CCI-induced neuropathic pain and neuronal activation were reversed by spinal inhibition of GPR30.

A Schematic illustration of CCI surgery for induction of neuropathic pain (Left) and diagram showing the timeline of CCI surgery, drug administration and behavioral tests (Right).

B Quantitative PCR analysis of Gper1 mRNA in SDH from sham and CCI mice (n = 3 mice for each group).

C-E Behavioral tests of basic nociception and 14 days after CCI or sham surgery along with intrathecal injection of antagonist of GPR30 or vehicle in Von Frey tests (C), Brush tests (D) and Heat tests (E) in mice of both genders (n = 6 mice for each group).

F Immunochemical detection of c-Fos (Green) and GPR30 (Red). Scale bars: 100 μm. Boxed area of images is enlarged on the right. Scale bars: 10 μm. White arrows indicate double-positive cells.

G, H Total number of c-Fos positive neurons in the SDH per section in female mice (G) and male mice (H) (n = 3-4 mice for each group).

I, J Percentage of c-Fos positive neurons expressing GPR30 in female mice (I) and male mice

(J). (n = 3-4 mice for each group).

Data information: in (B), *P < 0.05 (Unpaired Student’s t-test). In (C, D), **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-way ANOVA with Turkey’s multiple comparisons test). In (E), **P < 0.01; ****P < 0.0001 (one-way ANOVA with Turkey’s multiple comparisons test). In (G-J), *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA with Turkey’s multiple comparisons test). All data are presented as mean ± SEM.

GPR30 was widely expressed in the CCK+ excitatory interneurons in the SDH.

A Schematic illustration of the strategy to identify the Camk2+ excitatory interneurons in spinal dorsal horn by AAV (left).

B Double staining of Camk2+ mCherry+ neurons (Red) with GPR30 (Green) by immunohistochemistry. Scale bars: 100 μm. Boxed area of images is enlarged on the right. Scale bars: 10 μm.

C CCKCre mice express tdTomato in a Cre-dependent manner.

D In situ hybridization showing CCK (Green) with tdTomato (Red) in the SDH. Scale bar: 100 μm. Boxed area of image is enlarged on the right. Scale bars: 10 μm.

E Double staining of tdTomato+ neurons (Red) with IB4 (Green) by immunochemistry. Scale bars: 100 μm.

F Immunofluorescence in SDH to verify the co-localization of tdTomato+ neurons (Red) with GPR30 (Green). Scale bars: 100 μm. Boxed area of images is enlarged on the right. Scale bars: 10 μm.

Knockdown of GPR30 in spinal CCK+ neurons alleviated neuropathic pain in CCI mice.

A Schematic illustration of the strategy to knock down Gper1 in the spinal CCK+ neurons in a Cre-dependent manner (Up) and the diagram showing the timeline of virus injection, CCI surgery and behavioral tests (Down).

B Immunochemical detection of the localization of virus expression (Green). Scale bars: 100 μm.

C Quantitative PCR analysis of Gper1 mRNA in SDH from sham and CCI mice with intraspinal virus injection (n = 3-4 mice for each group).

D-F Behavioral tests of basic nociception, 21 days after spinal virus injection and 14 days after CCI or sham surgery along with intrathecal injection of antagonist of GPR30 or vehicle in Von Frey tests (D), Brush tests (E) and Heat tests (F) in mice of both genders (n = 6-7 mice for each group).

G Schematic illustration of real-time place escape/avoidance (PEA) test.

H, I Representative spatial tracking maps showing the location of a Scramble + CCI group mice (H) and a shGper1 + CCI group mice (I) before, during and after mechanical stimulation of hind paw in the chambers.

J Quantification of time the Scramble + CCI group mice spent in preferred chamber before, during and after stimulation (n = 8 mice).

K Quantification of time the shGper1 + CCI group mice spent in preferred chamber before, during and after stimulation (n = 8 mice).

L Quantification of the aversion time of mice. (time spent in preferred chamber after stimulation minus the time spent before stimulation) (n = 8 mice for each group).

