LC→SDH-NA neurons mediate stress-induced mechanical hypersensitivity

(A) Schematic illustration of an experiment to investigate the effects of acute exposure to restraint stress (1 hour) on mechanosensory behavior in mice, using von Frey (vF) filaments. (B) Change in paw withdrawal threshold (PWT) measured by vF filaments in wild-type mice after restraint stress (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P <0.0001 vs. no-restraint stress group). (C) Expression of GCaMP6s (GC; green) in the LC at 3 weeks after intra-LC injection of AAV-flex[GCaMP6s] in NET-Cre mice. TH immunofluorescence is shown in magenta. Dashed line indicates the location of the implanted optic fiber. Scale bar, 100 μm. (D and E) Representative traces and change in the frequency of GCaMP6s signals in LC-NA neurons (n = 6 mice; Friedman test with post hoc Dunn’s multiple comparisons test; **P < 0.01 vs. the data of ‘Before’). Traces shown at the top, middle, and bottom (D) indicate Ca2+ signals before, during, and after restraint stress, respectively. (F) Schematic illustration of the strategy of ablating LC-NA neurons using AAV vectors incorporating DTR (fused with EGFP) injected into the LC in NET-Cre mice. (G) TH immunofluorescence (magenta) and GFP (green) in the LC (left) and NET immunofluorescence (magenta) in the SDH (right) after administration of DTX (10 µg/kg, i.p., two injections 24 h-apart) in control mice (top) and DTR-expressing mice (bottom). Scale bars, 100 μm. (H) Effects of ablation of LC-NA neurons on PWT changes after acute restraint stress (n = 7 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; **P < 0.01 vs. control group). (I) Schematic illustration of the strategy of ablating LC→SDH-NA neurons using a retrograde AAV vector incorporating Cre injected into the SDH and an AAV vector incorporating DTR (fused with EGFP) injected into the LC in wild-type mice. (J) Representative images of LC→SDH - NA neurons in control or DTR-expressing mice treated with vehicle or DTX administration, respectively. GFP (green) and TH (magenta). Scale bar, 100 μm. (K) Effect of ablation of LC→SDH - NA neurons on PWT changes after restraint stress (n = 11 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; **P < 0.01 vs. control group). (L) Schematic illustration of the strategy for activating LC→SDH-NA neuronal axons/terminals using an AAV vector incorporating ChrimsonR (fused with tdTomato) injected into the LC in NET-Cre mice and of an optic cannula implanted in the SDH. (M) Representative images of TH (green) and tdTomato (magenta) expression in the LC (top) and NET (green) and tdTomato (magenta) expression in the SDH (bottom) at 3 weeks after intra-LC injection of AAV-flex[ChrimsonR-tdTomato] in NET-Cre mice. Scale bars, 100 μm (top) and 50 μm (bottom). (N) PWT before and after optogenetic stimulation (opto-stim.) in LC→SDH-NA axons/terminals (625 nm, 2 mW, 10 Hz, 5-ms pulse duration, 5-s light on, 15-s light off, 10 cycles) (Control, n = 4 mice; ChrimsonR, n = 5 mice; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; ****P <0.0001 vs. control group). Data represent mean ± SEM. This figure was created using BioRender.com. See also Figure S1 and S2.

α1ARs in Hes5+ SDH astrocytes are required for stress-induced mechanical hypersensitivity

(A) Schematic illustration of intra-SDH microinjection of AAV-gfaABC1D-GRABNE1m or - GCaMP6m and intra-LC microinjection of AAV-flex[ChrimsonR-tdTomato] in NET-Cre mice. (B) Representative traces of GRABNE1m signals by fluorescence imaging using spinal cord slices. Each trace represents the GRABNE1m signal before and after optogenetic stimulation (625 nm, 1 mW, 10 Hz, 5 ms pulse duration, 1– 20 s). (C) Quantitative analysis of the peak amplitude of GRABNE1m ΔF/F after optogenetic stimulation in LC→SDH-NA axons/terminals (n = 4 slices; one-way ANOVA with post hoc Dunnett’s multiple comparisons test; **P < 0.01, ****P <0.0001). (D) Representative traces of astrocytic GCaMP6m signals by fluorescence imaging using spinal cord slices. Each trace represents the GCaMP6m signal before and after optogenetic stimulation (as described in B). (E) Quantitative analysis of the peak amplitude of GCaMP6m ΔF/F after optogenetic stimulation in LC→SDH-NA axons/terminals (n = 133 cells, 4 slices, 4 mice; Friedman test with post hoc Dunn’s multiple comparisons test; ****P <0.0001). (F) Effect of silodosin (40 nM) on astrocytic Ca2+ responses in the SDH after optogenetic stimulation (10 s) in LC→SDH-NA axons/terminals (Control, n = 83 cells, 4 slices, 4 mice; Silodosin, n = 53 cells, 4 slices, 4 mice; Mann-Whitney test; ****P <0.0001). (G) Effect of intrathecal silodosin (3 nmol) on mechanical hypersensitivity induced by optogenetic stimulation in LC→SDH-NA axons/terminals (Vehicle, n = 5 mice; Silodosin, n = 6 mice; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; *P < 0.05, ***P < 0.001 vs. vehicle group). (H) Change in PWT at 30 min after intrathecal injection of NA (0.1 nmol) in control (Adra1aflox/flox) and Hes5+ astrocyte-selective α1AR conditional knockout mice [Hes5-CreERT2;Adra1aflox/flox mice treated with tamoxifen (TAM) (Hes5+ astrocyte–α1AR cKO mice) (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; ****P <0.0001). (I and J) Stress-induced mechanical hypersensitivity in Hes5+ astrocyte–α1AR cKO mice [I: Control (Adra1aflox/flox), n = 7 mice; Hes5+ astrocyte–α1AR cKO, n = 8 mice] or wild-type mice with intrathecal DCK (10 nmol) (J: n = 5 mice per group) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; **P < 0.01, ***P < 0.001 vs. control or vehicle group). Data represent mean ± SEM. This figure was created using BioRender.com.

