Single Synaptic Vesicles Research Articles

Nanoscale architecture of synaptic vesicles and scaffolding complexes revealed by cryo-electron tomography

The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.

Fusion pore flux controls the rise-times of quantal synaptic responses.

The release of neurotransmitter from a single synaptic vesicle generates a quantal response, which at excitatory synapses in voltage-clamped neurons is referred to as a miniature excitatory postsynaptic current (mEPSC). We analyzed mEPSCs in cultured mouse hippocampal neurons and in HEK cells expressing postsynaptic proteins enabling them to receive synaptic inputs from cocultured neurons. mEPSC amplitudes and rise-times varied widely within and between cells. In neurons, mEPSCs with larger amplitudes had longer rise-times, and this correlation was stronger in neurons with longer mean rise-times. In HEK cells, this correlation was weak and unclear. Standard mechanisms thought to govern mEPSCs cannot account for these results. We therefore developed models to simulate mEPSCs and assess their dependence on different factors. Modeling indicated that longer diffusion times for transmitters released by larger vesicles to reach more distal receptors cannot account for the correlation between rise-time and amplitude. By contrast, incorporating the vesicle size dependence of fusion pore expulsion time recapitulated experimental results well. Larger vesicles produce mEPSCs with larger amplitudes and also take more time to lose their content. Thus, fusion pore flux directly contributes to mEPSC rise-time. Variations in fusion pores account for differences among neurons, between neurons and HEK cells, and the correlation between rise-time and the slope of rise-time versus amplitude plots. Plots of mEPSC amplitude versus rise-time are sensitive to otherwise inaccessible properties of a synapse and offer investigators a means of assessing the role of fusion pores in synaptic release.

Metabolic regulation of single synaptic vesicle exo- and endocytosis in hippocampal synapses

Glucose has long been considered a primary energy source for synaptic function. However, it remains unclear to what extent alternative fuels, such as lactate/pyruvate, contribute to powering synaptic transmission. By detecting individual release events in hippocampal synapses, we find that mitochondrial ATP production regulates basal vesicle release probability and release location within the active zone (AZ), evoked by single action potentials. Mitochondrial inhibition shifts vesicle release closer to the AZ center and alters the efficiency of vesicle retrieval by increasing the occurrence of ultrafast endocytosis. Furthermore, we uncover that terminals can use oxidative fuels to maintain the vesicle cycle during trains of activity. Mitochondria are sparsely distributed along hippocampal axons, and we find that terminals containing mitochondria display enhanced vesicle release and reuptake during high-frequency trains. Our findings suggest that mitochondria not only regulate several fundamental features of synaptic transmission but may also contribute to modulation of short-term synaptic plasticity.

Synapsin 2a tetramerisation selectively controls the presynaptic nanoscale organisation of reserve synaptic vesicles

Neurotransmitter release relies on the regulated fusion of synaptic vesicles (SVs) that are tightly packed within the presynaptic bouton of neurons. The mechanism by which SVs are clustered at the presynapse, while preserving their ability to dynamically recycle to support neuronal communication, remains unknown. Synapsin 2a (Syn2a) tetramerization has been suggested as a potential clustering mechanism. Here, we used Dual-pulse sub-diffractional Tracking of Internalised Molecules (DsdTIM) to simultaneously track single SVs from the recycling and the reserve pools, in live hippocampal neurons. The reserve pool displays a lower presynaptic mobility compared to the recycling pool and is also present in the axons. Triple knockout of Synapsin 1-3 genes (SynTKO) increased the mobility of reserve pool SVs. Re-expression of wild-type Syn2a (Syn2aWT), but not the tetramerization-deficient mutant K337Q (Syn2aK337Q), fully rescued these effects. Single-particle tracking revealed that Syn2aK337QmEos3.1 exhibited altered activity-dependent presynaptic translocation and nanoclustering. Therefore, Syn2a tetramerization controls its own presynaptic nanoclustering and thereby contributes to the dynamic immobilisation of the SV reserve pool.

Frequency of Spontaneous Neurotransmission at Individual Boutons Corresponds to the Size of the Readily Releasable Pool of Vesicles.

