Descending locus coeruleus noradrenergic signaling to spinal astrocyte subset is required for stress-induced mechanical pain hypersensitivity
- Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Japan
- Department of Neurophysiology, Hyogo Medical University, Japan
- Laboratory for Circuit and Behavioral Physiology, RIKEN Center for Brain Science, Japan
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Japan
- Viral Vector Core, Gunma University Initiative for Advanced Research (GIAR), Japan
- Super-network Brain Physiology, Graduate School of Life Sciences, Tohoku University, Japan
- Division of Brain Sciences, Institute for Advanced Medical Research, Keio University School of Medicine, Japan
- Kyushu University Institute for Advanced Study, Japan
Figures
Figure 1 with 7 supplements
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 hr) 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 Slc6a2-Cre mice. TH immunofluorescence is shown in magenta. Dashed line indicates the location of the implanted optic fiber. Scale bar, 100 μm. (D, 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 Slc6a2-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 hr apart) in control mice (top) and DTR-expressing mice (bottom). Scale bars, 100 μm. (H) Effect 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 Slc6a2-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 Slc6a2-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. See also Figure 1—figure supplements 1–6. Some figure elements were created with BioRender.com.
-
Figure 1—source data 1
Raw numerical values for Figure 1 plots.
- https://cdn.elifesciences.org/articles/104453/elife-104453-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Mechanical hypersensitivity is weak in mice with longer exposure (2 hr) to restraint stress.
PWT change at 30 min after various exposure periods of restraint stress (15 min, 30 min, 1 hr, and 2 hr) 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.
-
Figure 1—figure supplement 1—source data 1
Raw numerical values for Figure 1—figure supplement 1 plots.
- https://cdn.elifesciences.org/articles/104453/elife-104453-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Behavioral responses to thermal and mechanical stimuli in stress models.
(A) Schematic illustration of an experiment to investigate the effects of acute exposure to restraint stress (1 and 2 hr) on thermosensory behavior in mice, using the hot-plate test. Change in paw withdrawal latency (PWL) measured by noxious heat stimulation in wild-type mice after restraint stress for 1 hr (B) or 2 hr (C) (B: n = 10 mice per group; C: n = 5 mice per group) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; *p < 0.05, **p < 0.01, ****p < 0.0001 vs. no-stress group). (D) Schematic illustration of an experiment to investigate the effects of acute exposure to forced swim stress (3 min, 32°C) on mechanosensory behavior in mice, assessed with the von Frey test. (E) PWT measured by mechanical stimulation in wild-type mice after forced swim stress (n = 5 mice per group; two-tailed paired t-test and two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). Data represent mean ± SEM. Some figure elements were created with BioRender.com.
-
Figure 1—figure supplement 2—source data 1
Raw numerical values for Figure 1—figure supplement 2 plots.
- https://cdn.elifesciences.org/articles/104453/elife-104453-fig1-figsupp2-data1-v1.xlsx
Figure 1—figure supplement 3
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.
Figure 1—figure supplement 4
Specific expression and ablation of LC→SDH-NA neurons.
(A) Representative of LC→SDH -NA neurons in DTR-expressing mice treated with vehicle (control) and DTX (ablated). Scale bar, 100 μm. (B) Number of TH+ GFP+ cells in multiple sections of the anterior and posterior regions of the LC (n = 3–4 mice; Mann Whitney test; *P<0.05). (C) Representative of LC→SDH -NA neurons in DTR-expressing mice (a retrograde AAV vector incorporating Cre injected into the SDH and an AAV vector incorporating DTR (fused with EGFP) injected into the LC). DTR expression was not observed in the A5 or A7 regions, indicating that DTR expression was specific to the A6 (LC) region (this image is the same as Control in panel A). Scale bar, 100 μm. Data show the mean ± SEM.
-
Figure 1—figure supplement 4—source data 1
Raw numerical values for Figure 1—figure supplement 4 plots.
- https://cdn.elifesciences.org/articles/104453/elife-104453-fig1-figsupp4-data1-v1.xlsx
Figure 1—figure supplement 5
Ablation of LC→SDH -NA neurons does not affect motor function or thermal sensation.
