Frontiers in Synaptic Neuroscience

Neuronal communication relies on synaptic vesicle recycling, which has long been investigated by live imaging approaches. Synapto-pHluorins, genetically encoded reporters that incorporate a pH-sensitive variant of GFP within the lumen of the synaptic vesicle, have been especially popular. However, they require genetic manipulation, implying that a tool combining their excellent reporter properties with the ease of use of classical immunolabeling would be desirable. We introduce this tool here, relying on primary antibodies against the luminal domain of synaptotagmin 1, decorated with secondary single-domain antibodies (nanobodies) carrying a pHluorin moiety. The application of the antibodies and nanobodies to cultured neurons results in labeling their recycling vesicles, without the need for any additional manipulations. The labeled vesicles respond to stimulation, in the expected fashion, and the pHluorin signals enable the quantification of both exo- and endocytosis. We conclude that pHluorin-conjugated secondary nanobodies are a convenient tool for the analysis of vesicle recycling.

Dendritic spines, small ([~]1 {micro}m) membrane protrusions from neuronal dendrites which receive most of the excitatory synaptic inputs in the mammalian brain, are widely considered the elementary computational units of the brain. Our understanding of electrical signalling in spines is currently being debated, primarily for methodological reasons. We combined the standard techniques of whole-cell recording and voltage imaging methods to study excitatory postsynaptic potentials evoked by two-photon glutamate uncaging (uEPSPs) on individual dendritic spines on basal dendrites in rat cortical slices. We analyzed the initiation, temporal summation, and propagation of uEPSPs from the spine head to the parent dendrites in three principal neocortical pyramidal neuron classes. Our measurements show no significant attenuation of uEPSPs across the spine neck in most tested mushroom spines on basal dendrites. This result implies that spine synapses are not electrically isolated from parent dendrites and that these spines do not serve a meaningful electrical role. Using the same high-sensitivity voltage imaging techniques, we characterized the temporal summation of uEPSPs induced by repetitive glutamate uncaging mimicking burst activity of presynaptic neurons. We found that responses to high-frequency repetitive quantal EPSPs are strictly limited in amplitude and waveform. This finding reveals a biophysical mechanism for preventing synaptic saturation. Significance StatementWe used an electrochromic voltage-sensitive dye, which acts as a transmembrane optical voltmeter, to define the electrical role of dendritic spines, small membrane protrusions that receive most of the excitatory synaptic inputs in the brain. The data argue that investigated spine synapses of principal neurons are not electrically isolated from the parent dendrites. We also found that the amplitude of temporal uEPSP summation during repetitive synaptic activation is restricted at the site of origin, preventing synaptic saturation. These results facilitate our understanding of how a complex assembly of receptors and ion channels in spines generates and processes electrical signals and mediate plasticity in response to the quantal release of chemical transmitters caused by patterned activity in presynaptic axons.

The subdivision of synaptic vesicles (SVs) into discrete pools is a leading concept of synaptic physiology. To better explain specific properties of transmission and plasticity, it has been suggested initially that the readily releasable pool (RRP) of SVs is subdivided into two parallel pools differing in their release probability. More recently, evidence was provided that sequential pools with a single RRP and a series-connected finite-size replacement pool (RP) inserted between the reserve pool (RSP) and RRP equally well or even better account for most aspects of transmission and plasticity. It was further suggest that a fraction of the presynaptic release sites (N) are initially unoccupied by SVs, with vesicle recruitment occurring rapidly during activity, and furthermore that the number of release sites itself changes with rapid dynamics during activity. Here we propose a framework that identifies specific signs of the presence of the series-connected RP, using a combination of two experimental electrophysiological standard methods, cumulative analysis (CumAna) and multiple probability fluctuation analysis (MPFA). In particular we show that if the y-intercept (y(0)) of CumAna is larger than N reported by MPFA (y(0) > NMPFA) this is a strong indication for a series-connected RP. This is due to the fact that y(0) reports the sum of RRP and RP. Our analysis further suggests that this result is not affected by unoccupied release sites, as such empty sites contribute to both estimates, y(0) and NMPFA. We discuss experimental findings and models in the recent literature in the light of our theoretical considerations.

