CTB muscle injections

Mice aged P14 were anesthetized using isoflurane and prepared for injections into muscle. An approximate total of 500–750 nl of cholera toxin subunit B (CTB) Alexa Fluor 488 or 647 conjugate (Invitrogen; Nanoject II, Drummond Scientific) was injected into two to three different locations in the left forepaw (IH MN pool) or hindpaw (IF MN pool) in 50.6 nl increments. For injections into the tibialis anterior (TA), gastrocnemius (GS), quadricep (Q), or adductor (Ad), the area of the skin above the muscle was shaved and 70% ethanol and betadine (Avrio Health L.P.) applied. An incision was made above the muscle and 500–750 nl of the CTB-conjugated Alexa Fluor was injected into three to four different locations directly into the muscle. The incision was closed with surgical glue (Henry Schein Medical). Carprofen (5 mg/kg) was administered daily 3 d after surgery. Spinal cords were harvested 5 d after injection. For injections at P0, mice were anesthetized with isoflurane and injected with <250 nl CTB-488 or 647 in one or two different locations of the forepaw and hindpaw and harvested 3 d later.