Activation of Hes5+ astrocytes reduces activity in Vgat+ INs in the SDH

(A and B) Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control (Adra1aflox/flox) and Vgat+ IN-selective α1AR conditional knockout mice [Vgat-Cre;Adra1aflox/flox mice (Vgat+ INs–α1AR cKO mice)] (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; n.s., not significant). (C) Changes in PWT after acute exposure to restraint stress in control (Adra1aflox/flox; n = 6 mice) and Vgat+ INs–α1AR cKO mice (n = 9 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). (D) Representative trace of membrane potentials in tdTomato+ (Vgat+) SDH neurons after application of NA (20 μM) to spinal cord slices from Vgat-Cre;Rosa-tdTomato mice. A1R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Vgat+ SDH neurons whose NA-evoked response was inhibited by CPA (n = 10 cells from 7 mice). (F) Representative images of Adora1 (A1R) mRNA expression (green) in Slc32a1 (Vgat)+ INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A1R-expressing Vgat+ cells. Scale bar, 25 μm. (G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2+ INs (cyan) at 3 weeks after intra-SDH injection of AAV-flex[SaCas9] and AAV-flex[mCherry]-U6-sgAdora1 in Vgat-Cre mice. Arrowheads indicate genome-editing Vgat+ cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Vgat+ INs after application of NA and CPA to spinal cord slices from Vgat-Cre mice with conditional knockdown of A1Rs in Vgat+ INs (SDH-Vgat+ IN–A1R cKD) and their controls (Control; Vgat-Cre mice with intra-SDH injection of AAV-flex[mCherry]). (I) Percentage of mCherry+ (Vgat+) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Vgat+ IN–A1R cKD, n = 8 cells from 5 mice). (J) Expression of hM3Dq (green, detected by HA-tag) in the SDH at 3 weeks after intra-SDH injection of AAV-flex[hM3Dq] in Hes5-CreERT2 mice treated with TAM. Dashed line indicates an outline of the gray matter of SDH. GFAP (magenta, bottom left), SOX9 (magenta, bottom right). Arrowheads indicate HA-tag+ astrocytes. Scale bars, 200 μm (top) and 25 μm (bottom). (K and L) Representative traces of sIPSCs (K) and quantitative analysis of their frequency (L) in SG neurons in spinal cord slices from Hes5-CreERT2;AAV-hM3Dq mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] (n = 7 cells from 7 mice; Wilcoxon signed-rank test; *P < 0.05). (M and N) Representative traces of sIPSCs (M) and quantitative analysis of their frequency (N) in SG neurons in spinal cord slices from Hes5-CreERT2;AAV-flex[hM3Dq] mice treated with TAM [Pre and CNO: before and after bath application of CNO with CPT (1 μM), respectively] (n = 13 cells from 13 mice; Wilcoxon signed-rank test; n.s., not significant). Data represent mean ± SEM.

Hes5+ astrocyte-mediated inhibitory signals to SDH-Vgat+ INs contribute to stress-induced mechanical hypersensitivity

(A and B) PWT before and 30 min after intrathecal administration of NA (0.1 nmol) in wild-type mice pretreated intrathecally with vehicle or CPT (3 nmol) (A: n = 5 mice per group) or in control (Vgat-Cre mice with intra-SDH of AAV-flex[mCherry]) and SDH-Vgat+ IN–A1R cKD mice (B: n = 9 mice per group) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; ***P < 0.001). (C and D) PWT before and after acute restraint stress in wild-type mice pretreated intrathecally with vehicle or CPT (C: Vehicle, n = 6 mice; CPT, n = 5 mice) or in control (Vgat-Cre mice with intra-SDH of AAV-flex[mCherry]) and SDH-Vgat+ IN–A1R cKD mice (D: Control, n = 6 mice; SDH-Vgat+ IN–A1R cKD, n = 7 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P <0.0001 vs. vehicle or control group). (E) Representative images of pERK (green) and IB4 (magenta) immunofluorescence in the SDH with or without Aβ fiber stimulation and/or restraint stress. CPT was intrathecally administered 30 min before stress exposure. (F) Quantitative analysis of the number of pERK+ cells in superficial laminae of the SDH in each group (n = 4–5 mice per group; one-way ANOVA with post hoc Tukey’s multiple comparisons test; *P < 0.05, ***P < 0.001). (G) Schematic illustration of the mechanisms of stress-induced pain facilitation highlighting NA signals from LC→SDH-NAergic terminals to Hes5+ astrocytes and Vgat+ INs. Data represent mean ± SEM. This figure was created using BioRender.com.

Mechanical hypersensitivity is weak in mice with longer exposure (2 hours) to restraint stress

PWT change at 30 min after various exposure periods of restraint stress (15 min, 30 min, 1 hour, and 2 hours) in wild-type mice (n = 5 mice; two-tailed paired t-test; ***P < 0.001; n.s., not significant vs. pre group). Data represent mean ± SEM.

Raw fluorescent signals in LC-NA neurons during restraint stress (in vivo fiber photometry)

Representative traces of GCaMP6s signals in LC-NA neurons during restraint stress. Traces shown at the top (blue), middle (purple), and bottom (green) indicate 465-nm, 415-nm, and corrected fluorescent signals, respectively.