Optical detection of three modes of endocytosis at hippocampal synapses

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Abstract

Coupling of synaptic vesicle fusion and retrieval constitutes a core mechanism ensuring maintenance of presynaptic function. Recent studies using fast-freeze electron microscopy and capacitance measurements reported an ultrafast mode of endocytosis operating at physiological temperatures. Here, using rat hippocampal neurons, we optically monitored single synaptic vesicle endocytosis with high time resolution using the vesicular glutamate transporter, synaptophysin and the V0a1 subunit of the vacuolar ATPase as probes. In this setting, we could distinguish three components of retrieval operating at ultrafast (~150-250 ms, ~20% of events), fast (~5-12s, ~40% of events) and ultraslow speeds (>20s, ~40% of events). While increasing Ca²⁺ slowed the fast events, increasing temperature accelerated their time course. In contrast, the kinetics of ultrafast events were only mildly affected by these manipulations. These results suggest that synaptic vesicle proteins can be retrieved with ultrafast kinetics, although a majority of evoked fusion events are coupled to slower retrieval mechanisms.

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All data generated or analysed during this study are included in the manuscript and supporting files as histograms or box plots.

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Natali L Chanaday

Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, United States

Competing interests
The authors declare that no competing interests exist.
Ege T Kavalali

Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, United States

For correspondence
ege.kavalali@utsouthwestern.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-1777-227X

Funding

National Institute of Mental Health (MH066198)

Ege T Kavalali

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal protocols were approved by the Institutional Care and Use Committee at UT Southwestern Medical Center. The work presented in this study is covered by the Animal Protocol Numbers APN 2016-101416.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.