Data information: in (C), *P < 0.05; **P < 0.01 (one-way ANOVA with Turkey’s multiple comparisons test). In (D, E), **P < 0.01; ****P < 0.0001 (two-way ANOVA with Turkey’s multiple comparisons test). In (F), **P < 0.01; ****P < 0.0001 (one-way ANOVA with Turkey’s multiple comparisons test). In (J, K), *P < 0.05; **P < 0.01; ***P < 0.001; ns = not significant. (one-way ANOVA with Turkey’s multiple comparisons test). In (L), ***P < 0.001 (Unpaired Student’s t-test). All data are presented as mean ± SEM.

Knockdown of GPR30 in spinal CCK+ neurons reverse the enhancement of sEPSC in CCI mice

A Schematic illustration of the strategy to knock down Gper1 in the spinal CCK+ neurons in a Cre-dependent manner (Left) and the diagram showing the timeline of virus injection, CCI surgery and electrophysiological tests (Right).

B Schematic illustration of spinal slice electrophysiological recordings from CCK-EGFP neurons located in the deep laminae of SDH.

C Representative spontaneous EPSCs in CCK-EGFP neurons.

D The frequency of spontaneous EPSCs.

E The peak amplitude spontaneous EPSCs (n = 10-16 cells from 5 mice).

F Schematic illustration of the strategy to visualize CCK+ neurons in a Cre-dependent manner (Up) and the diagram showing the timeline of virus injection and electrophysiological tests (Down).

G Schematic illustration of spinal slice electrophysiological recordings from CCK-EGFP+ neurons located in the deep laminae of SDH during electric stimulation of the deep laminae of SDH to simulate APMA currents.

H Representative AMPA-current in CCK-EGFP+ neurons before and after administration of G-1.

I The peak amplitude AMPA currents before and after administration of G-1 (n = 8 cells from 4 mice).

Data information: in (D, E), *P < 0.05; **P < 0.01; ns = not significant (one-way ANOVA with Turkey’s multiple comparisons test). In (I), *P < 0.05 (Paired Student’s t-test). All data are presented as mean ± SEM.

GPR30 was expressed in the spinal CCK+ neurons receiving direct projection from S1 cortex.

A Schematic illustration of the strategy to retrograde tracing the projections innervating spinal dorsal horn via CTB-555.

B Fluorescence image showing the site of CTB-555 injection in the spinal dorsal horn. Scale bars: 500 μm.

C Coronal brain section showing the location of tdTomato+ neurons in the S1HL cortex. Scale bars: 300 μm. Boxed area of image is enlarged on the right. Scale bars: 100 μm.

D Schematic illustration of the strategy to anterograde tracing the projections from S1HL cortex.

E Coronal brain section showing the site of AAV injection in the S1HL cortex. Scale bars: 300 μm.

F Fluorescence image showing the S1HL-SDH tract terminates in the deep laminae of spinal dorsal horn. Scale bars: 500 μm. Boxed area of image is enlarged on the right. Scale bars: 100 μm.

G Schematic illustration of the strategy for identifying the spinal post-synaptic neurons of S1-SDH projections (Left) and diagram showing the timeline of AAV2/1 injection in the S1HL cortex, AAV2/9 injection in the spinal dorsal horn and immunofluorescence (Right). H Representative image showing the mCherry+ post-synaptic neurons of the S1HL-SDH projections in the deep laminae of SDH. Scale bars: 100 μm.

I In situ hybridization showing CCK (Green) with RFP (Red) by immunofluorescence in the spinal dorsal horn. Scale bars: 100 μm. Boxed area of image is enlarged on the right. Scale bars: 10 μm. White arrows indicate double-positive cells.

J About 28.13% of RFP+ post-synaptic neurons of S1HL-SDH projections expressing CCK (n = 3 mice).

