fm1-43 and vesamicol

At a variety of fast chemical synapses, spent synaptic vesicles are recycled via a large 'reserve' vesicle pool at high stimulus frequencies, and via fast 'local cycling' near release sites (e.g. 'kiss and run' transmitter release) at low stimulus frequencies. We have investigated recycling at the snake neuromuscular junction (NMJ), specifically seeking evidence for local cycling. Activity-dependent staining and destaining of the endocytic probe FM1-43 were directly compared to transmitter release over a range of stimulus frequencies. We found a fixed proportionality between staining/destaining and summed endplate potentials (EPPs) representing total transmitter release. There was no direct dependence of staining or destaining on stimulus frequency, as would be expected if local cycling (and consequent altered FM1-43 retention) were more prevalent at one frequency than another. In other experiments the drug vesamicol was used to abolish refilling of vesicles with transmitter, thereby blocking EPPs contributed by recycled vesicles. Control and vesamicol-treated NMJs had identical quantal content for the first 10 min of 1 Hz stimulation. Afterwards EPP amplitudes at vesamicol-treated NMJs declined at a rate consistent with use of a large pool containing approximately 130,000 vesicles. Finally, calibrated paired stimulations show that regenerated vesicles have poorer than random probability of re-release. Our findings are inconsistent with local cycling and suggest that the snake motor terminal utilizes exclusively a single large vesicle pool.

Topics: Animals; Colubridae; Electric Stimulation; Fluorescent Dyes; In Vitro Techniques; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Piperidines; Pyridinium Compounds; Quaternary Ammonium Compounds; Synaptic Transmission; Synaptic Vesicles; Time Factors

We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.

Topics: Acetylcholine; Animals; Carrier Proteins; Colubridae; Electric Stimulation; Exocytosis; Fluorescent Dyes; In Vitro Techniques; Membrane Transport Proteins; Motor Endplate; Motor Neurons; Nerve Endings; Piperidines; Pyridinium Compounds; Quaternary Ammonium Compounds; Synaptic Membranes; Synaptic Vesicles; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins