Abstract
There is a general consensus that synaptic vesicular release by a full collapse process is the primary machinery of synaptic transmission. However, competing view suggests that synaptic vesicular release operates via a kiss-and-run mechanism. By monitoring the release dynamics of a synaptic vesicular marker, FM1-43 from individual synapses in hippocampal neurons, we found evidence that the release of synaptic vesicle was delayed by several seconds after the start of field stimulation. This phenomenon was associated with modified opening kinetics of fusion pores. Detailed analysis revealed that some synapses were completely inactive for a few seconds after stimulation, despite immediate calcium influx. This delay in vesicular release was modulated by various stimulation protocols and different frequencies, indicating an activity-dependent regulation mechanism for neurotransmitter exocytosis. Staurosporine, a drug known to induce “kiss-and-run” exocytosis, increased the proportion of delayed synapses as well as the delay duration, while fluoxetine acted contrarily. Besides being a serotonin reuptake inhibitor, it directly enhanced vesicle mobilization and reduced synaptic fatigue. Exocytosis was never delayed, when it was monitored with pH-sensitive probes, synaptopHlourin and αSyt-CypHerE5 antibody, indicating an instantaneous formation of a fusion pore that allowed rapid equilibration of vesicular lumenal pH but prevented FM1-43 release because of its slow dissociation from the inner vesicular membrane. Our observations suggest that synapses operate via a sequential “kiss-and-run” and “full-collapse” exocytosis mechanism. The initially narrow vesicular pore allows the equilibration of intravesicular pH which then progresses toward full fusion, causing FM1-43 release.
Highlights
The predominant neurotransmitter exocytosis mechanism “kiss-and-run” or “full-collapse fusion” (Heuser and Reese, 1973; Ceccarelli and Hurlbut, 1980) in the brain is still under debate, despite a wealth of evidence collected for both models in many different cell- and vesicle types (Albillos et al, 1997; Schikorski and Stevens, 2001; Richards et al, 2005)
The kinetics of FM1-43 release from individual synaptic boutons appeared to be highly variable the average of several hundreds of fluorescence traces generally followed a single or second-order exponential decay function, which was the result of mutual compensatory effects of fast and slow destaining synapses
A special type of synapses was discovered and defined as “Delayed synapse” (DelSyn), whose release kinetics belonged to either of the classes’ (1–3), but whose onset of release was considerably delayed after the start of the stimulation
Summary
The predominant neurotransmitter exocytosis mechanism “kiss-and-run” or “full-collapse fusion” (Heuser and Reese, 1973; Ceccarelli and Hurlbut, 1980) in the brain is still under debate, despite a wealth of evidence collected for both models in many different cell- and vesicle types (Albillos et al, 1997; Schikorski and Stevens, 2001; Richards et al, 2005). Delayed Release of FM1-43 both mechanisms occur in mast- and neuro-endocrine cells as well as large dense core vesicles (Elhamdani et al, 2006; Xia et al, 2009; Balseiro-Gomez et al, 2016), either as separate mechanisms or in combinations of both, their functional significance for CNSsynapses with small synaptic vesicles is much less conclusive (Harata et al, 2006; Granseth et al, 2009; Zhang et al, 2009; Rudling et al, 2018) It is not clear yet, whether the variation in the process of vesicular release is due to the difference in types of synapses (GABAergic, glutamatergic, etc.), difference in the regulation of release of two and more transmitters from a single synapse (Jonas et al, 1998), or the change in the exocytosis kinetics of a specific synapse class (Gan and Watanabe, 2018). Since the mechanism remained unknown, we used a selection of activity-dependent staining techniques and drugs to investigate variability, timing, and kinetic mechanisms of exoand endocytosis in individually visualized synapses in order to experimentally document a consistent model of vesicle fusion
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