Differential Release of Exocytosis Marker Dyes Indicates Stimulation-Dependent Regulation of Synaptic Activity

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.

Bridging the gap between presynaptic hair cell function and neural sound encoding

“…In the framework of the univesicular hypothesis of spontaneous release, the flickering of the fusion pore, prior to or instead of full collapse fusion of the SV, might be favored in low SR synapses, leading to a lower percentage of monophasic sEPSCs. Such heterogeneity of fusion pore dynamics has been reported in chromaffin cells, calyx of Held and hippocampal neurons (Chang et al, 2021; Henkel et al, 2019; Shin et al, 2020, 2018).…”

“…In the framework of the univesicular hypothesis of spontaneous release, the flickering of the fusion pore, instead of a fusion pore opening followed directly to full vesicle collapse, might be favored in low SR synapses, leading to a lower percentage of monophasic sEPSCs. Such heterogeneity of fusion pore dynamics has been reported in chromaffin cells, calyx of Held and hippocampal neurons (Chang et al, 2021;Henkel et al, 2019;Shin et al, 2018Shin et al, , 2020. However, the relation between fusion pore dynamics and spontaneous release rate has not yet been suggested.…”

Platelet C3G: a key player in vesicle exocytosis, spreading and clot retraction

“…After staining, when vesicles undergo exocytosis in dye-free medium, FM1-43 molecules dissociate from the membrane, thus fluorescence is lost [ 30 ]. The FM1-43 signal only remains in recycled vesicles, i.e., in those undergoing kiss-and-run exocytosis, but is lost in full-collapse secretion [ 31 , 32 ]. Platelets (1 × 10 7 ) were incubated with 10 µM FM1-43 (Table S2 ) at 37 °C for 30 min.…”

Current molecular approaches to investigate pre‐synaptic dysfunction

“…Both cypher and pHrodo dyes can be chemically coupled to other molecules allowing production of bespoke reporters, for example coupling of cypHer to phospholipids allows integration into the plasma membrane and internalization in much the same way as FM dyes, with the advantage that fluorescence increases the more acidic the compartment becomes (Kahms & Klingauf, 2018). CypHer can also be covalently linked to antibodies in order to report internalization of specific reporters such as synaptotagmin, for example (Henkel et al 2019). CypHer and pHrodo can also be coupled to generic molecules like dextran, creating a fluid-phase marker which can be used to mark endocytic compartments (e.g.…”

“…CypHer can also be covalently linked to antibodies in order to report internalization of specific reporters such as synaptotagmin, for example (Henkel et al. 2019). CypHer and pHrodo can also be coupled to generic molecules like dextran, creating a fluid‐phase marker which can be used to mark endocytic compartments (e.g.…”