A small pool of vesicles maintains synaptic activity in vivo

Abstract

Chemical synapses contain substantial numbers of neurotransmitter-filled synaptic vesicles, ranging from approximately 100 to many thousands. The vesicles fuse with the plasma membrane to release neurotransmitter and are subsequently reformed and recycled. Stimulation of synapses in vitro generally causes the majority of the synaptic vesicles to release neurotransmitter, leading to the assumption that synapses contain numerous vesicles to sustain transmission during high activity. We tested this assumption by an approach we termed cellular ethology, monitoring vesicle function in behaving animals (10 animal models, nematodes to mammals). Using FM dye photooxidation, pHluorin imaging, and HRP uptake we found that only approximately 1-5% of the vesicles recycled over several hours, in both CNS synapses and neuromuscular junctions. These vesicles recycle repeatedly, intermixing slowly (over hours) with the reserve vesicles. The latter can eventually release when recycling is inhibited in vivo but do not seem to participate under normal activity. Vesicle recycling increased only to ≈5% in animals subjected to an extreme stress situation (frog predation on locusts). Synapsin, a molecule binding both vesicles and the cytoskeleton, may be a marker for the reserve vesicles: the proportion of vesicles recycling in vivo increased to 30% in synapsin-null Drosophila. We conclude that synapses do not require numerous reserve vesicles to sustain neurotransmitter release and thus may use them for other purposes, examined in the accompanying paper.pools | styryl dyes | vesicle cycle | shibire |

The reserve pool of synaptic vesicles acts as a buffer for proteins involved in synaptic vesicle recycling

“…Stimulating synaptic preparations in vitro at physiological frequencies results in the repetitive use of only a fraction of the vesicles (up to ≈20%), termed the recycling pool (1,2). Similarly, we found that living animals only recycled very few vesicles over a few hours (3), which, in contrast to the in vitro situation, constituted only 1-5%. These observations suggest that the bulk of the vesicle population is not involved in recycling at any one time point and therefore may serve another function.…”

Ratiometric Imaging of pH Probes

“…The pHluorin signal decreases again as endocytosis and vesicle acidification occur. This experimental strategy has revealed the kinetics and retrieval of vesicle dynamics (Granseth et al, 2006), vesicle fusion events in motor neurons in Drosophila larvae (Denker et al, 2011), and endocytosis in situ (Poskanzer, Marek, Sweeney, & Davis, 2003).…”

“…• Plasmid encoding a genetically encoded pH biosensor, such as mCherrypHluorin (Denker et al, 2011;Koivusalo et al, 2010) • Polypropylene round-bottom tube, 5 ml (Falcon, catalog number 352063) • Warm media to 37 C in a water bath before use.…”

Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion

“…One of the most widelyused methods exploits FM-dyes, fluorescent reporters that readily bind to lipid membrane and are taken up into recycling vesicles during endocytosis to provide a functional readout of vesicle turnover [21][22][23] . One dye variant, FM1-43FX, also efficiently drives the polymerization of diaminobenzidine (DAB) when photoactivated, leading to the formation of an osmiophilic precipitate [24][25][26][27][28][29][30][31] . In this way, functional vesicle pools that were previously labelled with FM-dye can be directly identified in electron micrographs.…”

“…In this way, functional vesicle pools that were previously labelled with FM-dye can be directly identified in electron micrographs. This approach has been used extensively and highly successfully in cultured neurons 7,10,24,28,30,[32][33][34] , a number of large, peripheral terminals [26][27][28][29]31,[35][36][37] , and large central release sites such as calyx of Held 25 revealing important information about organizational principles of vesicle pools. However, only recently 38 has this method been successfully applied to small central synapses in native brain tissue, where neurons are retained in relevant circuits with defined cytoarchitecture.…”