Latency to fall in the rotarod test (left) and paw withdrawal latency (PWL) in the paw-flick test (right) using mice with DTR expression in LC→SDH-NA neurons before and after injection of DTX or PBS (n = 5 mice per group; two-tailed paired t-test and two-way repeated measures ANOVA with Bonferroni’s multiple comparisons test). Data show the mean ± SEM.
-
Figure 1—figure supplement 5—source data 1
Raw numerical values for Figure 1—figure supplement 5 plots.
- https://cdn.elifesciences.org/articles/104453/elife-104453-fig1-figsupp5-data1-v1.xlsx
Figure 1—figure supplement 6
Expression of ChrimsonR in the superficial and deeper SDH.
Representative images of NET (green) and tdTomato (magenta) expression in the SDH at 3 weeks after intra-LC injection of AAV-FLEx[ChrimsonR-tdTomato] in Slc6a2-Cre mice. Scale bar, 200 µm (left) and 50 µm (right).
Figure 1—video 1
In vivo Ca²+ imaging of LC neurons in mice using fiber photometry during acute restraint stress.
Figure 2
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 Slc6a2-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, 5ms 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, 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. Some figure elements were created with BioRender.com.
-
Figure 2—source data 1
Raw numerical values for Figure 2 plots.
- https://cdn.elifesciences.org/articles/104453/elife-104453-fig2-data1-v1.xlsx
Figure 3 with 2 supplements
Activation of Hes5+ astrocytes reduces activity in Slc32a1+ INs in the SDH.
Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control (Adra1aflox/flox) and Slc32a1+ IN-selective α1AR conditional knockout mice [Slc32a1-Cre;Adra1aflox/flox mice (Slc32a1+ 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 Slc32a1+ 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+ (Slc32a1+) SDH neurons after application of NA (20 μM) to spinal cord slices from Slc32a1-Cre;Rosa26-LSL-tdTomato mice. A1R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Slc32a1+ 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+ INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A1R-expressing Slc32a1+ 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 Slc32a1-Cre mice. Arrowheads indicate genome-editing Slc32a1+ cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Slc32a1+ INs after application of NA and CPA to spinal cord slices from Slc32a1-Cre mice with conditional knockdown of A1Rs in Slc32a1+ INs (SDH-Slc32a1+ IN–A1R cKD) and their controls (Control; Slc32a1-Cre mice with intra-SDH injection of AAV-FLEx[mCherry]). (I) Percentage of mCherry+ (Slc32a1+) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Slc32a1+ 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). Representative traces of spontaneous inhibitory postsynaptic currents (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). 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. See also Figure 3—figure supplements 1 and 2.
-
Figure 3—source data 1
Raw numerical values for Figure 3 plots.
- https://cdn.elifesciences.org/articles/104453/elife-104453-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Effects of CNO on Ca22+ response in astrocytes and IPSCs in SG neurons in spinal cord slices from mice with or without hM3Dq expression.
(A) Representative images of the astrocytic Ca2+ response by NA (10 µM) or CNO (100 µM). Fluorescence intensity of GCaMP6m in wild-type; AAV-mCherry mice with GCaMP6m expression in SDH astrocytes. (B) Effect of CNO (1, 10, and 100 µM) on the astrocytic Ca2+ response in Hes5-CreERT2;AAV-FLEx[hM3Dq] mice with GCaMP6m expression in SDH astrocytes. (C) Frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in SG neurons in the SDH from Hes5-CreERT2;AAV-mCherry mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] (n = 8 cells from 8 mice; Wilcoxon signed-rank test; p = 0.5781). Data show the mean ± SEM.
-
Figure 3—figure supplement 1—source data 1
Raw numerical values for Figure 3—figure supplement 1 plots.
- https://cdn.elifesciences.org/articles/104453/elife-104453-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
Knockdown of A1Rs in Slc32a1+ neurons tends to increase the proportion of Slc32a1+ neurons with NA-induced depolarizing responses.
Slc32a1+ neurons were recorded in spinal cord slices of Slc32a1-Cre mice with AAV-FLEx[SaCas9] and AAV-FLEx[mCherry]-U6-sgAdora1 or -FLEx[mCherry]-U6-sgControl, and were classified as depolarizing based on the peak change in membrane potential following bath application of NA (sgControl: n = 38 from 13 mice; sgAdora1: n = 17 from 7 mice; Fisher’s exact test; p = 0.2127).
