Individual Synaptic Vesicles Research Articles

Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy.

Understanding the organization of biomolecules into complexes and their dynamics is crucial for comprehending cellular functions and dysfunctions, particularly in neuronal networks connected by synapses. In the last two decades, various powerful super-resolution (SR) microscopy techniques have been developed that produced stunning images of synapses and their molecular organization. However, current SR microscopy methods do not permit multicolor fluorescence imaging with 20 to 30nm spatial resolution. We developed a method that enables 4-color fluorescence imaging of synaptic proteins in neurons with 20 to 30nm lateral resolution. We used post-expansion immunolabeling of eightfold expanded hippocampal neurons in combination with Airyscan and structured illumination microscopy (SIM). We demonstrate that post-expansion immunolabeling of approximately eightfold expanded hippocampal neurons enables efficient labeling of synaptic proteins in crowded compartments with minimal linkage error and enables in combination with Airyscan and SIM four-color three-dimensional fluorescence imaging with 20 to 30nm lateral resolution. Using immunolabeling of Synaptobrevin 2 as an efficient marker of the vesicle pool allowed us to identify individual synaptic vesicles colocalized with Rab3-interacting molecule 1 and 2 (RIM1/2), a marker of pre-synaptic fusion sites. Our optimized expansion microscopy approach improves the visualization and location of pre- and post-synaptic proteins and can thus provide invaluable insights into the spatial organization of proteins at synapses.

Co-packaging of opposing neurotransmitters in individual synaptic vesicles in the central nervous system.

Many mammalian neurons release multiple neurotransmitters to activate diverse classes of postsynaptic ionotropic receptors. Entopeduncular nucleus somatostatin (EP Sst+) projection neurons to the lateral habenula (LHb) release both glutamate and GABA, but it is unclear whether these are packaged into the same or segregated pools of synaptic vesicles. Here, we describe a method combining electrophysiology, spatially patterned optogenetics, and computational modeling designed to analyze the mechanism of glutamate/GABA co-release in mouse brain. We find that the properties of postsynaptic currents elicited in LHb neurons by optogenetically activating EP Sst+ terminals are only consistent with co-packaging of glutamate/GABA into individual vesicles. Furthermore, presynaptic neuromodulators that weaken EP Sst+ to LHb synapses maintain the co-packaging of glutamate/GABA while reducing vesicular release probability. Our approach is applicable to the study of multi-transmitter neurons throughout the brain, and our results constrain the mechanisms of neuromodulation and synaptic integration in LHb.

Direct imaging of rapid tethering of synaptic vesicles accompanying exocytosis at a fast central synapse

A high rate of synaptic vesicle (SV) release is required at cerebellar mossy fiber terminals for rapid information processing. As the number of release sites is limited, fast SV reloading is necessary to achieve sustained release. However, rapid reloading has not been observed directly. Here, we visualize SV movements near presynaptic membrane using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, SVs appeared in the TIRF-field and became tethered to the presynaptic membrane with unexpectedly rapid time course, almost as fast as SVs disappeared due to release. However, such stimulus-induced tethering was abolished by inhibiting exocytosis, suggesting that the tethering is tightly coupled to preceding exocytosis. The newly tethered vesicles became fusion competent not immediately but only 300 ms to 400 ms after tethering. Together with model simulations, we propose that rapid tethering leads to an immediate filling of vacated spaces and release sites within <100 nm of the active zone by SVs, which serve as precursors of readily releasable vesicles, thereby shortening delays during sustained activity.

Dedicated Setup for the Photoconversion of Fluorescent Dyes for Functional Electron Microscopy.

Here, we describe a cost-effective setup for targeted photoconversion of fluorescent signals into electron dense ones. This approach has offered invaluable insights in the morphology and function of fine neuronal structures. The technique relies on the localized oxidation of diaminobenzidine (DAB) mediated by excited fluorophores. This paper includes a detailed description of how to build a simple photoconversion setup that can increase reliability and throughput of this well-established technique. The system described here, is particularly well-suited for thick neuronal tissue, where light penetration and oxygen diffusion may be limiting DAB oxidation. To demonstrate the system, we use Correlative Light and Electron Microscopy (CLEM) to visualize functionally-labeled individual synaptic vesicles released onto an identified layer 5 neuron in an acute cortical slice. The setup significantly simplifies the photoconversion workflow, increasing the depth of photoillumination, improving the targeting of the region of interest and reducing the time required to process each individual sample. We have tested this setup extensively for the photoconversion of FM 1-43FX and Lucifer Yellow both excited at 473 nm. In principle, the system can be adapted to any dye or nanoparticle able to oxidize DAB when excited by a specific wavelength of light.