Synapses maintain two forms of neurotransmitter release to support communication in the brain. First, evoked neurotransmitter release is triggered by the invasion of an action potential (AP) across en passant boutons that form along axons. The probability of evoked release (Pr) varies substantially across boutons, even within a single axon. Such heterogeneity is the result of differences in the probability of a single synaptic vesicle (SV) fusing (Pv) and in the number of vesicles available for immediate release, known as the readily releasable pool (RRP). Spontaneous release (also known as a mini) is an important form of neurotransmission that occurs in the absence of APs. Because it cannot be triggered with electrical stimulation, much less is known about potential heterogeneity in the frequency of spontaneous release between boutons. We utilized a photostable and bright fluorescent indicator of glutamate release (iGluSnFR3) to quantify both spontaneous and evoked release at individual glutamatergic boutons. We found that the rate of spontaneous release is quite heterogenous at the level of individual boutons. Interestingly, when measuring both evoked and spontaneous release at single synapses, we found that boutons with the highest rates of spontaneous release also displayed the largest evoked responses. Using a new optical method to measure RRP at individual boutons, we found that this heterogeneity in spontaneous release was strongly correlated with the size of the RRP, but not related to Pv. We conclude that the RRP is a critical and dynamic aspect of synaptic strength that contributes to both evoked and spontaneous vesicle release.

Single synaptic vesicle tracking in TIRFM-amenable hippocampal synapses.

Single synaptic vesicle (SV) tracking is used to explore the dynamics of SV movement close to active zones (AZ) by total internal reflection fluorescence microscopy (TIRFM). To do so, we used cultured hippocampal ‘xenapses’, purely presynaptic boutons formed by neurons on micro-patterned coverslips, functionalized with synaptogenic cell adhesion proteins. For single SV labeling we developed surface-modified single-color quantum dots with improved photostability and pH-sensitivity for single SV tracking. Obtained single SV movies are analyzed with an improved single-particle tracking software, which combines Automatic Correction for sCMOS-related Noise algorithm (Nat. Commun., Mandracchia et al., 2020) for denoising, U-Track (Nat. Methods, Jaqaman et al., 2008) for frame-to-frame particle linking, and a newly developed single-particle localization algorithm. We yield a localization accuracy of 0.1229 ± 0.1004 pixels (110 nm pixel size) and are able to resolve merging and splitting events of two crossing particle trajectories even though they have low signal-to-noise ratio of ∼1.2. In hippocampal xenapses with active zones (AZ) facing the coverslip, SVs appear to jump between discrete levels of axial distance to AZs, and these discrete levels are remarkably stable over tens of seconds. As SVs in electron micrographs appear to be interconnected by short tethers, these discrete levels might arise from a layered structure of the SV cluster above the AZ.

Mechano-fluorescence actuation in single synaptic vesicles with a DNA framework nanomachine.

Biomimetic machines that can convert mechanical actuation to adaptive coloration in a manner analogous to cephalopods have found widespread applications at various length scales. At the nanoscale, a transmutable nanomachine with adaptive colors that can sense and mediate cellular or intracellular interactions is highly desirable. Here, we report the design of a DNA framework nanomachine (DFN) that can autonomously change shape in response to pH variations in single synaptic vesicles, which, in turn, displays adaptive fluorescent colors with a mechano-fluorescence actuation mechanism. To construct a DFN, we used a tetrahedral DNA nanostructure as the framework to incorporate an embedded pH-responsive, i-motif sequence tagged with a Förster resonance energy transfer pair and an affinity cholesterol moiety targeting vesicular membranes. We found that endocytosed DFNs are individually trapped in single endocytic vesicles in living synaptic cells due to the size-exclusion effect. The adaptive fluorescence coloration of DFNs enabled single-vesicle quantification of resting pH values in a processive manner, allowing long-term tracking of the exocytosis and fusion dynamics in intracellular processes and cell-cell communications.

Regulation of the mammalian-brain V-ATPase through ultraslow mode-switching.

Vacuolar-type adenosine triphosphatases (V-ATPases)1-3 are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases4,5. They hydrolyse ATP to establish electrochemical proton gradients for a plethora of cellular processes1,3. In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by about one V-ATPase molecule per synaptic vesicle6,7. To shed light on this bona fide single-molecule biological process, we investigated electrogenic proton-pumping by single mammalian-brain V-ATPases in single synaptic vesicles. Here we show that V-ATPases do not pump continuously in time, as suggested by observing the rotation of bacterial homologues8 and assuming strict ATP-proton coupling. Instead, they stochastically switch between three ultralong-lived modes: proton-pumping, inactive and proton-leaky. Notably, direct observation of pumping revealed that physiologically relevant concentrations of ATP do not regulate the intrinsic pumping rate. ATP regulates V-ATPase activity through the switching probability of the proton-pumping mode. By contrast, electrochemical proton gradients regulate the pumping rate and the switching of the pumping and inactive modes. A direct consequence of mode-switching is all-or-none stochastic fluctuations in the electrochemical gradient of synaptic vesicles that would be expected to introduce stochasticity in proton-driven secondary active loading of neurotransmitters and may thus have important implications for neurotransmission. This work reveals and emphasizes the mechanistic and biological importance of ultraslow mode-switching.

