G-Protein-Coupled Estrogen Receptor (GPER) in the Rostral Ventromedial Medulla Is Essential for Mobilizing Descending Inhibition of Itch

Nonserotonergic but GABAergic GPER⁺ neurons were significantly activated in DCP-induced chronic contact dermatitis rat and mouse models. A, Distinctive localization of GPER in RVM of female WT rat. Scale bar, 200 µm. B, High-power image of boxed area in A. Scale bar, 50 µm. C, Percentage of GABAergic and non-GABAergic GPER⁺ neurons in the RVM of WT rats. Nine slides from three rats. D, E, Double IHC of GPER (green) and 5-HT (red; D); GPER (green) and GAD67 (red; E) in the RVM of female naive rats. Scale bar, 20 µm. F, G, Double IHC showing the coexpression of GPER (green) and c-fos (red) in the RVM of contact dermatitis female rat (F) and mouse (G) models. Scale bar, 20 µm.

Selective ablation of RVM GPER⁺ neurons aggravates acute and chronic itch. A, Schematic showing the injection of AAV-DIO-taCasp3 into the RVM of female WT or Gper-Cre mice. B, C, Western blot showing the decreased expression of GPER protein in the RVM of Gper-Cre mice 35 d after AAV-DIO-taCasp3 injection; n = 4 mice for each group; one-way ANOVA and Tukey's post hoc test were used to assess statistical differences. F = 36.92, p = 0.0003 for WT::taCasp3 versus Gper-Cre::taCasp3, p < 0.0001 for WT versus Gper-Cre::taCasp3. D, Apoptotic neurons in the RVM were examined using TUNEL staining. Scale bar, 50 µm. E, The number of apoptotic neurons in the RVM of female WT or Gper-Cre mice injected with AAV-CAG-DIO-taCasp3-TEVp; n = 8–9 slides from three mice, unpaired Student's t test. p < 0.0001, t = 7.671, df = 15. F, Acute mechanical itch in GPER⁺ neuron-ablated Gper-Cre mice and control mice; n = 10–11 mice for each group; two-way ANOVA followed by Bonferroni's post hoc test. F_(1,133) = 26.65; 0.08 g, p = 0.0299; 0.3 g, p = 0.0316. G, H, Ablation of RVM GPER⁺ neurons increased the scratching behavior in response to chloroquine (150 µg/20 µL; G) and histamine (500 µg/20 µL; H); n = 8–10 mice for each group; unpaired Student's t test. p < 0.0001, t = 5.855, df = 14 (G); p = 0.0215, t = 2.548, df = 16 (H). I, Ablation of RVM GPER⁺ neurons increased the scratch bouts induced by DCP on every testing day; n = 7–9 mice for each group. Difference in area under curve (AUC) was compared by unpaired Student's t test. p = 0.0115, t = 2.908, df = 14. J, K, Representative photograph of neck skin in DCP-treated female WT (J) and Gper-Cre (K) mice injected with AAV-DIO-taCasp3.

Chemogenetic inhibition or activation of RVM GPER⁺ neurons differentially modulates itch-related behaviors. A, Schematic showing the injection of AAV-DIO-hM4Di-mCherry into the RVM of female Gper-Cre mice. B, Expression of hM4Di-mCherry in the RVM after viral infection. Scale bar, 50 µm. C, Acute mechanical itch test 30 min following saline or CNO injection (1 mg/kg, i.p.) in mice expressing hM4Di receptors in RVM GPER⁺ neurons; n = 12 mice for each group; two-way ANOVA followed by Bonferroni's post hoc test. F_(1,154) = 99.52; 0.013 g, p < 0.0001; 0.04 g, p = 0.0001; 0.08 g, p < 0.0001; 0.1 g, p = 0.0356; 0.3 g, p = 0.0100. D, E, Effect of pharmacogenetic inhibition of GPER⁺ neurons in the RVM on scratching behavior induced by intradermal injection of chloroquine (150 µg/20 µL; D) and histamine (500 µg/20 µL; E); n = 10–12 mice for each group; unpaired Student's t test. p = 0.0028, t = 3.382, df = 21 (D). p = 0.0021, t = 3.523, df = 20 (E). F, Time-dependent changes in scratching bouts of DCP-treated Gper-Cre mice after viral infection, CNO were injected intraperitoneally on days 5, 7, and 10 (gray frame), and the control group were injected with saline intraperitoneally; n = 5 mice for each group. Note CNO-treated group had higher scratching bouts than the saline group on the days of treatment (day 5, 7, and 10), although statistical significance was not reached (a: p = 0.1022, t = 0.1845 df = 8; b: p = 0.5456, t = 0.6310, df = 8; c: p = 0.2258, t = 1.312, df = 8). G, Schematic showing the injection of AAV-DIO-hM3Dq-eGFP into the RVM of female Gper-Cre mice. H, Expression of hM3Dq-eGFP in the RVM after viral infection. Scale bar, 50 µm. I, Acute mechanical itch test following saline or CNO injection in mice expressing hM3Dq receptors in RVM GPER⁺ neurons; n = 9 mice for each group; two-way ANOVA followed by Bonferroni post hoc test. F_(1,112) = 7.877; 0.013 g, 0.04 g, 0.08 g, 0.1 g, 0.2 g and 0.8 g, p > 0.9999; 0.3 g, p = 0.4622. J, K, Pharmacogenetic activation of RVM GPER⁺ neurons decreased scratching behavior induced by chloroquine (200 µg/20 µL; J) and histamine (600 µg/20 µL; K); n = 6–9 mice for each group; CNO (1 mg/kg, i.p.); unpaired Student's t test. p = 0.0012, t = 4.062, df = 14 (J). p = 0.0005, t = 4.916, df = 11 (K). L, Pharmacogenetic activation of RVM GPER⁺ neurons attenuated spontaneous scratching behavior on day 15 of contact dermatitis mouse models; n = 4–5 mice for each group, unpaired Student's t test. p = 0.0339, t = 2.630, df = 7. NS, Not significant.

