Pulled glass micropipette

For CT and EO muscle injections, adult (age 6–8 weeks) Nts^Cre mice were used that had been previously injected as described above with AAVDJ-hSyn-FLEX-mGFP-2A-Synaptophysin-mRuby into RAm 8 weeks before the muscle injections. Mice were anesthetized with isoflurane (3% for induction and 1–2% for maintenance) and then pre-treated with analgesic (carprofen 5 mg kg⁻¹ and buprenorphine SR 0.5–1.0 mg kg⁻¹, subcutaneous). For CT injections, a 1-cm incision was made in the ventral neck, and the CT muscles were exposed by dissection of the overlying strap muscles. A pulled glass micropipette (Drummond Scientific, 5-000-2005) was then used to inject 200–300 nl of 1% CTB solution (Sigma-Aldrich, C9903, diluted in PBS + 0.05% Fast Green dye) into the left and right CT muscles. The overlying skin was sutured, and the mouse was placed in a heated recovery cage. For EO injections, 400–1,000 nl of 1% CTB was similarly injected into the left and right EO muscles through an incision in the overlying skin that was sutured after injection. Mice recovered for 3 d (CT) or 7 d (EO) before perfusion and immunostaining.

Injections were performed using our established procedures [24 (link)]. Briefly, Dbh^Cre+/- mice at 6 weeks of age were anesthetized with isoflurane (4% induction, 1–2% maintenance) and stabilized in a custom stereotaxic apparatus with body temperature being maintained by a heating pad. A small craniotomy was made over each target of interest and a pulled glass micropipette (Drummond, tip diameter: 15–20 μm), beveled sharp and loaded with the injectant, was lowered into the brain. Pipettes were front-filled with AAV (serotype 2/1) expressing either CAG-FLEX-GFP (UPenn, lot# V0827) or pCAG-FLEX-tdTomato-WPRE (Addgene cat# 51503). 3 min after reaching the target, 30–50 nL of virus was dispensed using a hydraulic injector (Narishige) followed by a 5-min waiting period. The pipette was retracted 0.3 mm, paused for 10 min, and then fully retracted. Up to two injections were made per mouse targeting the locus coeruleus bilaterally (in mm relative to Bregma; 4.80 posterior, +/- 0.75 lateral, 4.00 ventral).