Individual synaptic vesicles mediate stimulated exocytosis from cochlear inner hair cells

Spontaneous excitatory postsynaptic currents (sEPSCs) measured from the first synapse in the mammalian auditory pathway reach a large mean amplitude with a high level of variance (CV between 0.3 and 1). This has led some to propose that each inner hair cell (IHC) ribbon-type active zone (AZ), on average, releases ∼6 synaptic vesicles (SVs) per sEPSC in a coordinated manner. If true, then the predicted change in membrane capacitance (Cm) for such multivesicular fusion events would equate to ∼300 attofarads (aF). Here, we performed cell-attached Cm measurements to directly examine the size of fusion events at the basolateral membrane of IHCs where the AZs are located. The frequency of events depended on the membrane potential and the expression of Cav1.3, the principal Ca2+-channel type of IHCs. Fusion events averaged 40 aF, which equates to a normal-sized SV with an estimated diameter of 37 nm. The calculated SV volumes showed a high degree of variance (CV > 0.6). These results indicate that SVs fused individually with the plasma membrane during spontaneous and evoked release and SV volume may contribute more variability in EPSC amplitude than previously assumed.

Kinetics of transmitter release at the calyx of Held synapse.

Synaptic contacts mediate information transfer between neurons. The calyx of Held, a large synapse in the mammalian auditory brainstem, has been used as a model system for the mechanism of transmitter release from the presynaptic terminal for the last 20 years. By applying simultaneous recordings from pre- and postsynaptic compartments, the calcium-dependence of the kinetics of transmitter release has been quantified. A single pool of readily releasable vesicles cannot explain the time course of release during repetitive activity. Rather, multiple pools of vesicles have to be postulated that are replenished with distinct kinetics after depletion. The physical identity of vesicle replenishment has been unknown. Recently, it has become possible to apply total internal reflection fluorescent microscopy to the calyx terminal. This technique allowed the visualization of the dynamics of individual synaptic vesicles. Rather than recruitment of vesicles to the transmitter release sites, priming of tethered vesicles in the total internal reflection fluorescent field limited the number of readily releasable vesicles during sustained activity.

VGLUT2 Trafficking Is Differentially Regulated by Adaptor Proteins AP-1 and AP-3.

Release of the major excitatory neurotransmitter glutamate by synaptic vesicle exocytosis depends on glutamate loading into synaptic vesicles by vesicular glutamate transporters (VGLUTs). The two principal isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in adult brain that broadly distinguishes cortical (VGLUT1) and subcortical (VGLUT2) systems, and correlates with distinct physiological properties in synapses expressing these isoforms. Differential trafficking of VGLUT1 and 2 has been suggested to underlie their functional diversity. Increasing evidence suggests individual synaptic vesicle proteins use specific sorting signals to engage specialized biochemical mechanisms to regulate their recycling. We observed that VGLUT2 recycles differently in response to high frequency stimulation than VGLUT1. Here we further explore the trafficking of VGLUT2 using a pHluorin-based reporter, VGLUT2-pH. VGLUT2-pH exhibits slower rates of both exocytosis and endocytosis than VGLUT1-pH. VGLUT2-pH recycling is slower than VGLUT1-pH in both hippocampal neurons, which endogenously express mostly VGLUT1, and thalamic neurons, which endogenously express mostly VGLUT2, indicating that protein identity, not synaptic vesicle membrane or neuronal cell type, controls sorting. We characterize sorting signals in the C-terminal dileucine-like motif, which plays a crucial role in VGLUT2 trafficking. Disruption of this motif abolishes synaptic targeting of VGLUT2 and essentially eliminates endocytosis of the transporter. Mutational and biochemical analysis demonstrates that clathrin adaptor proteins (APs) interact with VGLUT2 at the dileucine-like motif. VGLUT2 interacts with AP-2, a well-studied adaptor protein for clathrin mediated endocytosis. In addition, VGLUT2 also interacts with the alternate adaptors, AP-1 and AP-3. VGLUT2 relies on distinct recycling mechanisms from VGLUT1. Abrogation of these differences by pharmacological and molecular inhibition reveals that these mechanisms are dependent on the adaptor proteins AP-1 and AP-3. Further, shRNA-mediated knockdown reveals differential roles for AP-1 and AP-3 in VGLUT2 recycling.

