Neurotransmitter release from synaptic vesicle fusion is the fundamental mechanism for neuronal communication at synapses. Evoked release following an action potential has been well characterized for its function in activating the postsynaptic cell, but the significance of spontaneous release is less clear. Using transgenic tools to image single synaptic vesicle fusion events at individual release sites (active zones) in Drosophila, we characterized the spatial and temporal dynamics of exocytotic events that occur spontaneously or in response to an action potential. We also analyzed the relationship between these two modes of fusion at single release sites. A majority of active zones participate in both modes of fusion, although release probability is not correlated between the two modes of release and is highly variable across the population. A subset of active zones is specifically dedicated to spontaneous release, indicating a population of postsynaptic receptors is uniquely activated by this mode of vesicle fusion. Imaging synaptic transmission at individual release sites also revealed general rules for spontaneous and evoked release, and indicate that active zones with similar release probability can cluster spatially within individual synaptic boutons. These findings suggest neuronal connections contain two information channels that can be spatially segregated and independently regulated to transmit evoked or spontaneous fusion signals.

Neurotransmitter release following synaptic vesicle (SV) fusion is the fundamental mechanism for neuronal communication. Synaptic exocytosis is a specialized form of intercellular communication that shares a common SNARE-mediated fusion mechanism with other membrane trafficking pathways. The regulation of synaptic vesicle fusion kinetics and short-term plasticity is critical for rapid encoding and transmission of signals across synapses. Several families of SNARE-binding proteins have evolved to regulate synaptic exocytosis, including Synaptotagmin (SYT) and Complexin (CPX). Here we demonstrate that Drosophila CPX controls evoked fusion occurring via the synchronous and asynchronous pathways. cpx−/− mutants show increased asynchronous release, while CPX overexpression largely eliminates the asynchronous component of fusion. We also find that SYT and CPX co-regulate the kinetics and Ca2+ cooperativity of neurotransmitter release. CPX functions as a positive regulator of release in part by coupling the Ca2+ sensor SYT to the fusion machinery and synchronizing its activity to speed fusion. In contrast, syt−/−; cpx−/− double mutants completely abolish the enhanced spontaneous release observe in cpx−/− mutants alone, indicating CPX acts as a fusion clamp to block premature exocytosis in part by preventing inappropriate activation of the SNARE machinery by SYT. CPX levels also control the size of synaptic vesicle pools, including the immediate releasable pool and the ready releasable pool – key elements of short-term plasticity that define the ability of synapses to sustain responses during burst firing. These observations indicate CPX regulates both spontaneous and evoked fusion by modulating the timing and properties of SYT activation during the synaptic vesicle cycle.

Neurons communicate through neurotransmitter release at specialized synaptic regions known as active zones (AZs). Using biosensors to visualize single synaptic vesicle fusion events at Drosophila neuromuscular junctions, we analyzed the developmental and molecular determinants of release probability (Pr) for a defined connection with ~300 AZs. Pr was heterogeneous but represented a stable feature of each AZ. Pr remained stable during high frequency stimulation and retained heterogeneity in mutants lacking the Ca2+ sensor Synaptotagmin 1. Pr correlated with both presynaptic Ca2+ channel abundance and Ca2+ influx at individual release sites. Pr heterogeneity also correlated with glutamate receptor abundance, with high Pr connections developing receptor subtype segregation. Intravital imaging throughout development revealed that AZs acquire high Pr during a multi-day maturation period, with Pr heterogeneity largely reflecting AZ age. The rate of synapse maturation was activity-dependent, as both increases and decreases in neuronal activity modulated glutamate receptor field size and segregation.

Association of Nwk with SNX16 promotes down-regulation of synaptic growth signaling at the interface between early and recycling endosomes.

