Ctb 555

Viral injections were performed using previously described procedures^{6 (link)} at the following stereotaxic coordinates: pPVT, –1.34 mm from Bregma, 0.05 mm lateral from midline, and 3.03 mm vertical from cortical surface; CeL, −1.22 mm from Bregma, 2.9 mm lateral from midline, and 4.6 mm vertical from cortical surface; BLA, –1.80 mm from Bregma, 3.4 mm lateral from midline, and 5.4 mm vertical from cortical surface. For pPVT injections we used a 6.5° angle to avoid damage of the superior sagittal sinus. Animals were kept on a heating pad throughout the entire surgical procedures and were brought back to their home cages after 24 h post-surgery recovery and monitoring. Postoperative care included intraperitoneal injection with 0.3–0.5 ml of lactated Ringers solution and metacam (meloxicam, 1–2 mg/kg) for analgesia and anti-inflammatory purposes. All AAVs and the CAV2-Cre were injected at a total volume of approximately 1 μl (except for the monosynaptic rabies viral tracing, see below), and were allowed at least two weeks for maximal expression. For retrograde tracing of amygdala-projecting pPVT cells, CTB-555 or CTB-488 (0.1-0.3 μl, 0.5% in PBS) (Invitrogen) was injected into CeL and BLA and allowed 3-5 days for sufficient retrograde transport.

Adult female Wistar rats were anesthetized with isoflurane until a surgical plane of anesthesia was obtained (induction 4–5%; maintenance 1–3%, both in 100% O₂). The TN was exposed at mid-thigh by a midline posterior incision (~1.5 cm) through the skin and underlying connective in the left hind limb. The TN was then crushed with fine #5 forceps for 10 s. After washing with 0.9% sterile saline, the wound was closed in layers and the animals removed from anesthesia. Buprenorphine (subcutaneous, 0.1 mg/kg) was delivered immediately and every 12 hr after surgery prophylactically for 48 hr to alleviate any possible pain and distress. Signs of pain or distress (lethargy, vocalizations, weight loss, absence of grooming) were closely monitored but not observed in any animals. One week prior to the animals being euthanized, the MG muscle of the left leg was exposed and injected with 10–20 μl of 0.1% Alexa 555 conjugated to recombinant cholera toxin subunit b (CTb-555, Invitrogen, ThermoFisher Scientific, Waltham, MA). The total volume was distributed throughout the muscle in four to five 2–5 μl injections. The control group did not undergo any nerve surgeries prior to the CTb-555 injections.