Abstract
Styryl dyes and genetically encoded pH-sensitive fluorescent proteins like pHluorin are well-established tools for the optical analysis of synaptic vesicle (SV) recycling at presynaptic boutons. Here, we describe the development of a new class of fluorescent probes based on pH-sensitive organic dyes covalently bound to lipids, providing a promising complementary assay to genetically encoded fluorescent probes. These new optical tracers allow a pure read out of membrane turnover during synaptic activity and visualization of multiple rounds of stimulation-dependent SV recycling without genetic perturbation. Measuring the incorporation efficacy of different dye-labeled lipids into budding SVs, we did not observe an enrichment of lipids with affinity for liquid ordered membrane domains. But most importantly, we found no evidence for a static segregation of SVs into recycling and resting pools. A small but significant fraction of SVs that is reluctant to release during a first round of evoked activity can be exocytosed during a second bout of stimulation, showing fast intermixing of SV pools within seconds. Furthermore, we found that SVs recycling spontaneously have a higher chance to re-occupy release sites than SVs recycling during high-frequency evoked activity. In summary, our data provide strong evidence for a highly dynamic and use-dependent control of the fractions of releasable or resting SVs.
Highlights
During synaptic transmission, neurotransmitters (NTs) stored in synaptic vesicles (SVs) are released by calcium-triggered exocytosis of membrane-docked vesicles
The cypHer5E dye exhibits maximal fluorescence at acidic pH of around 5 and is quenched at neutral to alkaline pH, providing a pH-dependency applicable to sense pH-changes associated with SV recycling (Figure 1B)
We stained primary hippocampal neurons with DMPE-cypher5E, incubated them for 20 min at 37◦C and challenged them by 900 action potentials (APs) to mobilize the entire recycling pool
Summary
Neurotransmitters (NTs) stored in synaptic vesicles (SVs) are released by calcium-triggered exocytosis of membrane-docked vesicles. Exocytosed synaptic membranes and proteins recycle by compensatory endocytosis (Ceccarelli et al, 1973; Heuser and Reese, 1973). A readily releasable pool (RRP) comprises 10–15 docked SVs at the active zone of presynaptic boutons which can be depleted by 40–50 action potentials (APs) (Schikorski and Stevens, 2001). The reserve pool of SVs refills docking sites upon prolonged activity and together, RRP and reserve pool constitute the recycling pool of SVs, which can released upon AP-mediated activity. A further population of SVs is reluctant to release upon electrical stimulation and is classified as the resting pool [for review see (Denker and Rizzoli, 2010)]
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