Different neuronal populations mediate inflammatory pain analgesia by exogenous and endogenous opioids
- Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
- Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, China
Figures
Figure 1 with 1 supplement
Distribution of Oprm1 in Vglut2+ and Vgat+ neurons in the mouse brain.
(a–f) Multiple in situ hybridization in wild-type mice shows the distribution of Oprm1 in glutamatergic and GABAergic neurons using RNAscope assay. Scale bars, 1 mm (a and d), 100 μm (b), (c), (e), …
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Figure 1—source data 1
Percentage of glutamatergic and GABAergic neurons in MOR expressing neurons in representative brain regions.
- https://cdn.elifesciences.org/articles/55289/elife-55289-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Multiple in situ hybridization in wild-type mice.
(a) Multiple in situ hybridization in wild-type mice showing the distribution of Oprm1, Vglut2 and Vgat by using RNAscope assay. Scale bar, 1 mm. (b) Enlarged images of the frames in (a). Scale bar, …
Figure 2 with 1 supplement
Generation and verification of the Oprm1KI/KI mouse line.
(a) Targeting strategy for generating the Oprm1KI/KI mouse line through inserting a stop cassette flanked by two loxP sites between Oprm1 exon 1 and exon 2. PPS, RNA polymerase pause site. (b and c) …
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Figure 2—source data 1
Raw data of the behavioral tests and MOR expression level in wild-type and Nestin-Cre/Oprm1-KI mice.
- https://cdn.elifesciences.org/articles/55289/elife-55289-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
MOR expression in Nestin-Cre/Oprm1KI/KI mice.
(a) MOR expression in representative brain regions in Nestin-Cre/Oprm1KI/KI mice compared to wild-type mice. Scale bar, 200 μm. (b) Quantification of MOR expression level in Nestin-Cre/Oprm1KI/KI …
Figure 3 with 2 supplements
MORs in Vglut2+ glutamatergic neurons mediate exogenous opioid analgesia.
(a) Schematic diagram for re-expression of MORs in glutamatergic neurons. Vglut2-Cre mice were crossed with Oprm1KI/KI mice to generate Vglut2-Cre/Oprm1KI/KI mice. Red hexagon: Stop cassette. (b) …
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Figure 3—source data 1
Raw data of the behavioral tests and MOR expression level in Vglut2-Cre/Oprm1-KI and Vglut2-Cre/Oprm1-fl groups of mice.
- https://cdn.elifesciences.org/articles/55289/elife-55289-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Expression pattern of the re-expressed MOR and functional role of MORs in Vglut2+ glutamatergic neurons in morphine analgesia.
(a and b) Immunofluorescent staining showing the expression of MOR and Vglut2 expression in the habenula and parabrachial nucleus of wild-type and Vglut2-Cre/Oprm1KI/KI mice. Scale bars, 100 μm. …
Figure 3—figure supplement 2
Functional role of MORs in glutamatergic neurons in opioid analgesia.
(a) Schematic diagram for conditional knockout MORs from glutamatergic neurons. Vglut2-Cre mice were crossed with Oprm1fl/fl mice to generate Vglut2-Cre/Oprm1fl/fl mice. (b) Immunostaining of MOR in …
Figure 4 with 2 supplements
MORs in GABAergic neurons mediate endogenous opioid analgesia.
(a) Schematic diagram for re-expression of MORs in GABAergic neurons. Vgat-Cre mice were crossed with Oprm1KI/KI mice to generate Vgat-Cre/Oprm1KI/KI mice. Red hexagon: Stop cassette. (b) Graphs …
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Figure 4—source data 1
Raw data of the behavioral tests and MOR expression level in Vgat-Cre/Oprm1-KI and Vgat-Cre/Oprm1-fl groups of mice.
- https://cdn.elifesciences.org/articles/55289/elife-55289-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Expression pattern of the re-expressed MOR and functional role of MORs in GABAergic neurons in morphine analgesia.
(a and b) Immunofluorescent staining showing the expression of MOR and GAD67 expression in the interpeduncular nucleus (IPN) and ventral pallidum (VP) of wild-type and Vgat-Cre/Oprm1KI/KI mice. …
Figure 4—figure supplement 2
Functional role of MORs in GABAergic neurons in opioid analgesia.
(a) Schematic diagram for conditional knockout MORs from GABAergic neurons. Vgat-Cre mice were crossed with Oprm1fl/fl mice to generate Vgat-Cre/Oprm1fl/fl mice. (b) Immunostaining of MOR in Oprm1fl/…
Figure 5 with 1 supplement
MORs in dorsal spinal cord mediate morphine analgesia.
(a) Schematic diagram for re-expression of MORs in dorsal spinal neurons. Lbx1-Cre mice were crossed with Oprm1KI/KI mice to generate Lbx1-Cre/Oprm1KI/KI mice. Red hexagon: Stop cassette. (b and c) …
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Figure 5—source data 1
Raw data of the behavioral tests in Lbx1-Cre/Oprm1-KI and Lbx1-Cre/Oprm1-fl groups of mice.
