Orexin neurons play contrasting roles in itch and pain neural processing via projecting to the periaqueductal gray

Abstract

Pain and itch are recognized as antagonistically regulated sensations; pain suppresses itch, whilst pain inhibition enhances itch. The neural mechanisms at the central nervous system (CNS) underlying these pain-itch interactions still need to be explored. Here, we revealed the contrasting role of orexin-producing neurons (ORX neurons) in the lateral hypothalamus (LH), which suppresses pain while enhancing itch neural processing, by applying optogenetics to the acute pruritus and pain model. We also revealed that the circuit of ORX neurons from LH to periaqueductal gray regions served in the contrasting modulation of itch and pain processing using optogenetic terminal inhibition techniques. Additionally, by using an atopic dermatitis model, we confirmed the involvement of ORX neurons in regulating chronic itch processing, which could lead to a novel therapeutic target for persistent pruritus in clinical settings. Our findings provide new insight into the mechanism of antagonistic regulation between pain and itch in the CNS.

Fig. 1. Optical inhibition of ORX neurons induces opposite modulation in itch and pain.

a Left, schematic illustration of ORX-tTA mice. Middle, schematic showing the injection of the AAV-TRE-ArchT-mCherry into the LH of ORX-tTA mice and implantation of the optical fiber. Right, the timeline of the experiments. b Left, Histological verification of viral injection and optical fiber implantation in a representative ORX-tTA mouse. Right, the quantification (inset pie chart) shows the co-expression of ArchT-mCherry with orexin in LH of ORX-tTA mice (n = 3 sections per animal from 7 animals). Scale bars, 50 μm. c Optical inhibition of ORX neurons did not alter scratching behaviors in the absence of pruritogen (left) and wiping behaviors in the absence of algogen (right) (n = 4 mice in each group). Paired Student’s t test. d Schematic illustration of scratching (itch-related behavior) induced by the chloroquine injection into the nape. e Time course of the number of scratching behaviors induced by intradermal chloroquine injection. The plots indicate the cumulative number of scratching bouts recorded every 5 min. f Optical inhibition of ORX neurons reduced the chloroquine-induced scratching behaviors (n = 6–7 mice in each group). (g) Schematic illustration of wiping (pain-related behavior) induced by the capsaicin injection into the right cheek. h Time course of the number of wiping behaviors induced by capsaicin injection into the cheek. The plots indicate the cumulative number of wiping events recorded every 3 min. i Optical inhibition of ORX neurons increased the capsaicin-induced wiping behaviors (n = 5–7 mice in each group). The data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test. hORX promoter, human prepro-orexin promoter; tTA, tetracycline transactivator; TRE, tetracycline response element; LH, lateral hypothalamus.

Fig. 2. Optical stimulation of orexin neurons does not affect itch and pain.

a Left, schematic showing the injection of the AAV-TRE-ChR2-mCherry into the LH of ORX-tTA mice and implantation of the optical fiber. Right, the timeline of the experiments. b Left, Histological confirmation of viral injection and optical fiber implantation in a representative ORX-tTA mouse. Right, the quantification (inset pie chart) shows the co-expression of ChR2-mCherry with orexin in LH of ORX-tTA mice (n = 3 sections per animal from 10 animals). Scale bars, 50 μm. c Optical activation of ORX neurons did not alter scratching behaviors in the absence of pruritogen (left) and wiping behaviors in the absence of algogen (right) (n = 4 mice in each group). Paired Student’s t test. d Time course of the number of scratching behaviors induced by intradermal chloroquine injection. The plots indicate the cumulative number of scratching bouts recorded every 5 min. e Optical activation of ORX neurons did not alter the chloroquine-induced scratching behaviors (n = 7–8 mice in each group). f Time course of the number of wiping behaviors induced by capsaicin injection into the cheek. The plots indicate the cumulative number of wiping events recorded every 3 min. g Optical activation of ORX neurons did not alter the capsaicin-induced wiping behaviors (n = 8–10 mice in each group). The data represent the mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test was done for statistical analysis. ORX, orexin; tTA, tetracycline transactivator; TRE, tetracycline response element; LH, lateral hypothalamus.

Fig. 3. Optical inhibition of the LH → PAG axis of orexin neurons induces opposite modulation in itch and pain.

a Top, schematic showing the injection of the AAV-TRE-ArchT-mCherry into the LH of ORX-tTA mice and implantation of the optical fiber into lPAG/vlPAG. Bottom, the timeline of the experiments. b Left, the quantification (inset pie chart) shows the co-expression of ArchT-mCherry with orexin in LH of ORX-tTA mice (n = 3 sections per animal from 8 animals). Right, histological confirmation of ArchT-mCherry expression and optical fiber implantation at lPAG/vlPAG in a representative ORX-tTA mouse. Scale bars, 50 μm. c Optical inhibition of LH^ORX → lPAG/vlPAG neuronal terminals did not alter scratching behaviors in the absence of pruritogen (left) and wiping behaviors in the absence of algogen (right) (n = 5 mice in each group). Paired Student’s t test. (d) Time course of the number of scratching behaviors induced by intradermal chloroquine injection. The plots indicate the cumulative number of scratching bouts recorded every 5 min. e Optical inhibition of ORX neurons reduced the chloroquine-induced scratching behaviors (n = 7–8 mice in each group). f Time course of the number of wiping behaviors induced by capsaicin injection into the cheek. The plots indicate the cumulative number of wiping events recorded every 3 min. g Optical inhibition of ORX neurons increased the capsaicin-induced wiping behaviors (n = 8–9 mice in each group). The data represent the mean ± SEM. **p < 0.01, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons tests. ORX, orexin; tTA, tetracycline transactivator; TRE, tetracycline response element; LH, lateral hypothalamus; Aq, aqueductal; lPAG, lateral periaqueductal gray; vlPAG, ventrolateral periaqueductal gray.

Fig. 4. Ablation of orexin neurons suppresses scratching behavior and skin barrier dysfunction in chronic pruritus model.

a Schematic illustration showing the specific ablation of orexin neurons using tet-off system in ORX-abl mice. In the absence of DOX, orexin neurons are specifically ablated by expressing DTA. b Experimental timeline of the DCP-induced chronic pruritus model. c Time course of the number of scratching behaviors in DCP-induced chronic itch model. The plots indicate the cumulative number of scratching bouts recorded every 10 min. d Scratching behaviors during DCP-induced chronic itch were significantly suppressed in ORX-abl mice (n = 5–10 for each group). e The rise of TEWL by repeated DCP application was decreased in ORX-abl mice compared to WT counterparts (n = 5–10 for each group). The data represent the mean ± SEM. **p < 0.01, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test. hORX promoter, human prepro-orexin promoter; tTA, tetracycline transactivator; DTA, diphtheria toxin A; DOX, doxycycline; DCP, diphenylcyclopropenone; TEWL, transepidermal water loss; DW, distilled water.

Fig. 5. The LH → PAG axis of orexin neurons is crucial for the diametric regulation of itch and pain processing.

A visual summary of our present findings. The ArchT-driven optical inhibition of LH^ORX → lPAG/vlPAG neuronal terminals resulted in a decrease in itch-related behavior and an increase in pain-related behavior (Fig. 3). This result aligns with those obtained from optogenetic inhibition at the cell body of ORX neurons in the LH (Fig. 1). Thus, our present findings support a pivotal role for the LH → PAG axis projection of ORX neurons in the opposing modulation of itch and pain neural processing. LH, lateral hypothalamus; lPAG, lateral periaqueductal gray; vlPAG, ventrolateral periaqueductal gray.