Activity-Dependence of Synaptic Vesicle Dynamics.

The proper function of synapses relies on efficient recycling of synaptic vesicles. The small size of synaptic boutons has hampered efforts to define the dynamical states of vesicles during recycling. Moreover, whether vesicle motion during recycling is regulated by neural activity remains largely unknown. We combined nanoscale-resolution tracking of individual synaptic vesicles in cultured hippocampal neurons from rats of both sexes with advanced motion analyses to demonstrate that the majority of recently endocytosed vesicles undergo sequences of transient dynamical states including epochs of directed, diffusional, and stalled motion. We observed that vesicle motion is modulated in an activity-dependent manner, with dynamical changes apparent in ∼20% of observed boutons. Within this subpopulation of boutons, 35% of observed vesicles exhibited acceleration and 65% exhibited deceleration, accompanied by corresponding changes in directed motion. Individual vesicles observed in the remaining ∼80% of boutons did not exhibit apparent dynamical changes in response to stimulation. More quantitative transient motion analyses revealed that the overall reduction of vesicle mobility, and specifically of the directed motion component, is the predominant activity-evoked change across the entire bouton population. Activity-dependent modulation of vesicle mobility may represent an important mechanism controlling vesicle availability and neurotransmitter release.SIGNIFICANCE STATEMENT Mechanisms governing synaptic vesicle dynamics during recycling remain poorly understood. Using nanoscale resolution tracking of individual synaptic vesicles in hippocampal synapses and advanced motion analysis tools we demonstrate that synaptic vesicles undergo complex sets of dynamical states that include epochs of directed, diffusive, and stalled motion. Most importantly, our analyses revealed that vesicle motion is modulated in an activity-dependent manner apparent as the reduction in overall vesicle mobility in response to stimulation. These results define the vesicle dynamical states during recycling and reveal their activity-dependent modulation. Our study thus provides fundamental new insights into the principles governing synaptic function.

Actin/Myosin-V- and Activity-Dependent Inter-synaptic Vesicle Exchange in Central Neurons

Vesicle sharing between synaptic boutons is an important component of the recycling process that synapses employ to maintain vesicle pools. However, the mechanisms supporting and regulating vesicle transport during the inter-synaptic exchange remain poorly understood. Using nanometer-resolution tracking of individual synaptic vesicles and advanced computational algorithms, we find that long-distance axonal transport of synaptic vesicles between hippocampal boutons is partially mediated by the actin network, with myosin V as the primary actin-dependent motor that drives this vesicle transport. Furthermore, we find that vesicle exit from the synapse to the axon and long-distance vesicle transport are both rapidly and dynamically regulated by activity. We corroborated these findings with two complementary modeling approaches of vesicle exit, which closely reproduced experimental observations. These findings uncover the roles of actin and myosin V in supporting the inter-synaptic vesicle exchange and reveal that this process is dynamically modulated in an activity-dependent manner.

Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction.

The quantitative relationship between presynaptic calcium influx and transmitter release critically depends on the spatial coupling of presynaptic calcium channels to synaptic vesicles. When there is a close association between calcium channels and synaptic vesicles, the flux through a single open calcium channel may be sufficient to trigger transmitter release. With increasing spatial distance, however, a larger number of open calcium channels might be required to contribute sufficient calcium ions to trigger vesicle fusion. Here we used a combination of pharmacological calcium channel block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo reaction-diffusion simulations in the adult frog neuromuscular junction, to show that release of individual synaptic vesicles is predominately triggered by calcium ions entering the nerve terminal through the nearest open calcium channel. Furthermore, calcium ion flux through this channel has a low probability of triggering synaptic vesicle fusion (∼6%), even when multiple channels open in a single active zone. These mechanisms work to control the rare triggering of vesicle fusion in the frog neuromuscular junction from each of the tens of thousands of individual release sites at this large model synapse.