Structural plasticity of dendritic spines is considered to be the basis of synaptic plasticity, learning and memory. Here, we performed ultrastructural analysis of spines undergoing LTP using a novel high throughput correlative light-electron microscopy approach. We found that the PSD displays rapid (< 3 min) reorganization of its nanostructure, including perforation and segmentation. This increased structural complexity is maintained over intermediate and late phases of LTP (20 and 120 min). In a few spines, segmented PSDs are connected to different presynaptic terminals, producing a multi-innervated spine in the intermediate and late phases. In addition, the area of extrasynaptic axon-spine interface (eASI) displayed a pronounced, rapid and sustained increase. Finally, presynaptic vesicle number increased slowly and became significantly higher at late phases of LTP. These rapid ultrastructural changes in PSD and surrounding membrane, together with the slow increase in presynaptic vesicle number, likely support the rapid and sustained increase in synaptic transmission during LTP.

Long-term potentiation (LTP) has become a standard model for investigating synaptic mechanisms of learning and memory. Increasingly, it is of interest to understand how LTP affects the synaptic information storage capacity of the targeted population of synapses. Here, structural synaptic plasticity during LTP was explored using three-dimensional reconstruction from serial section electron microscopy. Storage capacity was assessed by applying a new analytical approach, Shannon information theory, to delineate the number of functionally distinguishable synaptic strengths. LTP was induced by delta-burst stimulation of perforant pathway inputs to the middle molecular layer of hippocampal dentate granule cells in adult rats. Spine head volumes were measured as predictors of synaptic strength and compared between LTP and control hemispheres at 30 min and 2 hr after the induction of LTP. Synapses from the same axon onto the same dendrite were used to determine the precision of synaptic plasticity based on the similarity of their physical dimensions. Shannon entropy was measured by exploiting the frequency of spine heads in functionally distinguishable sizes to assess the degree to which LTP altered the number of bits of information storage. Outcomes from these analyses reveal that LTP expanded storage capacity; the distribution of spine head volumes was increased from 2 bits in controls to 3 bits at 30 min and 2.7 bits at 2 hr after the induction of LTP. Furthermore, the distribution of spine head volumes was more uniform across the increased number of functionally distinguishable sizes following LTP, thus achieving more efficient use of coding space across the population of synapses. SignificanceEstablishing relationships between structure, function, and information storage capacity provides a new approach to assessing network strength from structural measurements. Long term potentiation (LTP) is a standard model for investigating synaptic mechanisms of learning and memory. Information is a retrievable quantity that is being stored in synapses as synaptic strength and is correlated with multiple structural components of synaptic strength. Structural synaptic plasticity was measured in 3D reconstructions from serial section electron microscopy of spine head volume, as a proxy for synapse strength, at 30 min and 2 hr after LTP induction. Outcomes indicate that LTP enhances information storage capacity for at least 2 hr by increasing the precision of the synaptic structure and expanding the range of synapse sizes.

Excitatory synapses are the most abundant synapse type in the brain. Being essential for behaviour and are implicated in hundreds of brain disorders, these synapses exhibit striking structural and functional diversity. Synaptome mapping at single-synapse resolution reveals that synaptic protein diversity is spatially organised along the dendritic tree of individual neurons and varies with age and cell-type. However, the cell biological mechanisms underlying the generation of these complex spatial synaptic patterns remain poorly understood. Potential mechanisms include somatic and dendritic protein synthesis, protein trafficking, and local regulatory mechanisms such as activity-dependent degradation. Here we developed computational models to test how combinations of these processes account for empirical synaptome data. We found that a combination of molecular transport mechanisms and local synaptic demand for proteins was sufficient to explain very complex profiles of synaptic protein distributions observed in young, mature and old mice and in different cell types. Our findings suggest the highly complex and dynamic synaptome architecture of the brain is an emergent property of a minimal set of cell biological processes. Our model sets the stage for simulations of brain tissue incorporating molecularly diverse neuronal and synaptic types in a synaptome and connectome architecture.