Nanoscopic distribution of VAChT and VGLUT3 in striatal cholinergic varicosities suggests colocalization and segregation of the two transporters in synaptic vesicles.

Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) and glutamate (Glut) to regulate the striatal network since they express vesicular transporters for ACh (VAChT) and Glut (VGLUT3). However, whether ACh and Glut are released simultaneously and/or independently from cholinergic varicosities is an open question. The answer to that question requires the multichannel detection of vesicular transporters at the level of single synaptic vesicle (SV). Here, we used super-resolution STimulated Emission Depletion microscopy (STED) to characterize and quantify the distribution of VAChT and VGLUT3 in CINs SVs. Nearest-neighbor distances analysis between VAChT and VGLUT3-immunofluorescent spots revealed that 34% of CINs SVs contain both VAChT and VGLUT3. In addition, 40% of SVs expressed only VAChT while 26% of SVs contain only VGLUT3. These results suggest that SVs from CINs have the potential to store simultaneously or independently ACh and/or Glut. Overall, these morphological findings support the notion that CINs varicosities can signal with either ACh or Glut or both with an unexpected level of complexity.

Detection of Glutamate Encapsulated in Liposomes by Optical Trapping Raman Spectroscopy.

The transmission of neuronal information is propagated through synapses by neurotransmitters released from presynapses to postsynapses. Neurotransmitters released from the presynaptic vesicles activate receptors on the postsynaptic membrane. Glutamate acts as a major excitatory neurotransmitter for synaptic vesicles in the central nervous system. Determining the concentration of glutamate in single synaptic vesicles is essential for understanding the mechanisms of neuronal activation by glutamate in normal brain functions as well as in neurological diseases. However, it is difficult to detect and quantitatively measure the concentration of glutamate in single synaptic vesicles owing to their small size, i.e., ∼40 nm. In this study, to quantitatively evaluate the concentrations of the contents in small membrane-bound vesicles, we developed an optical trapping Raman spectroscopic system that analyzes the Raman spectra of small objects captured using optical trapping. Using artificial liposomes encapsulating glutamate that mimic synaptic vesicles, we investigated whether spontaneous Raman scattered light of glutamate can be detected from vesicles trapped at the focus using optical forces. A 575 nm laser beam was used to simultaneously perform the optical trapping of liposomes and the detection of the spontaneous Raman scattered light. The intensity of Raman scattered light that corresponds to lipid bilayers increased with time. This observation suggested that the number of liposomes increased at the focal point. The number of glutamate molecules in the trapped liposomes was estimated from the calibration curve of the Raman spectra of glutamate solutions with known concentration. This method can be used to measure the number of glutamate molecules encapsulated in synaptic vesicles in situ.

Real-time three-dimensional tracking of single vesicles reveals abnormal motion and pools of synaptic vesicles in neurons of Huntington's disease mice

SummaryAlthough defective synaptic transmission was suggested to play a role in neurodegenerative diseases, the dynamics and vesicle pools of synaptic vesicles during neurodegeneration remain elusive. Here, we performed real-time three-dimensional tracking of single synaptic vesicles in cortical neurons from a mouse model of Huntington's disease (HD). Vesicles in HD neurons had a larger net displacement and radius of gyration compared with wild-type neurons. Vesicles with high release probability (Pr) were interspersed with low-Pr vesicles in HD neurons, whereas high-Pr vesicles were closer to fusion sites than low-Pr in wild-type neurons. Non-releasing vesicles in HD neurons had an abnormally high prevalence of irregular oscillatory motion. These abnormal dynamics and vesicle pools were rescued by overexpressing Rab11, and the abnormal irregular oscillatory motion was rescued by jasplakinolide. Our studies reveal the abnormal dynamics and pools of synaptic vesicles in the early stages of HD, suggesting a possible pathogenic mechanism of neurodegenerative diseases.

A Novel Neuron-Specific Regulator of the V-ATPase in Drosophila.

