4.1. Tissue Preparation

Details of the tissue preparation of synapses on skeletal muscles of frogs can be found elsewhere [45,47]. Briefly, paired cutaneous pectoris muscles of two adult Rana pipiens (5 cm nose rump length, male), obtained in summer (Hazen JM Frog Co., Alburg, VT, USA) and sacrificed by double-pithing, were pinned out in a Petri dish containing Ringer’s solution (115 mM NaCl, 2 mM KCl, 2.2 mM CaCl₂, 1 mM NaHPO₄⋅H₂O; 220–230 milliosmoles (mOsM), pH 7.2). The cut end of the nerve was drawn into a suction electrode. Stimulation parameters were established by passing single current pulses through the suction electrode, while using a dissecting microscope to monitor muscle contractions induced by synaptic transmission. The threshold for contraction of the entire muscle occurred at a current amplitude of ~1 μA for 1 msec. The Ringer’s solution was then replaced for more than 5 min by Ringer’s solution containing 10⁻⁵ g/mL (+)-Tubocurarine chloride hydrate (Sigma-Aldrich, Inc., St. Louis, MO, USA) to block muscle contractions. The (+)-Tubocurarine-containing Ringer’s solution was replaced by Ringer’s solution containing 0.8–1% glutaraldehyde (220–230 mOsM total; pH 7.2), as the resting terminals that were not electrically stimulated were fixed in previous studies, and nerve stimulation simultaneously began at 10 Hz with a current amplitude 10 times greater than threshold. Stimulation continued for 2 min; it was previously observed under a dissecting microscope that in stimulated nerve–muscle preparations not exposed to (+)-Tubocurarine, contractions of all muscle fibers ceased after 2 min in the fixative, indicating all of the NMJ’s were fixed at that time. After stimulation, the muscles remained in fixative for 40 min. They were further processed at room temperature for electron tomography, according to the method used for preparing resting terminals. They were further fixed and stained for 1 h in 1% OsO₄ in phosphate buffer (220–230 mOsM total, pH 7.2), washed for one hour in H₂O, stained one hour in saturated aqueous uranyl acetate, dehydrated in increasing concentrations of ethanol, and embedded flat in a 1-mm-thick wafer of Eponate 12 (Ted Pella). The animal experimentation described here was approved by Stanford University’s (Protocol Number 10505, 24 January 2008) and Texas A&M University (AUP Number 2011-8, 23 May 2011) administrative panels on laboratory animal care (IACUC), which oversee the use of animals according to U.S. federal regulations.