Neurons receive thousands of inputs onto their dendritic arbour, where individual synapses undergo activitydependent changes in strength. The durable forms of synaptic strength change, long-term potentiation (LTP) and long-term depression (LTD) require calcium entry through N-methyl-D-aspartate receptors (NMDARs) that triggers downstream protein signalling cascades in the dendrite. Notably, changes in postsynaptic strengths associated with LTP and LTD are correlated to changes in spine head volume, referred to as structural LTP (sLTP) and structural LTD (sLTD). Intriguingly, LTP and LTD, including sLTP and sLTD, are not necessarily restricted to the active, targeted synapses (homosynapses), and the changes in synaptic strength can spread and affect the strengths of inactive or non-stimulated synapses (heterosynapses) on the same cell. Moreover, the plasticity outcome at both homo- and heterosynapses can depend on the number of stimulated sites when eliciting multi-spine plasticity. Precisely how neurons allocate resources for implementing the changes in strength at individual synapses depending on their proximity to input activity across space and time remains an open question. In order to gain insights into the elementary processes underlying multi-spine plasticity that engages both homosynaptic and heterosynaptic changes, we have combined experimental and mathematical modelling approaches. On the one hand, we used glutamate uncaging to precisely and systematically stimulate variable numbers of homosynapses sharing the same dendritic branch whilst monitoring tens of other heterosynapses on the same dendrite. Homosynaptic potentiation of clusters of dendritic spines leads to heterosynaptic changes that are dependent on NMDAR, CaMKII and calcineurin. On the other hand, inspired by the Ca2+ levels hypothesis where different amounts of Ca2+ lead to either growth or shrinkage of spines, we have built a model based on a dual-role Ca2+-dependent protein that induces sLTP or sLTD. Comparing our experimental results with model predictions, we find that (i) both collaboration and competition among spines for protein resources are key drivers of heterosynaptic plasticity and (ii) the temporal and spatial distance between simultaneously stimulated spines impact the resulting spine dynamics. Moreover, our model can reconcile disparate experimental reports of sLTP and sLTD at homo- and heterosynaptic spines. Our results provide a quantitative description of the heterosynaptic footprint over minutes and hours post-stimulation across tens of microns of dendritic space. This broadens our knowledge about the operation of non-linear dendritic summation rules and how they impact spiking decisions.

Neurotransmitter release and membrane retrieval at active zones require precise spatial and temporal coordination relying on an intricate molecular machinery. However, the exact nano-structural organization of this machinery is not yet fully elucidated. Here, we used 3D MINFLUX combined with both spectral demixing and DNA-PAINT to analyze the positioning of the scaffolding protein Bassoon at presynaptic active zones of glutamatergic spine synapses of hippocampal neurons, achieving a localization precision of 5 nm in 3D. This approach allowed us to visualize directly the distribution of N-terminal and the C-terminal regions of Bassoon, and demonstrates that Bassoon exhibits an orientation at the active zone, where the C-terminal region is directed toward the synaptic cleft and the N-terminal region towards synaptic vesicles. Having demonstrated this spatial configuration for endogenous and recombinant Bassoon molecules, our study paves the way towards molecular-scale resolution analysis of other prominent proteins of the presynaptic release machinery.