K Schematic illustration of the strategy for identifying the spinal CCK+ post-synaptic neurons of S1HL-SDH projections in CCKCre mice (Left) and diagram showing the timeline of AAV2/1 injection in the S1HL cortex, AAV2/9 injection in the spinal dorsal horn and immunofluorescence (Right).

L Representative image showing the EGFP+ CCK+ post-synaptic neurons of the S1-SDH projections in the spinal dorsal horn with DAPI. Scale bars: 100 μm.

M Double staining of EGFP+ neurons with GPR30 by immunohistochemistry. Scale bars: 100 μm. Boxed area of image is enlarged on the right. Scale bars: 10 μm.

N About 81.7% of CCK+ post-synaptic neurons of S1HL-SDH projections expressing GPR30 (n = 3 mice).

Data information: in (J, N), data are presented as mean ± SEM.

Chemogenetic activation of S1-SDH post-synaptic neurons mimicked neuropathic pain symptoms, which were reversed by spinal inhibition of GPR30.

A Schematic illustration of the strategy for identifying the post-synaptic neurons of S1HL-SDH projections in SDH (Left) and diagram showing the timeline of AAV2/1 injection in the S1HL cortex, AAV2/9 injection in the spinal dorsal horn and behavioral tests (Right).

B Spontaneous pain induced by intraperitoneal injection of CNO within 20 min (n = 6 mice for each group).

C-E Behavioral tests of basic nociception, 28 days after brain virus injection along with intraperitoneal injection of CNO and intrathecal injection of antagonist of GPR30 or vehicle in Von Frey tests (C), Brush tests (D) and Heat tests (E) in mice of both genders (n = 6 mice for each group).

F Immunochemical detection of c-Fos (Green) and m-Cherry+ post-synaptic neurons (Red). Scale bars: 100 μm. Boxed area of images is enlarged on the right. Scale bars: 10 μm.

G Total number of c-Fos positive neurons in the SDH per section (n = 3 mice for each group).

H Percentage of c-Fos positive neurons expressed in m-Cherry (n = 3 mice for each group). Data information: in (B), *P < 0.05 (Unpaired Student’s t-test). In (C, D), **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-way ANOVA with Turkey’s multiple comparisons test). In (E), ***P < 0.001; ****P < 0.0001 (one-way ANOVA with Turkey’s multiple comparisons test). In (G, H), ***P < 0.001; ****P < 0.0001. (one-way ANOVA with Turkey’s multiple comparisons test). All data are presented as mean ± SEM.

CCI-induced neuropathic pain was attenuated by knock-down of GPR30 in S1-SDH post-synaptic neurons.

A Schematic illustration of the strategy to knock down Gper1 in the post-synaptic neurons of S1HL-SDH projections in a Cre-dependent manner (Up) and the diagram showing the timeline of virus injection, CCI surgery and behavioral tests (Down).

B-D Behavioral tests of basic nociception and 14 days after CCI surgery in Von Frey tests (B), Brush tests (C) and Heat tests (D) in mice of both genders (n = 6 mice for each group).

E Immunochemical detection of the localization of virus expression (Green). Scale bars: 100 μm.

F Quantitative PCR analysis of Gper1 mRNA in SDH (n = 3 mice for each group).

Data information: in (B, C), **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-way ANOVA with Turkey’s multiple comparisons test). In (D), *P < 0.05; **P < 0.01; ****P < 0.0001 (one-way ANOVA with Turkey’s multiple comparisons test). In (F), *P < 0.05 (one-way ANOVA with Turkey’s multiple comparisons test). All data are presented as mean ± SEM.

Spinal inhibition of GPR30 did not change the basic nociception while spinal activation of GPR30 mimicked neuropathic pain symptoms in naïve mice

A, B Behavioral tests of nociception before and after intrathecal injection of G-15 or vehicle in Von Frey tests (A) and Heat tests (B) in naive mice of both genders (n = 6 mice for each group).

C Time course of Von Frey tests after intrathecal injection of G-15 or vehicle in CCI mice (n = 6 mice for each group).