- https://cdn.elifesciences.org/articles/55289/elife-55289-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Functional role of MORs in dorsal spinal neurons in opioid analgesia.
(a) Schematic diagram for re-expression of MORs in dorsal spinal neurons. Lbx1-Cre mice were crossed with Oprm1KI/KI mice to generate Lbx1-Cre/Oprm1KI/KI mice. (b–d) Basal pain threshold tests with …
Figure 6 with 1 supplement
Activation of MORs expressed in spinal GABAergic neurons induced hyperalgesia.
(a) Expression of Oprm1, Vglut2 and Vgat mRNA in the spinal cord of wild-type mice. Scale bar, 200 μm. (b) Quantification of Oprm1+ cells in the dorsal spinal cord. (c) Expression of Oprm1, Vglut2 …
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Figure 6—source data 1
Raw data of the behavioral tests in Vglut2-Cre/Oprm1-KI and Vgat-Cre/Oprm1-KI groups of mice with intrathecal morphine injection.
- https://cdn.elifesciences.org/articles/55289/elife-55289-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Functional role of MORs in primary neurons in opioid analgesia.
(a) Schematic diagram for re-expression of MORs in DRG neurons. SNS-Cre mice were crossed with Oprm1KI/KI mice to generate SNS-Cre/Oprm1KI/KI mice. (b) Co-immunostaining of MOR with IB4 and CGRP in …
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Figure 6—figure supplement 1—source data 1
Raw data of the behavioral tests in SNS-Cre/Oprm1-KI mice.
- https://cdn.elifesciences.org/articles/55289/elife-55289-fig6-figsupp1-data1-v2.xlsx
Figure 7 with 2 supplements
Functional role of MORs expressed in the PBN in opioid analgesia.
(a) Schematic diagram showing stereotaxic injection into PBN and representative expression of AAV-hSyn-DIO-GCaMP6s virus in MOR-iCreERT2 mice. Dashed line outlines the track of optical fiber. Scale …
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Figure 7—source data 1
Raw data of the fiberphotometry recording in Oprm1-GCaMP6s mice and behavioral tests in PBN-Cre/Oprm1-KI mice.
- https://cdn.elifesciences.org/articles/55289/elife-55289-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Generation and verification of MOR-iCreERT2 mice.
(a) Targeting strategy for generating MOR-iCreERT2 mice. (b) Schematic diagram for labeling MOR+ neurons. (c–e) Double in situ hybridization in MOR-iCreERT2×Ai9 mice shows the distribution of Oprm1 …
Figure 7—figure supplement 2
Functional role of MORs expressed in the PBN in morphine analgesia.
(a) Schematic diagram of bilateral PBN stereotaxic injection of AAV-hSyn-Cre-EGFP or AAV-hSyn-EGFP virus. (b–d) Effects of morphine (10 mg/kg, s.c.) on pain tests with tail immersion (50°C), hot …
Author response image 1
MOR and CGRP staining in DRG of several MOR-rescue mouse lines.
Scale bar, 100 μm.
Videos
Video 1
Intrathecal morphine-induced nocifensive behaviors in Vgat-Cre/Oprm1KI/KI mice.
Left, Oprm1KI/KI mice. Right, Vgat-Cre/Oprm1KI/KI mice. Morphine (1.0 nmol/ 5 μl) was intrathecally injected.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus. musculus) | Oprm1KI/KI | This paper | See Figure 2a | |
| Genetic reagent (Mus. musculus) | Oprm1fl/fl | This paper and Zhang et al., 2020 | See Figure 3—figure supplement 2a | |
| Genetic reagent (Mus. musculus) | MOR-iCreERT2 | This paper | See Figure 7—figure supplement 1a | |
| Genetic reagent (Mus. musculus) | B6.Cg-Tg(Nes-cre)1Kln/J (Nestin-Cre) | Jackson Laboratory | Stock#: 003771 RRID:MGI:2174506 | |
| Genetic reagent (Mus. musculus) | STOCK Slc17a6tm2(cre)Lowl/J (Vglut2-ires-Cre) | Jackson Laboratory | Stock#: 016963 RRID:MGI:5300532 | |
| Genetic reagent (Mus. musculus) | STOCK Slc32a1tm2(cre)Lowl/J (Vgat-ires-Cre) | Jackson Laboratory | Stock#: 016962 RRID:MGI:5300525 | |
| Genetic reagent (Mus. musculus) | B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Ai9) | Jackson Laboratory | Stock#: 007909 RRID:MGI:3813511 | |
| Genetic reagent (Mus. musculus) | Lbx1-Cre | Sieber et al., 2007 | Dr. Yang Liu (Hangzhou Normal University) | |