In the early stages of development, most excitatory synapses are formed directly on dendritic shafts. As neurons mature, these sites gradually shift from the shaft to dendritic spines. In fully developed excitatory neurons, the majority of glutamatergic postsynaptic sites containing the postsynaptic density (PSD) molecules reside on dendritic spines. However, some glutamatergic synapses remain as shaft synapses, yet their characteristics have remained unexplored. Here, we show that the molecular composition of the shaft PSDs closely resembles that of spine PSDs. Key components such as AMPARs, NMDARs, CaV1.2 channels, and F-actin interacting proteins, SynGAP, as well as cortactin, are present in comparable amounts in both synapse types. The major distinction between shaft and spine PSDs lies in the lower abundance of the scaffold proteins Shanks and Homer in shaft PSDs. Shaft synapses are not merely passive structures but actively participate in synaptic transmission. Their structure and function are modulated by changes in neuronal activity. Long-term live imaging combined with a cLTP protocol revealed that shaft PSDs were potentiated but rarely underwent a transition to spine synapses. In contrast, during LTD, shaft PSDs were eliminated more frequently than their spine counterparts. Together, these findings highlight excitatory shaft synapses as a distinct, and notably less stable, synapse type.

Vesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain neurons in mammalian brain release multiple transmitters, the prevalence, physiology of such pluralism and if the release occurs from same or distinct vesicle pools is not clear. Using quantitative single vesicle imaging, we show that a small population of neuronal SVs indeed contain different VTs to accomplish corelease. Surprisingly, this population is highly diverse (27 types), expressing distinct dual transporters suggesting corelease of various combinations of neurotransmitters. Using glutamatergic vesicles as an example, we demonstrate that transporter colocalization not only determine the transmitter type but also influences the SV content and synaptic quantal size. Thus, presence of diverse transporters on the same vesicle is bona-fide and, depending on the VT types, this may act as one mechanism to regulate neurotransmitter type, content and release in space and time.

Connectomics is generating an ever-increasing deluge of data, which challenges us to develop new methods for analyzing and extracting insights from these data. We introduce here a powerful method for analyzing three-dimensional reconstruction from serial section electron microscopy (3DEM) to measure synaptic information storage capacity (SISC) and apply it to data following in vivo long-term potentiation (LTP). Connectomic researchers have focused on the pattern of connectivity between neurons. The strengths of synapses have also been studied by quantifying the sizes of synapses. Importantly, synapses from the same axon onto the same dendrite have a common history of coactivation, making them a candidate for measuring the precision of synaptic plasticity based on the similarity of their dimensions. Quantifying precision is fundamental to understanding information storage and retrieval in neural circuits. We quantify this precision with Shannon information theory, which is a more reliable estimate than prior analyses based on signal detection theory because there is no overlap between states, and outliers do not artificially bias the outcome. Spine head volumes are well correlated with other measures of synaptic weight, thus SISC can be determined by identifying the non-overlapping clusters of dendritic spine head volumes to determine the number of distinguishable synaptic weights. SISC analysis of spine head volumes in the stratum radiatum of hippocampal area CA1 revealed 24 distinguishable states (4.1 bits). In contrast, spine head volumes in the middle molecular layer of control dentate gyrus occupied only 5 distinguishable states (2 bits). Thus, synapses in different hippocampal regions had significantly different SISCs. Moreover, these were not fixed properties but increased by 30 min following induction of LTP in the dentate gyrus to occupy 10 distinguishable states (3 bits), and this increase lasted for at least 2 hours. We also observed a broader and nearly uniform distribution of spine head volumes across the increased number of states, suggesting the distribution evolved towards the theoretical upper bound of SISC following LTP. For dentate granule cells these findings show that the spine size range was broadened by the interplay among synaptic plasticity mechanisms. SISC provides a new analytical measure to probe these mechanisms in normal and diseased brains.

Cultures of dissociated hippocampal neurons display a stereotypical development of network activity patterns within the first three weeks of maturation. During this process, network connections develop and the associated spiking patterns range from increasing levels of activity in the first two weeks to regular bursting activity in the third week of maturation. Characterization of network structure is important to examine the mechanisms underlying the emergent functional organization of neural circuits. To accomplish this, confocal microscopy techniques have been used and several automated synapse quantification algorithms based on (co)localization of synaptic structures have been proposed recently. However, these approaches suffer from the arbitrary nature of intensity thresholding and the lack of correction for random-chance colocalization. To address this problem, we developed and validated an automated synapse quantification algorithm that requires minimal operator intervention. Next, we applied our approach to quantify excitatory and inhibitory synaptogenesis using confocal images of dissociated hippocampal neuronal cultures captured at 5, 8, 14 and 20 days in vitro, the time period associated with the development of distinct neuronal activity patterns. As expected, we found that synaptic density increased with maturation, coinciding with increasing spiking activity in the network. Interestingly, the third week of the maturation exhibited a reduction in excitatory synaptic density suggestive of synaptic pruning that coincided with the emergence of regular bursting activity in the network.