D-F Behavioral tests of nociception before and after intrathecal injection of G-1 or vehicle in Von Frey tests (D), Brush tests (E) and Heat tests (F) in naive mice of both genders (n = 6 mice for each group).

G Time course of Von Frey tests after intrathecal injection of G-1 or vehicle in naive mice (n = 6 mice for each group).

H Immunochemical detection of c-Fos (Green) and GPR30 (Red). Scale bars: 100 μm. Boxed area of images is enlarged on the right. Scale bars: 10 μm. White arrows indicate double-positive cells.

I Total number of c-Fos positive neurons in the SDH per section (n = 3 mice for each group).

J Percentage of c-Fos positive neurons expressing GPR30 (n = 3 mice for each group).

Data information: in (A), ns = not significant (two-way ANOVA with Turkey’s multiple comparisons test). In (B), ns = not significant (Unpaired Student’s t-test). In (C), **P < 0.01; ****P < 0.0001; ns = not significant (two-way ANOVA with Turkey’s multiple comparisons test). In (D, E), ***P < 0.001; ****P < 0.0001; ns = not significant. (two-way ANOVA with Turkey’s multiple comparisons test). In (F), **P < 0.01; ****P < 0.0001 (Unpaired Student’s t-test). In (G), *P < 0.05; ***P < 0.001; ****P < 0.0001; ns = not significant (two-way ANOVA with Turkey’s multiple comparisons test). In (I, J), **P < 0.01 (Unpaired Student’s t-test).All data are presented as mean ± SEM.

GPR30 was mainly expressed in the spinal neurons.

A Double-staining of GPR30 (Green) with Nissl (Red) in male (up) and female mice (down). Scale bars: 100 μm. Boxed area of images is enlarged on the right. Scale bars: 10 μm.

B Double-staining of GPR30 (Red) with GFAP (Green) in male (up) and female mice (down). Scale bars: 100 μm. Boxed area of images is enlarged on the right. Scale bars 10 μm.

C Double-staining of GPR30 (Red) with IBA1 (Green) in male (up) and female mice (down). Scale bars: 100 μm. Boxed area of images is enlarged on the right. Scale bars: 10 μm.

D Percentage of GPR30 expressed in neurons, astrocytes and microglia in both genders of mice (n = 3 mice for each group).

Data information: in (D), data are presented as mean ± SEM.

Knockdown of GPR30 in spinal CCK+ neurons did not change the sIPSC in CCI mice.

A Immunohistochemical detection of GPR30 (Red) and shRNA (Green) in mice with virus injection. Note that the intensity of GPR30 fluorescence is less in shGper1-EGFP+ cells than that in scramble-EGFP+ cells. Scale bars: 100 μm. Boxed area of image is enlarged on the right. Scale bars: 10 μm.

B Representative spontaneous IPSCs in CCK-EGFP neurons.

C The frequency of spontaneous IPSCs (n = 12-16 cells from 5 mice).

D The peak amplitude spontaneous IPSCs (n = 12-16 cells from 5 mice).

Data information: in (C, D), ns = not significant (Unpaired Student’s t-test). All data are presented as mean ± SEM.

CCI-induced neuropathic pain was relieved by chemogenetic inhibition of S1-SDH post-synaptic neurons.

A Schematic illustration of the strategy for identifying the post-synaptic neurons of S1HL-SDH projections in SDH (Left) and diagram showing the timeline of AAV2/1 injection in the S1HL cortex, AAV2/9 injection in the spinal dorsal horn, CCI surgery and behavioral tests (Right).

B-D Behavioral tests of basic nociception, 14 days after CCI surgery along with intraperitoneal injection of CNO in Von Frey tests (B), Brush tests (C) and Heat tests (D) in mice (n = 6 mice per group).

Data information: In (B, C), ***P < 0.001; ****P < 0.0001. (two-way ANOVA with Turkey’s multiple comparisons test). In (D), **P < 0.01; ****P < 0.0001 (one-way ANOVA with Turkey’s multiple comparisons test). All data are presented as mean ± SEM