Parvalbumin Neurons in Zona Incerta Regulate Itch in Mice

Abstract

Pain and itch are intricately entangled at both circuitry and behavioral levels. Emerging evidence indicates that parvalbumin (PV)-expressing neurons in zona incerta (ZI) are critical for promoting nocifensive behaviors. However, the role of these neurons in itch modulation remains elusive. Herein, by combining FOS immunostaining, fiber photometry, and chemogenetic manipulation, we reveal that ZI PV neurons act as an endogenous negative diencephalic modulator for itch processing. Morphological data showed that both histamine and chloroquine stimuli induced FOS expression in ZI PV neurons. The activation of these neurons was further supported by the increased calcium signal upon scratching behavior evoked by acute itch. Behavioral data further indicated that chemogenetic activation of these neurons reduced scratching behaviors related to histaminergic and non-histaminergic acute itch. Similar neural activity and modulatory role of ZI PV neurons were seen in mice with chronic itch induced by atopic dermatitis. Together, our study provides direct evidence for the role of ZI PV neurons in regulating itch, and identifies a potential target for the remedy of chronic itch.

Keywords: chemogenetics; fiber photometry; itch; parvalbumin; zona incerta.

FIGURE 1

Acute itch stimuli and Chronic itch stimuli increase FOS expression in the ZIv. (A) Representative images of FOS staining in the ZI after saline (the upper panel), histamine (the middle panel), and chloroquine (the lower panel) injection. Scale bar = 200 μm. (B) Quantification of FOS-expressing neurons in response to saline, histamine, and chloroquine injection showed both histamine and chloroquine stimuli increased FOS expression in the ZIv instead of other ZI subregions. n = 3 mice per group, three sections per mouse. One-way ANOVA with post hoc LSD test for multiple comparisons. For the analysis in ZIv, F(2,6) = 17.378, P = 0.003. (C) Representative images of FOS staining in the ZI in control (the upper panel) and AD (the lower panel) mice. Scale bar = 200 μm. (D) Quantification of FOS-expressing neurons in control and AD mice showed AD increased FOS expression in the ZIv instead of other ZI subregions. n = 3 mice per group, three sections per mouse. Unpaired T-test. For the analysis in ZIv, t = –4.759, P = 0.009. ZIr, the rostral ZI; ZIv, the ventral ZI; ZId, the dorsal ZI; ZIc, caudal ZI; AD, atopic dermatitis. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 2

Acute and chronic itch stimuli increase FOS expression in ZI PV neurons. (A–C) Representative images of FOS and PV staining in ZI after saline (the upper panel), histamine (the middle panel), and chloroquine (the lower panel) injection. (B) Representative images of FOS and PV staining in ZI in control (the upper panel) and AD (the lower panel) mice. Scale bar = 100 μm. (C) Quantification of FOS-expressing neurons in response to saline, histamine, and chloroquine injections showed both histamine and chloroquine stimuli increased FOS expression in ZI PV neurons. n = 3 mice per group, three sections per mouse. One-way ANOVA with post hoc LSD test for multiple comparisons. F(2,6) = 73.642, P < 0.001. (D) Quantification of FOS-expressing neurons in control and AD mice showed chronic itch stimuli increased FOS expression in ZI PV neurons. n = 3 mice per group, three sections per mouse. Unpaired T-test. t = –7.03, P = 0.002. ^∗∗∗∗P < 0.0001, ^∗∗P < 0.01.

FIGURE 3

Increased activity of ZI PV neurons during acute itch-induced scratching behavior. (A) The fiber photometry setup. (B) Schematic showing the viral targeting of AAV-DIO-GCaMP6s-EYFP and AAV-DIO-EYFP into the ZI of PV-Cre mice. (C) Histological verification of viral expression (green) and optical fiber implantation in the ZI in a representative mouse. (D) Representative photographs showing the expression of PV (red) in GCaMP6s-positive (green) neurons in the ZI of PV-Cre mice. The framed area in d1 was magnified in d2-4. Scale bars represent 100 μm in d1 and 50 μm in d2-4. (E) Representative GCaMP6s fluorescence trace (top) and behavioral trace (bottom) recorded simultaneously in ZI PV neurons in response to histamine injection. (F) Heatmap illustrating GCaMP6s fluorescence aligned to the beginning of individual scratching trains in all the mice in response to histamine stimuli. Each row represents Ca²⁺ signals corresponding to one scratching train. The color scale at the right indicates ΔF/F. (G) Mean fluorescent signal in response to histamine stimuli in all the mice recorded, with shaded areas indicating the SEM. The black and gray lines 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). The vertical dotted line indicates the scratching bout. (H) Area under the curve showing fluorescence changes of ZI PV neurons in the mice with AAV-GCaMP6s and AAV-EYFP injection in both pre-scratching and post-scratching periods under the condition of histamine stimuli. Repeated ANOVA followed by simple effects analysis. n = 5 mice in EYFP group and 6 mice in GCaMP6s group, F(1,9) = 27.892, P = 0.001. (I–L) Same conventions as (E–H) but for recording in response to chloroquine stimuli. n = 5 mice in EYFP group and 6 mice in GCaMP6s group, F(1,9) = 36.586, P < 0.001. ^∗∗∗P < 0.001, ^∗∗P < 0.01. PMT, photomultiplier.

FIGURE 4

ZI PV neurons negatively regulate scratching behaviors during acute itch. (A) Schematic illustration of viral injection for chemogenetic modulation of ZI PV neurons. (B) Histological verification of viral expression in the ZI in a representative mouse with AAV-DIO-hM3Dq-mCherry injection. Scale bar = 250 μm. (C) Representative photographs showing the expression of PV (green) in hM3Dq-positive (red) neurons in the ZI of PV-Cre mice. (D) Representative photographs showing the expression of FOS (green) in hM3Dq-positive (red) neurons in the ZI of PV-Cre mice. (E) Quantification of FOS-expressing neurons in mice with AAV-hM3Dq and AAV-mCherry injection showed CNO injection increased FOS expression in ZI PV neurons in the mice in AAV-hM3Dq group. n = 3 mice per group, three sections per mouse. Unpaired T-test. t = –14.44, P < 0.001. (F,G) Chemogenetic activation of ZI PV neurons reduces scratching behaviors induced by histamine (F) and chloroquine (G), while inhibition of them exerts no effect on scratching behaviors. One-way ANOVA with post hoc LSD test for multiple comparisons. For the analysis of histamine-induced itch, F(2,6) = 10.599, P = 0.001. n = 6 mice in EGFP and hM4Di groups and 7 in hM3Dq group. For the analysis of chloroquine-induced itch, F(2,6) = 13.341, P < 0.001. n = 6 mice in EGFP and hM4Di groups and 7 in hM3Dq group. n = 7 mice in EGFP and hM4Di groups and 8 in hM3Dq group. ^∗∗∗P < 0.001, ^∗∗P < 0.01.

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.