Relating dendritic spine morphology to synaptic organization in brain tissue is essential for understanding excitatory synaptic transmission and plasticity. Single-molecule localization microscopy (SMLM) offers the spatial precision needed to study the synaptic protein distribution at the nanoscale. However, the widefield setup required for SMLM produces diffraction-limited images with poor contrast and resolution in thick brain slices (> 30 m), making accurate segmentation of dendritic spines challenging. To overcome this challenge, we developed an automated 3D segmentation approach tailored to this condition by combining two existing machine-learning models. We integrated this strategy with SMLM-based localization of synaptic proteins to map post-synaptic protein PSD-95 within spines at nanoscale resolution. This framework, named ISEPLA (Integrated Spine Extraction and Protein Localization Analysis), revealed a hierarchical organization of synaptic proteins: spines contain multiple nanomodules, each composed of smaller nanodomains. Larger spines contain more nanomodules, and larger nanomodules comprise more nanodomains. Therefore, our method enables precise morphological and molecular analysis under physiologically relevant imaging conditions, providing new insights into the synaptic organization in spines.

Moderate noise exposure is a common experience, yet its impact on central auditory synapses remains poorly understood. We study this issue at the first synapses in the central auditory pathway formed by auditory nerve afferents onto bushy cells in the cochlear nucleus, called endbulbs of Held. Non-traumatic noise exposure alters endbulb properties, decreasing the probability of vesicle release and enlarging the pool of releasable vesicles as assessed using electrophysiological methods and immunolabelling. These changes appear homeostatic, to maintain synaptic efficacy during periods of high activity. To identify structural changes underlying the larger vesicle pool, we used serial blockface electron microscopy of endbulbs from control and noise-exposed mice to quantitatively assess synaptic morphology. We observed no differences in the juxtapositional area between endbulbs and bushy cells, nor in the number or density of active zones and postsynaptic densities. Images of endbulb terminals were significantly darker after noise exposure, indicating an increase in the density of synaptic vesicles. These results suggest that moderate noise exposure induces an activity-dependent increase in presynaptic vesicle numbers, consistent with the observed physiological changes in neurotransmitter release. This work sets the stage for high-resolution studies to quantify docked and reserve vesicles. Significance statementNoise exposure is a fact of everyday life, and it is important to understand how noise affects function in the auditory pathway in the brain to understand the full consequences of noise exposure. Electrophysiological experiments indicate that noise triggers a homeostatic increase in the releasable pool of vesicles at auditory nerve synapses. We examined the cellular basis for this change using serial blockface electron microscopy of auditory nerve synapses with and without noise exposure. We reconstructed a number of bushy cells and their presynaptic auditory nerve terminals. After noise exposure, there was no significant increase in the area of synaptic contact or the number or density of synaptic release sites. There was an increase in the number of vesicles near release sites, which may account for the physiological changes. These results emphasize the importance of detailed anatomical studies to study the effects of noise exposure and thus determine the best mechanistic approach for therapies and treatments of noise-induced hearing loss.