| Genetic reagent (Mus. musculus) | SNS-Cre | Agarwal et al., 2004 | Dr. Rohini Kuner (University of Heidelberg) | |
| Genetic reagent (Mus. musculus) | C57BL/6J | SLAC Laboratory | ||
| Ggenetic reagent (Dependoparvovirus) | AAV2/9-hSyn-DIO-GCaMP6s-EYFP | Shanghai Taitool Inc | 4.60 × 1012 v.g./ml | |
| Genetic reagent (Dependoparvovirus) | AAV2/9-Ef1α-DIO-EYFP | Shanghai Taitool Inc | 3.83 × 1012 v.g./ml | |
| Genetic reagent (Dependoparvovirus) | AAV2/8-hSyn-Cre-EGFP | Shanghai Taitool Inc | 5.99 × 1012 v.g./ml | |
| Genetic reagent (Dependoparvovirus) | AAV2/8-hSyn-EGFP | Shanghai Taitool Inc | 4.66 × 1012 v.g./ml | |
| Antibody | Anti-Mu Opioid Receptor antibody (Rabbit Monoclonal) | Abcam | Cat#: ab134054 | 1:500 for brain and spinal cord, 1:1000 for DRG |
| Antibody | Anti-CGRP (Goat Polyclonal) | Abcam | Cat#: ab36001 RRID:AB_725807 | 1:500 |
| Antibody | anti-c-Fos (Rabbit Polyclonal) | Synaptic System | Cat#: 226003 RRID:AB_2231974 | 1:15000 |
| Antibody | FITC-IB4 | Sigma | Cat#: L2895 | 1:200 |
| Antibody | Anti-GAD67 (Mouse Monoclonal) | Millipore | Cat#: MAB5406 RRID:AB_2278725 | 1:2000 |
| Antibody | Anti-VGluT2 (Guinea pig Polyclonal) | Millipore | Cat#: AB2251-I RRID:AB_2665454 | 1:500 |
| Antibody | Cy5 AffiniPure Donkey Anti-Goat IgG (H+L) | Jackson ImmunoResearch Laboratories | Cat#: 705-175-147 RRID:AB_2340415 | 1:200 |
| Antibody | Cy3 AffiniPure Donkey Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch Laboratories | Cat#: 711-165-152 RRID:AB_2307443 | 1:200 |
| Antibody | Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch Laboratories | Cat#: 711-545-152 RRID:AB_2313584 | 1:200 |
| Antibody | Biotin-SP AffiniPure Goat Anti-Rabbit Polyclonal IgG (H+L) | Jackson ImmunoResearch Laboratories | Cat#: 111-065-003 RRID:AB_2337959 | 1:200 |
| Antibody | Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (H+L) | Jackson ImmunoResearch Laboratories | Cat#: 715-545-150 RRID:AB_2340846 | 1:200 |
| Antibody | Alexa Fluor 647 AffiniPure Donkey Anti-Guinea Pig IgG (H+L) | Jackson ImmunoResearch Laboratories | Cat#: 706-605-148 RRID:AB_2340476 | 1:200 |
| Sequence-based reagent | RNAscope Probe- Oprm1-O3 | Advanced Cell Diagnostics | Cat#: 493251 | |
| Sequence-based reagent | RNAscope Probe-tdTomato-C3 | Advanced Cell Diagnostics | Cat#: 317041 | |
| Sequence-based reagent | RNAscope Probe- Slc32a1-C2 (Vgat) | Advanced Cell Diagnostics | Cat#: 319191 | |
| Sequence-based reagent | RNAscope Probe- Slc17a6-C3 (Vglut2) | Advanced Cell Diagnostics | Cat#: 319171 | |
| Sequence-based reagent | RNAscope Probe- Slc17a7-C3 (Vglut1) | Advanced Cell Diagnostics | Cat#: 416631 | |
| Sequence-based reagent | RNAscope Probe- 3-Plex Negative Control | Advanced Cell Diagnostics | Cat#: 320871 | |
| Commercial assay or kit | VECTASTAIN ABC-Peroxidase Kit | Vector | Cat#: PK-4000 RRID:AB_2336818 | 1:100 |
| Commercial assay or kit | RNAscope Multiplex Fluorescent Reagent Kit v2 | Advanced Cell Diagnostics | Cat#: 323100 | |
| Chemical compound, drug | CFA | Sigma | Cat#: F5881 | 50% (vol/vol) |
| Chemical compound, drug | Formaldehyde | Sigma | Cat#: F1635 | 5% (vol/vol) |
| Chemical compound, drug | Tamoxifen | Sigma | Cat#: T5648 | 150 mg/kg |
| Chemical compound, drug | β-FNA | Tocris | Cat#: 0926 | 10 mg/kg |
| Chemical compound, drug | Red beads | Lumaflour | Cat#: Retrobeads IX | 1:10 diluted in PBS |
| Software, algorithm | Image J | NIH | ||
| Software, algorithm | LabState | Anilab | ||
| Software, algorithm | Fscope | BiolinkOptics |
Additional files
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Source code 1
Code for scorevideo.
- https://cdn.elifesciences.org/articles/55289/elife-55289-code1-v2.m
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Source code 2
Code for fiber photometry.
- https://cdn.elifesciences.org/articles/55289/elife-55289-code2-v2.m
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55289/elife-55289-transrepform-v2.docx
