and hM4Di group, respectively. ^∗∗∗P < 0.001, ^∗∗P < 0.01.