Spontaneous synaptic transmission has been established as essential for the maintenance of synaptic weights during action potential-induced transmission. However, spontaneous transmission also changes during synaptic plasticity and has been shown to, in part, mediate changes in synaptic weights. Despite decades of research, a coherent framework for understanding the complex molecular processes that support presynaptic spontaneous transmission during maintenance and plasticity has remained elusive. We show here that presynapses modulate spontaneous transmission frequency during the early time-course of plasticity following entropic force theory. We use live primary hippocampal cultures as a model system and induce plasticity using an established Long-Term Potentiation (LTP) protocol. We then use a combination of electron microscopy, fluorescence microscopy, and computational modeling to show how spontaneous release frequency dynamically changes during early plasticity. We use our entropic force theory to show how the dynamically changing synaptic vesicle pool structure mediates spontaneous release changes. Lastly, we show how these changes are altered in the presence of P301L tau leading to degeneration. The results from this study provide new insights that not only help understand normal synaptic function but also aid in understanding neurodegeneration.

Hippocampal neurons in tissue-culture were exposed to the trivalent cation lanthanum for short periods (15 to 30 minutes) and prepared for electron microscopy (EM), to evaluate the stimulatory effects of this cation on synaptic ultrastructure. Not only were characteristic ultrastructural changes of exaggerated synaptic vesicle turnover seen within the presynapses of these cultures - - including synaptic vesicle depletion and proliferation of vesicle-recycling structures - - but the overall architecture of a large proportion of the synapses in the cultures was dramatically altered, due to large postsynaptic bulges or herniations into the presynapses. Moreover, in most cases these postsynaptic herniations or protrusions produced by lanthanum were seen by EM to distort or break or perforate the so-called postsynaptic densities (PSDs) that harbor receptors and recognition-molecules essential for synaptic function. These dramatic EM-observations lead us to postulate that such PSD-breakages or perforations could very possibly create essential substrates or tags for synaptic growth, simply by creating fragmented free-edges around the PSDs, into which new receptors and recognition-molecules could be recruited more easily, and thus they could represent the physical substrate for the important synaptic-growth process known as "long-term potentiation" (LTP). All of this was created simply in hippocampal tissue-cultures, and simply by pushing synaptic vesicle recycling way beyond its normal limits with the trivalent cation lanthanum; but we argue in this report that such fundamental changes in synaptic architecture - - given that they can occur at all - - could also occur at the extremes of normal neuronal activity, which are presumed to lead to learning and memory.

The glutamate delta 1 receptor (GluD1) represents a unique subtype of ionotropic glutamate receptors that is strongly expressed in the mammalian striatum. Disruptions of the GRID1 gene, which encodes GluD1, have been associated with neuropsychiatric disorders, including schizophrenia and autism spectrum disorder; however, the role of GluD1 in the brain remains poorly understood. Previous studies in mice have demonstrated that the knockout of striatal GluD1 led to fear-conditioning deficits and depressive-like behaviors. Furthermore, these mice exhibited reduced excitatory input to the striatum due to a loss of thalamostriatal innervation, whereas corticostriatal innervation was unaffected. In this study, we examined whether changes in synapse morphology contribute to the observed functional deficits. We found that the ablation of GluD1 does not affect synaptic targeting patterns of corticostriatal and thalamostriatal terminals, using transmission electron microscopy. We further utilized three-dimensional reconstruction to obtain quantitative data on synapse ultrastructure and found no significant changes in corticostriatal and thalamostriatal synaptic components, including the presynaptic terminal volume, postsynaptic density area and morphology, and postsynaptic dendritic spine volume. These findings support a model in which GluD1 regulates input-specific circuit organization and synaptic connectivity rather than the structural morphology of individual synapses.

Neuromuscular transmission, from spontaneous release to facilitation and depression was accurately reproduced by a mechanistic kinetic model of sequential maturation transitions in the molecular fusion complex. The model incorporates three predictions. First, sequential calcium-dependent forward transitions take vesicles from docked to pre-primed to primed states, followed by fusion. Second, pre-priming and priming are reversible. Third, fusion and recycling are unidirectional. The model was fed with experimental data from previous studies while the backward ({beta}) and recycling ({rho}) rate constant values were fitted. Classical experiments were successfully reproduced when every forward () rate constant had the same value, and both backward rate constants were 50-100 times larger. Such disproportion originated an abruptly decreasing gradient of resting vesicles from docked to primed states. Simulations also predict that: i. Spontaneous release reflects primed to fusion spontaneous transitions. ii. Calcium elevations synchronize the series of forward transitions that lead to fusion. iii Facilitation reflects a transient increase of priming following calcium-dependent transitions. iv. Backward transitions and recycling restore the resting state. v. Depression reflects backward transitions and slow recycling after intense release. Such finely-tuned kinetics offers a mechanism for collective non-linear transitional adaptations of a homogeneous vesicle pool to an ever-changing pattern of electrical activity.

