Vesicles in snake motor terminals comprise one functional pool and utilize a single recycling strategy at all stimulus frequencies

Abstract

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 ∼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.

Preferred Sites of Exocytosis and Endocytosis Colocalize during High- But Not Lower-Frequency Stimulation in Mouse Motor Nerve Terminals

“…Moreover, the rapid onset of spot appearance is consistent with the time course of bulk endocytosis in central nerve terminals (Clayton et al , 2008). Finally, the slow dissipation of FM 4-64 spots, well after complete internalization of synaptic vesicle membrane (provided by the spH signal) is also consistent with the slow regeneration and scattering of synaptic vesicles from structures created by bulk endocytosis (Richards et al , 2000; 2003; Lin et al , 2005), although not necessarily measurements of endosome dissipation (Teng et al , 2007). If the FM 4-64 spots were not large endosomes, but rather clusters of synaptic vesicles, then the low mobility of these vesicles would likely make them less fusion competent (Gaffield and Betz, 2007).…”

Fast Synaptic Vesicle Reuse Slows the Rate of Synaptic Depression in the CA1 Region of Hippocampus

“…However, in contrast to our observations in acute hippocampal slices, RRP vesicles in most other preparations readily mix with a reserve pool over time (Pyle et al, 2000;de Lange et al, 2003). Interestingly, recent studies in the cone photoreceptor terminals (Rea et al, 2004) or in snake motor nerve terminals (Lin et al, 2005) revealed a more streamlined vesicle recycling consistent with a single functional pool of vesicles. Moreover, RRP vesicles in the frog neuromuscular junction also follow a rather isolated recycling pathway and only mix with a reserve pool after very strong and sustained stimulation (Richards et al, 2003;Rizzoli and Betz, 2005).…”

Sustained synaptic-vesicle recycling by bulk endocytosis contributes to the maintenance of high-rate neurotransmitter release stimulated by glycerotoxin

“…Considering that each amphibian motor nerve terminal contains approximately 700 active zones (Soyoun Cho and S.D.M., unpublished observations), the frequency of spontaneous fluctuations in Ca 2+ entry would be about 10/nerve terminal/second. In this light, it is likely that the increase in Ca 2+ fluctuations leads to the reported general elevation of MEPP frequency, and that the strong Ca 2+ entry events might instead trigger the large synchronous bursts in MEPP frequency observed in addition to the general increase in asynchronous MEPP frequency (Betz et al, 1992;de Paiva et al, 1999;Henkel et al, 1996;Lin et al, 2005;Ribchester et al, 1994;Smith and Betz, 1996).…”

“…Styryl dyes such as FM1-43 have been widely used to investigate synaptic-vesicle recycling at the NMJ (Betz et al, 1992;de Paiva et al, 1999;Henkel et al, 1996;Lin et al, 2005;Ribchester et al, 1994;Smith and Betz, 1996). FM1-43 was applied for 5 minutes in the absence of stimulation before washing the preparation with cold Ca 2+ -free medium.…”