-
Figure 3—figure supplement 2—source data 1
Raw numerical values for Figure 3—figure supplement 2 plots.
- https://cdn.elifesciences.org/articles/104453/elife-104453-fig3-figsupp2-data1-v1.xlsx
Figure 4
Hes5+ astrocyte-mediated inhibitory signals to SDH-Slc32a1+ INs contribute to stress-induced mechanical hypersensitivity.
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 (Slc32a1-Cre mice with intra-SDH of AAV-FLEx[mCherry]) and SDH-Slc32a1+ IN–A1R cKD mice (B: n = 9 mice per group) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; ***p < 0.001). 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 (Slc32a1-Cre mice with intra-SDH of AAV-FLEx[mCherry]) and SDH-Slc32a1+ IN–A1R cKD mice (D: Control, n = 6 mice; SDH-Slc32a1+ 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–H) Representative images of c-FOS (green, E), pERK (green, G), and IB4 (magenta, E, G) immunofluorescence in the SDH with or without Aβ fiber stimulation and/or restraint stress. CPT was intrathecally administered 30 min before stress exposure. Quantitative analysis of the number of c-FOS+ (F) and pERK+ (H) 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, ****p < 0.0001). (I) Schematic illustration of the mechanisms of stress-induced mechanical pain facilitation highlighting NA signals from LC→SDH-NAergic terminals to Hes5+ astrocytes and Slc32a1+ INs. Data represent mean ± SEM. Some figure elements were created with BioRender.com.
-
Figure 4—source data 1
Raw numerical values for Figure 4 plots.
- https://cdn.elifesciences.org/articles/104453/elife-104453-fig4-data1-v1.xlsx
Author response image 1
Expression of HA-tag and mCherry in inhibitory neurons (a different sample from Figure 3G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2+ inhibitory neurons (cyan) at 3 weeks after intra-SDH injection of AAV-FLEx[SaCas9-HA] and AAV-FLEx[mCherry]-U6-sgAdora1 in Vgat-Cre mice.
Arrowheads indicate genome-editing Vgat+ cells. Scale bar, 25 µm.
Author response image 2
Ex vivo imaging of GRAB-ATP and GRAB-Ado sensors.
(a) Representative images of GRABATP1.0 (left, green) or GRABAdo1.0 (right, green) expression in the SDH at 3 weeks after SDH injection of AAV-hSyn-GRABAdo1.0 or AAV-hSyn-GRABAdo1.0 in Hes5-CreERT2 mice. Scale bar, 200 µm. (b) Left: Representative fluorescence images showing GRABATP1.0 responses before and after perfusion with NA or ATP. Right: Representative traces showing responses to ATP (0.1 and 1 µM) or NA (10 µM). (c) Left: Representative fluorescence images showing GRABAdo1.0 responses before and after perfusion with NA or adenosine (Ado). Right: Representative traces showing responses to Ado (0.01, 0.1, and 1 µM), NA (10 µM), or no application (negative control).
Author response image 3
The observed "dip" in astrocyte Ca2+ signals was not affected by pretreatment with the α1AR-specific antagonist silodosin.
Representative traces of astrocytic GCaMP6m signals in response to optogenetic stimulation of LC-NAe→SDHrgic axons/terminals in a spinal cord slice. Each trace shows the GCaMP6m signal before and after optogenetic stimulation (625 nm, 1 mW, 10 Hz, 5 ms pulse duration, 10 s). Slices were pretreated with silodosin (40 nM) for 5 min prior to stimulation.
Additional files
-
Supplementary file 1
Summary of anti- and pro-nociceptive effects of acute restraint stress reported in previously published studies.
Previously reported changes in pain-related behaviors induced by acute restraint stress were summarized along with information on animal species, strain, sex, duration of restraint, and pain tests.
- https://cdn.elifesciences.org/articles/104453/elife-104453-supp1-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/104453/elife-104453-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Descending locus coeruleus noradrenergic signaling to spinal astrocyte subset is required for stress-induced mechanical pain hypersensitivity
eLife 14:RP104453.
https://doi.org/10.7554/eLife.104453.3