Noncanonical Neurotransmission at the Neuromuscular Junction

Large-scale proteomic analyses of heterogeneous CNS preparations, such as isolated CNS synaptic vesicles, have provided a wealth of information related to synapse specific proteins, and synaptic signaling pathways. However, these studies under-represent, or lack entirely, many vesicle and neurotransmitter signaling pathways. In particular the neuromodulatory adrenergic and cholinergic pathways are often under-represented. In order to better understand the molecules and signaling pathways involved at cholinergic synapses, we have focused on the classic preparation of the electric lobe and electric organ of Torpedo californica. The abundant macromolecular material available from this preparation has contributed greatly to our understanding of the synthesis, storage, and release of the neurotransmitter acetylcholine and its postsynaptic activation of the nicotinic acetylcholine receptor. Many lines of evidence support the idea that in addition to acetylcholine, additional neurotransmitters and/or neuromodulators are also released from cholinergic synapses. Utilizing a single-vesicle imaging approach, we have found that individual synaptic vesicles from the electric ray possess neurotransmitter transporters for glutamate, ATP, and acetylcholine. In addition to those transporters, cholinergic synaptic vesicles from the electric ray also possess an orphaned transporter from the bile acid transport family, SLC10A4. We are now expanding our research to identify the molecules transported by SLC10A4 using multiple biochemical approaches and a mouse knockout model.

Readily releasable vesicles recycle at the active zone of hippocampal synapses.

During the synaptic vesicle cycle, synaptic vesicles fuse with the plasma membrane and recycle for repeated exo/endocytic events. By using activity-dependent N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide dye uptake combined with fast (<1 s) microwave-assisted fixation followed by photoconversion and ultrastructural 3D analysis, we tracked endocytic vesicles over time, "frame by frame." The first retrieved synaptic vesicles appeared 4 s after stimulation, and these endocytic vesicles were located just above the active zone. Second, the retrieved vesicles did not show any sign of a protein coat, and coated pits were not detected. Between 10 and 30 s, large labeled vesicles appeared that had up to 5 times the size of an individual synaptic vesicle. Starting at around 20 s, these large labeled vesicles decreased in number in favor of labeled synaptic vesicles, and after 30 s, labeled vesicles redocked at the active zone. The data suggest that readily releasable vesicles are retrieved as noncoated vesicles at the active zone.

Individual synaptic vesicles from the electroplaque ofTorpedo californica, a classic cholinergic synapse, also contain transporters for glutamate and ATP

The type of neurotransmitter secreted by a neuron is a product of the vesicular transporters present on its synaptic vesicle membranes and the available transmitters in the local cytosolic environment where the synaptic vesicles reside. Synaptic vesicles isolated from electroplaques of the marine ray, Torpedo californica, have served as model vesicles for cholinergic neurotransmission. Many lines of evidence support the idea that in addition to acetylcholine, additional neurotransmitters and/or neuromodulators are also released from cholinergic synapses. We identified the types of vesicular neurotransmitter transporters present at the electroplaque using immunoblot and immunofluoresence techniques with antibodies against the vesicle acetylcholine transporter (VAChT), the vesicular glutamate transporters (VGLUT1, 2, and 3), and the vesicular nucleotide transporter (VNUT). We found that VAChT, VNUT, VGLUT 1 and 2, but not 3 were present by immunoblot, and confirmed that the antibodies were specific to proteins of the axons and terminals of the electroplaque. We used a single‐vesicle imaging technique to determine whether these neurotransmitter transporters were present on the same or different populations of synaptic vesicles. We found that greater than 85% of vesicles that labeled for VAChT colabeled with VGLUT1 or VGLUT2, and approximately 70% colabeled with VNUT. Based upon confidence intervals, at least 52% of cholinergic vesicles isolated are likely to contain all four transporters. The presence of multiple types of neurotransmitter transporters – and potentially neurotransmitters – in individual synaptic vesicles raises fundamental questions about the role of cotransmitter release and neurotransmitter synergy at cholinergic synapses.

Sorting of the Vesicular GABA Transporter to Functional Vesicle Pools by an Atypical Dileucine-like Motif

Increasing evidence indicates that individual synaptic vesicle proteins may use different signals, endocytic adaptors, and trafficking pathways for sorting to distinct pools of synaptic vesicles. Here, we report the identification of a unique amino acid motif in the vesicular GABA transporter (VGAT) that controls its synaptic localization and activity-dependent recycling. Mutational analysis of this atypical dileucine-like motif in rat VGAT indicates that the transporter recycles by interacting with the clathrin adaptor protein AP-2. However, mutation of a single acidic residue upstream of the dileucine-like motif leads to a shift to an AP-3-dependent trafficking pathway that preferentially targets the transporter to the readily releasable and recycling pool of vesicles. Real-time imaging with a VGAT-pHluorin fusion provides a useful approach to explore how unique sorting sequences target individual proteins to synaptic vesicles with distinct functional properties.

