Thermostatically controlled heating plate

During electrophysiological and muscle contractility measurements, mice were anesthetized with inhaled isoflurane using the Kent Scientific SomnoSuite System (induction: 2.5%–3.5%, maintenance: 1%–2%) (Torrington, CT, USA). During electrophysiological and SFEMG recordings, animal body temperature was maintained at 37 °C using a thermostatically controlled heating plate (World Precision Instruments, Sarasota, FL, USA). During the muscle contractility measurements, a warm water bath HTP-1500 heat therapy pump (Adroit Medical Systems, Loudon, TN, USA) was set to maintain temperature of the testing stage at 37 °C. During all procedures performed under anesthesia, lubricating ointment was applied to the eyes to prevent corneal dryness (Dechra Veterinary Products, Overland Park, KS, USA). All measurements were performed on the right hindlimb which was shaved before the studies. All procedures that required anesthesia lasted for no more than 20 minutes.

During in vivo electrophysiological and muscle contractility measurements, mice were anesthetized with inhaled isoflurane (3–5% for induction, and 1.5–2.5%) for maintenance using a digital inhaled anesthesia system (Kent Scientific, Torrington, CT, USA). Animal temperature was maintained at 37 °C, with a warm water bath-heated stage (HTP-1500, Androit Medical System, Loudan, TN, USA) for muscle physiology tests and with a thermostatically controlled heating plate (World Precision Instruments, Sarasota, FL, USA) for electrophysiology. The duration of experiments was kept under 20 minutes. During anesthesia, a petroleum-based eye ointment (Paralube Vet Ointment, Dechra Veterinary Products, UK) was applied to prevent corneal dryness. All procedures were performed on the right hind limb which was shaved using clippers (Remington VPG6530, Middleton, WI). Muscle physiology experiments were performed the day following the electrophysiology studies to minimize duration of anesthesia on a single day. At the final study time point, single fiber electromyography (SFEMG) was performed to assess NMJ transmission. Following SFEMG, mice were euthanized. The triceps brachii, gastrocnemius, and soleus muscles were collected and weighed.