Optical detection of three modes of endocytosis at hippocampal synapses.

Natali L Chanaday,Ege T Kavalali

doi:10.7554/elife.36097

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 (~5-12 s, ~40% of events) and ultraslow speeds (>20 s, ~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.

Highlights

Synapses form the basic computational units in the brain as they transfer and process information with exquisite temporal and spatial precision
The vesicular glutamate transporter 1 bound to the pH sensitive derivative of GFP, pHluorin has been widely used to track synaptic vesicle trafficking based on its low plasma membrane expression and high signal-to-noise ratio (Voglmaier et al, 2006; Balaji and Ryan, 2007; Leitz and Kavalali, 2011)
In this study, using a combination of fluorescent reporters based on the vesicular glutamate transporter, synaptophysin or the V0a1 subunit of the vacuolar ATPase, we were able to identify three kinetically different pathways that retrieve individual synaptic vesicles after single action potential (AP) driven fusion

Summary

Introduction

Synapses form the basic computational units in the brain as they transfer and process information with exquisite temporal and spatial precision In presynaptic terminals, this is achieved through fast neurotransmitter release via fusion of synaptic vesicles within hundreds of microseconds upon the arrival of a presynaptic action potential (Sudhof, 2013). Experiments using fast-freeze EM (Watanabe et al, 2013) or improved capacitance measurements (Delvendahl et al, 2016) produced estimates within 100—500 ms for synaptic vesicle retrieval in central synapses. This so-called ultrafast synaptic endocytosis appeared to predominantly occur near physiological temperatures (~36 ̊C) (Watanabe and Boucrot, 2017). Experiments using synaptic vesicle proteins labeled with pHluorin (a pH-sensitive derivative of GFP) to optically monitor single synaptic vesicle endocytosis gave rise to variable and often contradicting results (Balaji and Ryan, 2007; Gandhi and Stevens, 2003; Granseth et al, 2006; Leitz and Kavalali, 2011; Zhu et al, 2009) with the extra complication that these studies were typically conducted at room temperature

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