Constitutive sharing of recycling synaptic vesicles between presynaptic boutons

Abstract

The synaptic vesicle cycle is vital for sustained neurotransmitter release. It has been assumed that functional synaptic vesicles are replenished autonomously at individual presynaptic terminals. Here we tested this assumption by using FM dyes in combination with fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured rat hippocampal neurons. After photobleaching, synapses acquired recently recycled FM dye-labeled vesicles originating from nonphotobleached synapses by a process requiring dynamic actin turnover. The imported vesicles entered the functional pool at their host synapses, as revealed by the exocytic release of the dye upon stimulation. FM1-43 photoconversion and ultrastructural analysis confirmed the incorporation of imported vesicles into the presynaptic terminal, where they mixed with the native vesicle pools. Our results demonstrate that synaptic vesicle recycling is not confined to individual presynaptic terminals as is widely believed; rather, a substantial proportion of recycling vesicles are shared constitutively between boutons.

Inhibitory Synapse Dynamics: Coordinated Presynaptic and Postsynaptic Mobility and the Major Contribution of Recycled Vesicles to New Synapse Formation

“…Retrospective immunocytochemistry revealed that contribution of vesicles from previously active terminals to nascent synapses represents the major mode of inhibitory presynaptic development. Whether recycled vesicles contribute to new excitatory synapse formation has not been determined, although recent work has shown that recycling vesicles can be shared among multiple preexisting neighboring excitatory boutons (Darcy et al, 2006;Staras et al, 2010). Further quantitative assays to determine the precise contributions of different vesicle pools to nascent inhibitory and excitatory synapses may prove insightful.…”

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.…”

“…Most vesicles can be readily categorized by visual assessment. However, there are also a number of established quantitative approaches to aid in vesicle classification based on comparisons of membrane and lumenal optical densities 24,29,30,32 .…”

Myosin Va Mediates Docking of Secretory Granules at the Plasma Membrane

“…Vesicle supply to release sites seems to be primarily governed by diffusion, a conclusion that we propose to extend to the case of SGs. Nevertheless, actin modulates the recovery from synaptic depression at the calyx of Held (Sakaba and Neher, 2003) and controls the exchange of synaptic vesicles between different boutons (Darcy et al, 2006). Actin, which is concentrated at the periphery of clusters, could thus participate in the retention of synaptic vesicles arriving from the axon or from the presynaptic membrane via the recycling pathway.…”