Synaptic vesicle fusion mediates communication between neurons and is triggered by rapid influx of Ca2+. The Ca2+-triggering step for fusion is regulated by the synaptic vesicle transmembrane protein Synaptotagmin 1 (Syt1). Syt1 contains two cytoplasmic C2 domains, termed C2A and C2B, which coordinate Ca2+ binding. Although C2A and C2B share similar topology, binding of Ca2+ ions to the C2B domain has been suggested as the only critical trigger for evoked vesicle release. If and how C2A domain function is coordinated with C2B remains unclear. In this study, we generated a panel of Syt1 chimeric constructs in Drosophila to delineate the unique and shared functions of each C2 domain in regulation of synaptic vesicle fusion. Expression of Syt 1 transgenes containing only individual C2 domains, or dual C2A-C2A or C2B-C2B chimeras, failed to restore Syt1 function in a syt1−/− null mutant background, indicating both C2A and C2B are specifically required to support fast synchronous release. Mutations that disrupted Ca2+ binding to both C2 domains failed to rescue evoked release, but supported synaptic vesicle docking and endocytosis, indicating these functions of Syt1 are Ca2+-independent. The dual C2 domain Ca2+-binding mutant also enhanced spontaneous fusion, while dramatically increasing evoked release when co-expressed with native Syt1. Taken together, these data indicate that synaptic transmission can be regulated by Syt1 multimerization, and that both C2 domains of Syt1 are uniquely required for modulating Ca2+-independent spontaneous fusion and Ca2+-dependent synchronous release.

Summary Synaptic plasticity is a fundamental feature of the nervous system that allows adaptation to changing behavioral environments. Most studies of synaptic plasticity have examined the regulated trafficking of postsynaptic glutamate receptors that generates alterations in synaptic transmission. Whether and how changes in the presynaptic release machinery contribute to neuronal plasticity is less clear. The SNARE complex mediates neurotransmitter release in response to presynaptic Ca++ entry. Here we show that the SNARE fusion clamp Complexin undergoes activity-dependent phosphorylation that alters the basic properties of neurotransmission in Drosophila. Retrograde signaling following stimulation activates PKA-dependent phosphorylation of the Complexin C-terminus that selectively and transiently enhances spontaneous release. Enhanced spontaneous release is required for activity-dependent synaptic growth. These data indicate that SNARE-dependent fusion mechanisms can be regulated in an activity-dependent manner and highlight the key role of spontaneous neurotransmitter release as a mediator of functional and structural plasticity.

Complexins are small α-helical proteins that modulate neurotransmitter release by binding to SNARE complexes during synaptic vesicle exocytosis. They have been found to function as fusion clamps to inhibit spontaneous synaptic vesicle fusion in the absence of Ca2+, while also promoting evoked neurotransmitter release following an action potential. Complexins consist of an N-terminal domain and accessory α-helix that regulate the activating and inhibitory properties of the protein, respectively, and a central α-helix that binds the SNARE complex and is essential for both functions. In addition, Complexins contain a largely unstructured C-terminal domain whose role in synaptic vesicle cycling is poorly defined. Here, we demonstrate that the C-terminus of Drosophila Complexin (DmCpx) regulates localization to synapses and that alternative splicing of the C-terminus can differentially regulate spontaneous and evoked neurotransmitter release. Characterization of the single DmCpx gene by mRNA analysis revealed expression of two alternatively expressed isoforms, DmCpx7A and DmCpx7B, which encode proteins with different C-termini that contain or lack a membrane tethering prenylation domain. The predominant isoform, DmCpx7A, is further modified by RNA editing within this C-terminal region. Functional analysis of the splice isoforms showed that both are similarly localized to synaptic boutons at larval neuromuscular junctions, but have differential effects on the regulation of evoked and spontaneous fusion. These data indicate that the C-terminus of Drosophila Complexin regulates both spontaneous and evoked release though separate mechanisms and that alternative splicing generates isoforms with distinct effects on the two major modes of synaptic vesicle fusion at synapses.

Synaptic vesicle (SV) size is one parameter that controls the amount of neurotransmitter released from individual SVs and, therefore, is fundamental to our understanding of synaptic function. The recently discovered variability of mean SV size among excitatory hippocampal synapses -- if actively regulated -- is a potential mechanism for the regulation of transmitter release. Here, we investigated which parameters influence mean SV size. First, we revealed that synapse-to-synapse variability of SV size is a general phenomenon in several species and brain regions. In addition, we determined the relationship between mean SV size and synaptic morphology. In three-dimensional reconstructions from serial ultrathin sections, we found that SV size did not correlate with the area of the postsynaptic density (a measure for synaptic size and synaptic cleft volume) nor with the total number of SVs within a bouton or bouton volume. We tested the long-held hypothesis that a change in osmotic pressure (potentially caused by a change in neurotransmitter concentration) affects SV size. When we reduced the osmotic pressure, SVs became significantly smaller; however, an increase in osmotic pressure had no effect on SV size. Furthermore, we found that SV size does not adapt to chronic changes in activity and that the SV cycle is capable of providing constant SV size during long-lasting, high-frequency stimulation.