Key solutions and reagentsAnesthesis reagent:Ketamine (!), Xylazine (!)

Ringer SolutionNaCl, KCl, NaHCO3, NaH2PO4, MgCl2, CaCl2, D-Glucose.

Safety Instructions

1. All biological materials should be handled and discarded as potentially bio-hazardous.

2. Personal protective equipment such as lab coats, gloves (nitrile), and surgical masks are necessary in experiments for safety reasons.

3. If chemical spill, chemical spill kit is available in safety shelf in E-765, if necessary, please contact lab emergency: 368-3333, lab safety services: 368-2907.

Experimental Procedures

CMAP Recording:

1. The mice are anesthetized with ketamine and xylazine cocktail (100 and 10mg/kg body weight respectively, ddH2O as solvent). Then alternatively, the mice can be maintained on a 37 heating pad.

2. The stimulation needle electrode (TECA, 092-DMF25-S) is inserted near the sciatic nerve of the thigh. The reference needle electrode is inserted into the Achilles tendon and the recording needle electrode is inserted into the middle of the gastrocnemius.

3. The reference and recording needle electrodes (CV203 BU HEADSTAGE) are connected to Axopatch 200B Amplifier (Molecular Devices). The stimulating electrode is connected to isolator (AMPI, ISO-Flex) and then, Digidata 1322A (Molecular Devices) that is also connected to the Axopatch 200B Amplifier.

4. We apply to sciatic nerve a series of 10 stimuli at 1, 5, 10, 20, 40Hz. CMAP is recorded by Digidata 1322A and analyzed by Clampfit 9.2 (Molecular Devices).

1. Digidata 1322A 2. Axopatch 200B Amplifier 3. Isolator (AMPI, ISO-Flex) 4. Charger (10 Batteries) 5. Stimulating electrode 6. Reference and recording electrodes 7. CV203 BU HEADSTAGE (connected to the amplifier)

mEPP and EPP Recording:

1. Mouse diaphragm together with ribs and endings of phrenic nerve is dissected, fixed on Sylgard gel and perfused in oxygenated (95% O2, 5% CO2), 26-28 Ringer Solution (137mM NaCl, 5mM KCl, 12mM NaHCO3, 1mM NaH2PO4, 1mM MgCl2, 2mM CaCl2, 11mM D-Glucose, PH 7.3).

2. To record mEPP, microelectrode (CV203 BU HEADSTAGE, 20-40mΩ, filled with 3 M KCl) connected to the amplifier as indicated before is inserted to the center of muscle fiber. mEPP is recorded when the resting membrane potential is stable (-65~-80mV). Note: Usually 5 muscle fibers are recorded per mouse, one trace per fiber for a 3 min period.

3. To record EPP, phrenic nerve is held by sucking and stimulated by platinum electrode, which is connected to the isolator to the amplifier and Digidata 1322A as described before. 2.5mM μ-conotoxin is used to block action potential and muscle contraction. Trigger signals (1-ms duration) are programmed in Clampex 9.2 and elicited from a Digidata 1322A digital output channel to stimulus isolator. Data are collected with Axopatch 200B Amplifier, Digidata 1322A and analyzed by Clamfit 9.2.

1. Digidata 1322A 2. Axopatch 200B Amplifier 3. Isolator (AMPI, ISO-Flex) 4. Charger (10 Batteries) 5. Platinum stimulating electrode 6. CV203 BU HEADSTAGE (connected to the amplifier, with recording microelectrode) 7. Microscope 8. MP 225 (adjust the position of microelectrode)

1. Microscope 2. Platinum stimulating electrode 3. CV203 BU HEADSTAGE (connected to the amplifier, with recording microelectrode) 4. Sylgard gel plate to fix diaphragm sample 5 6 7. Perfusion system to recycle the oxygenated Ringer solution 8. Pump