Nanojector 2

Mice (8–12-week-old) were anaesthetized (with 2%-5% isoflurane for induction and 0.8%–2% for maintenance), and mounted on a stereotaxic frame (David Kopf Instruments) with heating pad placed underneath. We bilaterally injected a volume of 200 nL containing retrograde tracer viruses into dPAG, lPAG, or MPOA of using a pulled glass capillary (World Precision Instruments) by pressure injection at a flow rate of 100 nL/min (Micoro4 controller, World Precision Instruments; Nanojector II, Drummond Scientific). The combination of retrogradely transported virus and mice used were as follows: 1) rAAV2-retro-EF1a-Cre (Tervo et al., 2016 (link)) in a Cre-reporter mouse line (Ai14^Tg/+ or Ai110^Tg/+) and 2) HSV1-LS1L-EYFP (rHSV; MGH Vector Core) in Vglut2^Cre/+ or Esr1^Cre/+ (Figure S1E). Stereotactic injection coordinates of dPAG, lPAG, and MPOA were obtained from the Paxinos and Franklin atlas (Franklin and Paxinos, 2008 ) (AP: −4.72, ML: ± 0.12, DV: −2.0 mm for dPAG; AP: −4.48, ML: ± 0.45, DV: −2.65 mm for lPAG; AP: 0.022, ML: ± 0.35, DV: −5.37 mm for MPOA). After a 2–3 week viral incubation, retrogradely labeled single cells were manually dissected from VMHvl, isolated by FACS, and subjected to scRNA-seq using SMART-seq as described above.

Mice were deeply anesthetized, and CTβ, AAVs or gΔRabies were stereotaxically delivered. All coordinates used follows the Paxinos and Franklin mouse atlas (Franklin and Paxinos ). A 10μl microcapillary pipette was pulled and loaded with the solution; Injections were performed using a microinjector (Nanojector II, Drummond Scientific Company). A heating pad was used to maintain the body temperature. Before and after surgery, systemic analgesics (buprenorphine, 0.1 mg/kg) were administrated. For anatomical analysis mice were perfused at different times post injection (3–4 days after CTβ injection, 4 weeks after AAVs injection, or 5 days after gΔRabies injection) and the brains were subsequently sectioned on a cryostat. In specific experiments, retinas were also dissected out after perfusion and post-fixed for 1 hour.