GoKawiilMice
All procedures were in accordance with the US National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and were approved by Stanford University’s Administrative Panel on Laboratory Animal Care. Mice (1.5 days–10 weeks) of both sexes were used in experiments. Genetically engineered mouse lines used in this study included Oprm1cre/+ and vGlut2-IRES-cre (JAX 016963). The Oprm1cre/+ knock-in mouse line was generated in the Stanford University Transgenic, Knockout and Tumor Model Center (TKTC) using conventional embryonic-stem-cell-targeting strategies. The Cre recombinase cDNA, followed by the rabbit β-globin poly-A signal, was introduced by homologous recombination immediately after the start codon in exon 1 of the mouse Oprm1 gene51 (Extended Data Fig. 1a). Heterozygous mice were generated by mating chimeric mice with C57BL/6 mice.
Viruses
The following viruses were produced and packaged in the laboratory and used in this study: AAV8retro-hSyn-H2B-Clover3-FLEX(LoxP)-H2B-mRuby3 (2.0 × 1013 genome copies (gc) per ml), AAV8retro-CAG-mCherry (5.0 × 1012 gc ml−1), AAV2retro-CAG-eGFP (5.0 × 1012 gc ml−1), AAV8-hSyn-FLEX(FRT)-mCherry (1.0 × 1013 gc ml−1), AAV8-hSyn-FLEX(FRT)-Clover3 (1.0 × 1013 gc ml−1), AAV8-hSyn-FLEX(FRT)-hM4D-IRES-EGFP (2.0 × 1013 gc ml−1), AAV8-hSyn-FLEX(FRT)-hM3D-EYFP (3.0 × 1013 gc ml−1), AAV8-hSyn-FLEX(FRT)-taCaspase3-TEVp (1.0 × 1013 gc ml−1), AAV8-hSyn-FLEX(LoxP)-Ruby3-FLEX(FRT)-Clover3 (1.0 × 1013 gc ml−1), AAV8retro-hSyn-FLEX-mTagBFP2-P2A-FlpO (5.0 × 1013 gc ml−1), AAV8retro-hSyn-mTagBFP2-P2A-Cre (5.0 × 1013 gc ml−1), AAV8-hSyn-FLEX(LoxP)-hM4D-IRES-GFP (5.0 × 1013 gc ml−1), AAV8-hSyn-FLEX(LoxP)-hM3D-IRES-mCherry (1.0 × 1013 gc ml−1), AAV8-hSyn-FLEX(LoxP)-hM3D-IRES- EGFP (1.0 × 1013 gc ml−1), AAV8-hSyn-FLEX(FRT)-jGCaMP7s (5.0 × 1013 gc ml−1), AAV1-hSyn-Cre (1.0 × 1013 gc ml−1), AAV8-hSyn-hM4D-mCherry (5.0 × 1012 gc ml−1), AAV8-hSyn-hM3D-mCherry (5.0 × 1012 gc ml−1), AAV8-EF1α-FLEX(LoxP)-mScarlet (1.0 × 1013 gc ml−1), AAV8-Ef1a-DIO-hChR2(H134R)-EYFP (5.0 × 1012 gc ml−1), AAV8-hSyn-FLEX(FRT)-EGFP-P2A-TVA-T2A-oG (5.0 × 1013 gc ml−1). SADΔG-mCherry(EnvA) (2.0 × 108 infectious unit (IU) ml−1) was purchased from Salk viral core, CVS-N2cΔG-mCherry(EnvA) (5.0 × 108 IU ml−1) was purchased from Jefferson Center for Vaccines. AAV5-Ef1a-DIO-hChR2(H134R)-mCherry (1.2 × 1013 gc ml−1, Addgene 20297) and AAV2-hSyn-DIO-hM4D(Gi)-mCherry (1.2 × 1013 gc ml−1, Addgene 44362) were purchased from Addgene.
Surgery
Stereotaxic injection and implantation of optical fibres and cannulas
Stereotaxic surgeries were performed on five- to seven-week old mice under ketamine and xylazine (100 mg kg−1 and 5 mg kg−1, intraperitoneally; i.p.) anaesthesia using a stereotaxic instrument (BenchMARK Digital, Leica). Virus was injected into the RVM (200 nl AAV, Bregma −5.60 mm, lateral ±0.1 mm, ventral 5.75 mm), lSuC (200 nl AAV, Bregma −3.45 mm, lateral ±1.65 mm, ventral 2.40 mm), PAG (250 nl AAV, Bregma −4.65 mm, lateral ±0.5 mm, ventral 3 mm), SSp and SSs (200 nl AAV at site 1: Bregma −1.45 mm, lateral ±3.75 mm, ventral −1.45 mm; 200 nl AAV at site 2: Bregma −1.65 mm, lateral ±3.85 mm, ventral −1.55 mm), MOa (200 nl AAV, Bregma +1.8 mm, lateral ±1.5 mm, ventral −1.35 mm), VPL (200 nl, Bregma −1.8 mm, lateral ±2.0 mm, ventral 3.5 mm), Po (200 nl, Bregma −1.8 mm, lateral ±1.6 mm, ventral 2.85 mm), DCN (70 nl per injection, at site 1: obex 0.0 mm, lateral ±0.6 mm, ventral −0.25 mm; site 2: obex 0.25 mm, lateral ±0.8 mm, ventral −0.3 mm; site 3: obex 0.5 mm, lateral ±1.1 mm, ventral −0.35 mm) with a pulled glass capillary at a slow rate (100 nl min−1) using a pressure microinjector (Micro4 system, World Precision Instruments). The injection capillary was removed 5 min after the end of the injection. For mice used for terminal chemogenetic manipulation or fibre photometry, an infusion cannula (PlasticsOne) or optical fibre (Inper) was placed at least 200 μm above the target brain region and cemented to the skull using dental cement (Lang Dental Manufacturing). After surgery, a dummy cannula was inserted and a cap was screwed on to keep the guide cannula from becoming occluded. Mice were allowed at least two weeks to recover and to express the virus before behavioural training commenced.
SNI
SNI surgery were performed as previously described21 on five- to seven-week old mice under ketamine and xylazine (100 mg kg−1 and 5 mg kg−1, i.p.) anaesthesia. In brief, after skin incision and blunt dissection to expose the sciatic nerve, the tibial and common peroneal branches of the sciatic nerve were ligated with a 5.0 silk suture and transected distally. The sural nerve was left intact. For sham surgery, only skin incision and blunt dissection were performed. After injury, skin was sutured, and mice were left to recover on a heated pad before being returned to their home cages. Mechanical and thermal thresholds were measured two days after the surgery.
CFA injection
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