Categorization and counts of NMJs following in-vivo two-color BTX experimentation

Mice were anesthetized via isoflurane inhalation and placed in a supine position. Neck hair was removed using a depilatory and the surgical site sterilized with povidone-iodine. An incision was made from chin to sternum and the skin retracted. The salivary glands were moved to the side to expose the STM. Surrounding connective tissue was gently removed and a non-saturating concentration (diluted 2 ug/mL in sterile lactated Ringers) of α-bungarotoxin conjugated to Alexa FluorTM 555 (ThermoFisher Scientific, Cat# 35451) applied to the surface of the muscles for 5 minutes (BTX-1). The neck cavity was then washed copiously with lactated Ringer’s ten times. For muscle injury experiments, superficial muscle fibers at areas close to the site of entry of the accessory nerve into the muscle were cut with a sterile #15 surgical scalpel. The contralateral muscle was also labeled with BTX-1, but not injured and used as an internal control (IC). For denervation experiments both STM muscles were labeled with BTX-1 and the accessory nerve of one muscle was severed near its insertion site into the muscle. The other muscle was spared and used as an IC. The salivary glands were moved back into place following labeling, washing, and injury. The skin was sutured with 8-0 silk and treated with 2% lidocaine hydrochloride jelly. Animals were allowed to convalesce for ten days post-surgery. Endpoints for the studies were P38, P66, P160, and P450. were counted and categorized using a Leica DMRX epifluorescence microscope and a 40X Oil objective (NA 1.4). Junctions were considered fragmented if they had 5 or more non-connected clusters of AChRs in close proximity to each other on the same myofiber. Junctions were categorized based of the robustness of a primary fluorescent bungarotoxin stain and a second label, as well as if they were continuous (< 5 AChR clusters), or fragmented (> 5 AChR clusters). They were classified as stable if BTX-1 and BTX-2 labels were equally robust, or as dynamic if BTX-1 label was mostly absent and BTX-2 label intense. In many cases dynamic junctions did not completely lose BTX-1, and small puncta of fluorescence was seen. It is likely that these puncta are BTX-1 labelled receptors tethered to the basal lamina and demarcate the initial synaptic site before myofiber degeneration. If both the BTX-1 and BTX-2 labels were faint, junctions were classified as lost, but this was rarely observed.