GPER deficiency results in hypersensitivity to itch in mice and rats. A, Western blot showing that GPER protein expression in the RVM was increased in female contact dermatitis rat models; n = 3 rats; unpaired Student's t test. p = 0.0010, t = 8.586, df = 4. B, Scratching bouts of WT and GPER^−/− rats in the normal physiological condition; n = 5–6 rats for each group; one-way ANOVA and Tukey's post hoc test were used to assess statistical differences. F = 0.9604. p =0.9543 for WT female versus GPER^−/− female; p = 0.4076 for WT male versus GPER^−/− male. C, Acute mechanical itch test of female WT and GPER^−/− rats; n = 12–15 rats; two-way ANOVA followed by Bonferroni's post hoc test. F_(1,225) = 66.01; 0.2 g, p < 0.0001; 0.3 g, p = 0.0029; 0.6 g, p = 0.0003; 0.8 g, p = 0.0136. D–F, Scratching bouts of female WT and GPER^−/− rats induced by intradermal injection of chloroquine (2.5 mg/50 µL; D), histamine (2.5 mg/50 µL; E), and 5-HT (200 µg/50 µL; F); n = 5–11 rats; unpaired Student's t test. Chloroquine, p = 0.0398, t = 2.266, df = 14; histamine, p = 0.0302, t = 2.487, df = 11; 5-HT, p = 0.0081, t = 2.938, df = 20. G, The number of scratching bouts of DCP-treated GPER^−/− rats increased significantly on every testing day compared with WT rats; n = 6 rats for each group. Difference in area under curve (AUC) was compared by unpaired Student's t test. p = 0.0014, t = 4.350, df = 10. H, GPER^−/− rats treated with DCP for 7 d showed increased spontaneous scratching behavior on the eighth day (without DCP treatment) compared with WT rats treated with DCP for 7 d; n = 16–18 rats; unpaired Student's t test. p = 0.0006, t = 3.789, df = 32. I, Photograph of the back neck of a WT rat (shaved) before DCP treatment (left), DCP-treated WT rat on day 15 (middle) and DCP-treated GPER^−/− rat on day 15 (right). J, HE-stained skin before and after DCP treatment of WT and GPER^−/− rats. Scale bar, 50 µm. K, The lesion severity score of neck skin in control rats, DCP-treated WT rats, and GPER^−/− rats; 15 slides from three rats; one-way ANOVA and Tukey's post hoc test were used to assess statistical differences. F = 53.14; p < 0.0001 for WT versus WT+DCP, WT+DCP versus GPER^−/− +DCP and GPER^−/− versus GPER^−/− +DCP.

GPER^−/− rats show increased scratching behavior than WT rats in imiquimod-induced psoriasis models. A, The timeline for imiquimod-induced psoriasis-like rat model. B, C, Scratching behavior of female WT and GPER^−/− rats on each day before (B) and after (C) painting with imiquimod cream; n = 5 rats for each group; area under curves (AUC) was compared by unpaired Student's t test. t = 2.149, df = 8 (B). p < 0.0001, t = 8.499, df = 8 (C). D, Photograph of the back neck of imiquimod-treated WT and GPER^−/− rats on day 8. E, HE-stained neck skin of naive and imiquimod-treatment female WT and GPER^−/− rats. Scale bar, 50 µm. F, The thickness of epidermis of neck skin in naive and imiquimod-treated female WT rats and female GPER^−/− rats; 15 slides from three rats; one-way ANOVA and Tukey's post hoc test were used to assess statistical differences. F = 99.64; p < 0.0001 for WT versus WT IMQ and GPER^−/− versus GPER^−/− IMQ. p = 0.0001 for WT IMQ versus GPER^−/− IMQ. IMQ, imiquimod.