The dopamine transporter (DAT) mediated DA reuptake is a major molecular mechanism for termination of dopaminergic signaling in the brain. Psychoactive substances such as cocaine act by inhibition of plasma membrane DAT function as well as by altering its expression. The precise manner and mechanism by which cocaine regulates DAT trafficking, especially at neuronal processes, are poorly understood. We have now engineered a novel pH-sensitive reporter for DAT by conjugating pHluorin to the second exofacial loop of human DAT. We show that DAT-pHluorin can be used to study DAT localization and its dynamic trafficking at neuronal processes. Using DAT-pHluorin we show that unlike neuronal soma and dendrites, which contain majority of the DATs in weakly acidic intracellular compartments, axonal DATs at both shafts and boutons are primarily (75%) localized to the plasma membrane, while varicosities contain abundant intracellular DAT within acidic intracellular structures. Using this novel reporter, we show, for the first time, that cocaine exposure leads to a brief DAT internalization followed by membrane reinsertion that lasts for days. We further show that the cocaine-induced DAT trafficking is sensitive to the activities of Synaptojanin1 phosphatase. Thus, our study using the newly engineered DAT optical reporter reveals the previously unknown dynamics and molecular regulation for cocaine-regulated DAT trafficking in neuronal processes.

Maintaining the balance between neuronal excitation and inhibition is essential for proper function of the central nervous system, with inhibitory synaptic transmission playing an important role. Although inhibitory transmission has higher kinetic demands compared to excitatory transmission, its properties are poorly understood. In particular, the dynamics and exocytosis of single inhibitory vesicles have not been investigated, due largely to both technical and practical limitations. Using a combination of quantum dots (QDs) conjugated to antibodies against the luminal domain of the vesicular GABA transporter (VGAT) to selectively label GABAergic (i.e., inhibitory) vesicles together with dual-focus imaging optics, we tracked the real-time three-dimensional position of single inhibitory vesicles up to the moment of exocytosis (i.e., fusion). Using three-dimensional trajectories, we found that inhibitory synaptic vesicles traveled a short distance prior to fusion and had a shorter time to fusion compared to synaptotagmin-1 (Syt1)-labeled vesicles, which were mostly from excitatory neurons. Moreover, our analysis revealed a close correlation between the release probability of inhibitory vesicles and both the proximity to their fusion site and the total travel length. Finally, we found that inhibitory vesicles have a higher prevalence of kiss-and-run fusion compared than Syt1-labeled vesicles. These results indicate that inhibitory synaptic vesicles have a unique set of dynamics and fusion properties to support rapid synaptic inhibition, thereby maintaining a tightly regulated balance between excitation and inhibition in the central nervous system. SignificanceDespite playing an important role in maintaining brain function, the dynamics of inhibitory synaptic vesicles are poorly understood. Here, we tracked the three-dimensional position of single inhibitory vesicles up to the moment of exocytosis in real time by loading single inhibitory vesicle with QDs-conjugated to antibodies against the luminal domain of the vesicular GABA transporter (VGAT). We found that inhibitory synaptic vesicles have a smaller total travel length before fusion, a shorter fusion time, and a higher prevalence of kiss-and-run than synaptotagmin-1-lableled vesicles. Our findings provide the first evidence that inhibitory vesicles have a unique set of dynamics and exocytosis properties to support rapid inhibitory synaptic transmission.