The V-ATPase is a highly conserved enzymatic complex that ensures appropriate levels of organelle acidification in virtually all eukaryotic cells. While the general mechanisms of this proton pump have been well studied, little is known about the specific regulations of neuronal V-ATPase. Here, we studied CG31030, a previously uncharacterized Drosophila protein predicted from its sequence homology to be part of the V-ATPase family. In contrast to its ortholog ATP6AP1/VhaAC45 which is ubiquitous, we observed that CG31030 expression is apparently restricted to all neurons, and using CRISPR/Cas9-mediated gene tagging, that it is mainly addressed to synaptic terminals. In addition, we observed that CG31030 is essential for fly survival and that this protein co-immunoprecipitates with identified V-ATPase subunits, and in particular ATP6AP2. Using a genetically-encoded pH probe (VMAT-pHluorin) and electrophysiological recordings at the larval neuromuscular junction, we show that CG31030 knock-down induces a major defect in synaptic vesicle acidification and a decrease in quantal size, which is the amplitude of the postsynaptic response to the release of a single synaptic vesicle. These defects were associated with severe locomotor impairments. Overall, our data indicate that CG31030, which we renamed VhaAC45-related protein (VhaAC45RP), is a specific regulator of neuronal V-ATPase in Drosophila that is required for proper synaptic vesicle acidification and neurotransmitter release.

Synaptobrevin-2 dependent regulation of single synaptic vesicle endocytosis.

Evidence from multiple systems indicates that vesicle SNARE (soluble NSF attachment receptor) proteins are involved in synaptic vesicle endocytosis, although their exact action at the level of single vesicles is unknown. Here we interrogate the role of the main synaptic vesicle SNARE mediating fusion, synaptobrevin-2 (also called VAMP2), in modulation of single synaptic vesicle retrieval. We report that in the absence of synaptobrevin-2, fast and slow modes of single synaptic vesicle retrieval are impaired, indicating a role of the SNARE machinery in coupling exocytosis to endocytosis of single synaptic vesicles. Ultrafast endocytosis was impervious to changes in the levels of synaptobrevin-2, pointing to a separate molecular mechanism underlying this type of recycling. Taken together with earlier studies suggesting a role of synaptobrevin-2 in endocytosis, these results indicate that the machinery for fast synchronous release couples fusion to retrieval and regulates the kinetics of endocytosis in a Ca2+-dependent manner.

Recordings from neuron-HEK cell cocultures reveal the determinants of miniature excitatory postsynaptic currents.

Spontaneous exocytosis of single synaptic vesicles generates miniature synaptic currents, which provide a window into the dynamic control of synaptic transmission. To resolve the impact of different factors on the dynamics and variability of synaptic transmission, we recorded miniature excitatory postsynaptic currents (mEPSCs) from cocultures of mouse hippocampal neurons with HEK cells expressing the postsynaptic proteins GluA2, neuroligin 1, PSD-95, and stargazin. Synapses between neurons and these heterologous cells have a molecularly defined postsynaptic apparatus, while the compact morphology of HEK cells eliminates the distorting effect of dendritic filtering. HEK cells in coculture produced mEPSCs with a higher frequency, larger amplitude, and more rapid rise and decay than neurons from the same culture. However, mEPSC area indicated that nerve terminals in synapses with both neurons and HEK cells release similar populations of vesicles. Modulation by the glutamate receptor ligand aniracetam revealed receptor contributions to mEPSC shape. Dendritic cable effects account for the slower mEPSC rise in neurons, whereas the slower decay also depends on other factors. Lastly, expression of synaptobrevin transmembrane domain mutants in neurons slowed the rise of HEK cell mEPSCs, thus revealing the impact of synaptic fusion pores. In summary, we show that cocultures of neurons with heterologous cells provide a geometrically simplified and molecularly defined system to investigate the time course of synaptic transmission and to resolve the contribution of vesicles, fusion pores, dendrites, and receptors to this process.

Counting the Number of Glutamate Molecules in Single Synaptic Vesicles

Analytical tools for direct quantitative measurements of glutamate, the principal excitatory neurotransmitter in brain have been lacking. Here, we introduce a new method for direct quantification of the number of glutamate molecules stored inside single synaptic vesicles. An ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential dependent manner when applying a constant negative potential. High-speed amperometry is used to record sub-millisecond current spikes, which represent glutamate release from single vesicles that burst open at the sensor surface and is amperometrically quantified. Our measurements show that a single synaptic vesicle encapsulates about the same amount of glutamate molecules and sometimes the partial amount as we monitor being released at single exocytotic events and at in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis and drug treatments for neuronal disorders where glutamate is involved.

