Correlation between quantal secretion and vesicle loss at the frog neuromuscular junction

1. We measured the rate of occurrence of miniature endplate potentials (MEPPs) at identified endplates in frog cutaneous pectoris muscles treated with crude black widow spider venom (BWSV) or purified alpha-latrotoxin (alpha-LTX) in calcium-free solutions, and we examined the relationship between the length of the nerve terminal and the total number of quanta secreted, and the relationship between the number of quanta secreted and the number of vesicles remaining at different times. 2. The venom, or toxin, was applied in a modified Ringer solution with tetrodotoxin, 1 mM-EGTA and no divalent cations, and quantal secretion was started by applying Ca2(+)-free solutions with Mg2+. This was done to synchronize the quantal discharge at the various junctions in a muscle. Ringer solution was applied after the MEPP rate had declined to low levels, and then the muscle fibre was injected with Lucifer Yellow, the endplate stained for acetylcholinesterase and the length of the nerve terminal and the length of a sarcomere were measured on the fluorescent fibre. 3. The total number of quanta secreted by a terminal was measured under a wide variety of experimental conditions: the weights of the frogs ranged from 13 to 68 g, the temperature from 9 to 28 degrees C, and the concentration of Mg2+ from 2 to 10 mM. In one series of experiments the Mg2+ was withdrawn after 3-4 min and reapplied 35-40 min later in order to divide the total output of quanta into two approximately equal bouts of secretion that were well separated in time. 4. The total number of MEPPs recorded at a junction was loosely correlated with the length of its nerve terminal, but it was not affected by the temperature, the concentration of Mg2+ or the division of secretion into well-separated bouts of quantal release. The average total secretion per unit length was about 3700 quanta/sarcomere or about 1200 quanta/microns. 5. The average time course of quantal secretion per micrometre of terminal was determined at single junctions in muscles held at 22-23 degrees C or at 9-10 degrees C. Other muscles were fixed at various times during the course of secretion at each temperature and the number of synaptic vesicles remaining in cross-sections of the terminals were counted on electron micrographs. The number of vesicles remaining per micrometre of terminal was determined from the number per cross-section and the section thickness.(ABSTRACT TRUNCATED AT 400 WORDS)