GPER deficiency results in fewer activation of RVM neurons in contact dermatitis female rat models. Representative IHC images and graph showing c-fos⁺ neurons and serotonergic neurons in the RVM of naive and DCP-treated WT and GPER^−/− rats. Scale bar, 100 µm. The scatter plots show number of c-fos⁺ neurons in 12 slides from three rats for naive groups and 23–24 slides from five rats for DCP-treated groups. Differences were compared using one-way ANOVA and Tukey's post hoc tests. F = 15.10; p < 0.0001 for WT naive versus WT DCP, p = 0.0445 for WT DCP versus GPER^−/− DCP, p = 0.0106 for GPER^−/− naive versus GPER^−/− DCP.

GPER^−/− mice are hypersensitive to chemical and mechanical itch. A, Scratch bouts of female WT and GPER^−/− mice induced by intradermal injection of compound 48/80 (50 µg/20 µL); n = 4–5 mice for each group; unpaired Student's t test. p = 0.0004, t = 6.308, df = 7. B, Systemic administration of G15 (0.5 mg/kg, i.p.) significantly increased scratching bouts of WT female mice induced by compound 48/80; n = 4–5 mice; unpaired Student's t test. p = 0.0005, t = 6.092, df = 7. C, Acute mechanical itch test of WT and GPER^−/− female mice; n = 8 mice for each group; two-way ANOVA followed by Bonferroni's post hoc test. F_(1,154) = 128.1; 0.013 g, p = 0.0232; 0.02 g, p = 0.0019; 0.025 g, 0.08 g, 0.1 g, and 0.2 g, p < 0.0001; 0.3 g, p = 0.0489. D, E, Scratching behavior of WT and GPER^−/− mice on each day before (D) and after (E) painting with imiquimod cream; n = 4 mice for each group. Area under the curves (AUC) was compared using unpaired Student's t test. p = 0.0613, t = 2.298, df = 6 (D). p = 0.0287, t = 2.863, df = 6 (E). F, Photograph of the back neck and HE-stained skin of imiquimod-treated WT and GPER^−/− mice. Scale bar, 50 µm. G, The lesion severity score of neck skin in imiquimod-treated WT and GPER^−/− mice; four slides from four mice; unpaired Student's t test. p = 0.5847, t = 0.5774, df = 6. H, Acute mechanical itch test of WT and GPER^−/− male mice; n = 8 mice for each group; two-way ANOVA followed by Bonferroni's post hoc test. F_(1,154) = 140.3; 0.013 g, 0.02 g, and 0.025 g, p < 0.0001; 0.04 g, p = 0.0007; 0.08 g, p = 0.0001; 0.1 g, p = 0.0185; 0.2 g, p = 0.0007. I, J, Scratch bouts of male WT and GPER^−/− mice induced by intradermal injection of chloroquine (I; 150 µg/20 µL) and 5-HT (J; 10 µg/20 µL); n = 6–8 mice for each group; unpaired Student's t test. p = 0.1569, t = 1.531, df = 10 (I); p = 0.1843, t = 1.402, df = 13 (J).

Male GPER^−/− rats are hypersensitive to mechanical and chemical itch. A, Male GPER^−/− rats responded to light mechanical probing of the back neck by hindpaw scratching more vigorously than their WT counterparts; n = 12 rats for each group; two-way ANOVA followed by Bonferroni's post hoc test. F_(1,198) = 110.6; 0.2 g, 0.6 g, 0.8 g, and 2 g, p < 0.0001; 0.3 g, p = 0.0003; 4 g, p = 0.0066. B, Photograph of neck skin in DCP-treated male rats on day 8. C, HE-stained skin of naive and DCP-treated male WT and GPER^−/− rats. Scale bar, 100 µm. D, Scratch bouts on every testing day of DCP-treated WT and GPER^−/− male rats; n = 3–6 rats for each group; area under the curves (AUC) was compared using unpaired Student's t test. p = 0.0070, t = 3.773, df = 7. E, Spontaneous scratching behavior on day 8 of WT and GPER^−/− male rats; n = 3–6 rats, unpaired Student's t test. p = 0.0409, t = 2.501, df = 7.

GPER modulates itch through regulating μ opioid signaling. A, Double IHC showing the coexpression of GPER (green) and MOR (red) in the RVM of WT rat. Scale bar, 50 µm. B, Double staining of GPER and Leu enkephalin in RVM of female rat. Scale bar, 10 µm. C–F, Western blot showing increased MOR phosphorylation in the contact dermatitis female WT rats but not female GPER^−/− rats; n = 3 rats for each group; one-way ANOVA and Tukey's post hoc test were used to assess statistical differences; p-MOR/MOR, F = 11.80, p = 0.0026 for WT versus WT+DCP; P-MOR/β-actin, F = 32.03, p = 0.0004 for WT versus WT+DCP; MOR/β-actin, F = 1.693.