Counting the Number of Glutamate Molecules in Single Synaptic Vesicles.

Analytical tools for quantitative measurements of glutamate, the principal excitatory neurotransmitter in the brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for thecounting of glutamate molecules stored inside single synaptic vesicles. In this method, an ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential-dependent manner at a constant negative potential. The continuous amperometric signals are sampled at high speed (10 kHz) to record sub-millisecond spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various glutamate concentrations. Our measurements show that an isolated single synaptic vesicle encapsulates about 8000 glutamate molecules and is comparable to the measured exocytotic quantal glutamate release in amperometric glutamate sensing in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis, and drug treatments for neuronal disorders where glutamate is involved.

In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties

Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.

Real-time three-dimensional tracking of single synaptic vesicles reveals that synaptic vesicles undergoing kiss-and-run fusion remain close to their original fusion site before reuse

The release of neurotransmitters via the fusion between synaptic vesicles and the presynaptic membrane is an essential step in synaptic transmission. Synaptic vesicles generally undergo two distinct modes of exocytosis called full-collapse fusion and kiss-and-run fusion. In kiss-and-run fusion, the fusion pore of the synaptic vesicle opens transiently without the vesicle collapsing fully into the plasma membrane; thus, each synaptic vesicle can be used multiple times to release neurotransmitters. Despite considerable research, the detailed mechanisms that underlie kiss-and-run fusion remain elusive, particularly the location of synaptic vesicles after kiss-and-run events. To address this question, we performed real-time three-dimensional tracking of single synaptic vesicles labeled with a single quantum dot in the presynaptic terminal of cultured hippocampal neurons and analyzed the three-dimensional trajectories of these vesicles undergoing kiss-and-run fusion. We found that the majority of these synaptic vesicles underwent another exocytosis event within 120 nm of their original fusion site and underwent a second exocytosis event within 10 s of the first fusion event. These results indicate that after kiss-and-run fusion, synaptic vesicles remain relatively close to their original fusion site and can release repeatedly at brief intervals, allowing neurons to maintain neurotransmitter release during bursting activity.

Heterogeneous Signaling at GABA and Glycine Co-releasing Terminals.

The corelease of several neurotransmitters from a single synaptic vesicle has been observed at many central synapses. Nevertheless, the signaling synergy offered by cotransmission and the mechanisms that maintain the optimal release and detection of neurotransmitters at mixed synapses remain poorly understood, thus limiting our ability to interpret changes in synaptic signaling and identify molecules important for plasticity. In the brainstem and spinal cord, GABA and glycine cotransmission is facilitated by a shared vesicular transporter VIAAT (also named VGAT), and occurs at many immature inhibitory synapses. As sensory and motor networks mature, GABA/glycine cotransmission is generally replaced by either pure glycinergic or GABAergic transmission, and the functional role for the continued corelease of GABA and glycine is unclear. Whether or not, and how, the GABA/glycine content is balanced in VIAAT-expressing vesicles from the same terminal, and how loading variability effects the strength of inhibitory transmission is not known. Here, we use a combination of loose-patch (LP) and whole-cell (WC) electrophysiology in cultured spinal neurons of GlyT2:eGFP mice to sample miniature inhibitory post synaptic currents (mIPSCs) that originate from individual GABA/glycine co-releasing synapses and develop a modeling approach to illustrate the gradual change in mIPSC phenotypes as glycine replaces GABA in vesicles. As a consistent GABA/glycine balance is predicted if VIAAT has access to both amino-acids, we test whether vesicle exocytosis from a single terminal evokes a homogeneous population of mixed mIPSCs. We recorded mIPSCs from 18 individual synapses and detected glycine-only mIPSCs in 4/18 synapses sampled. The rest (14/18) were co-releasing synapses that had a significant proportion of mixed GABA/glycine mIPSCs with a characteristic biphasic decay. The majority (9/14) of co-releasing synapses did not have a homogenous phenotype, but instead signaled with a combination of mixed and pure mIPSCs, suggesting that there is variability in the loading and/or storage of GABA and glycine at the level of individual vesicles. Our modeling predicts that when glycine replaces GABA in synaptic vesicles, the redistribution between the peak amplitude and charge transfer of mIPSCs acts to maintain the strength of inhibition while increasing the temporal precision of signaling.

Characterization of developmental and molecular factors underlying release heterogeneity at Drosophila synapses.

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.