The real catecholamine content of secretory vesicles in the CNS revealed by electrochemical cytometry

Resolution of synaptic vesicle neurotransmitter content has mostly been limited to the study of stimulated release in cultured cell systems, and it has been controversial as to whether synaptic vesicle transmitter levels are saturated in vivo. We use electrochemical cytometry to count dopamine molecules in individual synaptic vesicles in populations directly sampled from brain tissue. Vesicles from the striatum yield an average of 33,000 dopamine molecules per vesicle, an amount considerably greater than typically measured during quantal release at cultured neurons. Vesicular content was markedly increased by L-DOPA or decreased by reserpine in a time-dependent manner in response to in vivo administration of drugs known to alter dopamine release. We investigated the effects of the psychostimulant amphetamine on vesicle content, finding that vesicular transmitter is rapidly depleted by 50% following in vivo administration, supporting the “weak base hypothesis” that amphetamine reduces synaptic vesicle transmitter and quantal size.

Properties of Ribbon and Non-Ribbon Release from Rod Photoreceptors Revealed by Visualizing Individual Synaptic Vesicles

Vesicle release from rod photoreceptors is regulated by Ca(2+) entry through L-type channels located near synaptic ribbons. We characterized sites and kinetics of vesicle release in salamander rods by using total internal reflection fluorescence microscopy to visualize fusion of individual synaptic vesicles. A small number of vesicles were loaded by brief incubation with FM1-43 or a dextran-conjugated, pH-sensitive form of rhodamine, pHrodo. Labeled organelles matched the diffraction-limited size of fluorescent microspheres and disappeared rapidly during stimulation. Consistent with fusion, depolarization-evoked vesicle disappearance paralleled electrophysiological release kinetics and was blocked by inhibiting Ca(2+) influx. Rods maintained tonic release at resting membrane potentials near those in darkness, causing depletion of membrane-associated vesicles unless Ca(2+) entry was inhibited. This depletion of release sites implies that sustained release may be rate limited by vesicle delivery. During depolarizing stimulation, newly appearing vesicles approached the membrane at ∼800 nm/s, where they paused for ∼60 ms before fusion. With fusion, vesicles advanced ∼18 nm closer to the membrane. Release events were concentrated near ribbons, but lengthy depolarization also triggered release from more distant non-ribbon sites. Consistent with greater contributions from non-ribbon sites during lengthier depolarization, damaging the ribbon by fluorophore-assisted laser inactivation (FALI) of Ribeye caused only weak inhibition of exocytotic capacitance increases evoked by 200-ms depolarizing test steps, whereas FALI more strongly inhibited capacitance increases evoked by 25 ms steps. Amplifying release by use of non-ribbon sites when rods are depolarized in darkness may improve detection of decrements in release when they hyperpolarize to light.

Synaptic Vesicles Isolated from the Electric Organ of Torpedo Californica Posses Multiple Classes of Neurotransmitter Transporters

The fusion of synaptic vesicles with the presynaptic membrane, and the subsequent release of small chemical neurotransmitters is the fundamental process by which neurons communicate at chemical synapses, and it has long been proposed that most neurons release a single type of small molecule neurotransmitter. The simplicity of one neuron, one neurotransmitter has come under intense scrutiny as examples of neurons that appear to co-release two or more neurotransmitters at single synapses (neurotransmitter synergy) have been identified. One such synapse is that of the electric organ of Torpedo californica; synaptic vesicles isolated from this single class of neurons appear to contain the neurotransmitter acetylcholine as well as the neurotransmitter ATP. We used immunofluorescence labeling in conjunction with single-molecule TIRF microscopy to observe whether one or more neurotransmitter transporters could be detected on single synaptic vesicles. We found that the vesicular acetylcholine transporter (VAChT) and several solute carrier proteins (SLC17A) co-localized to the same vesicles. The presence of multiple types of neurotransmitter transporters - and potentially neurotransmitters - in individual synaptic vesicles raises fundamental questions about chemical synaptic transmission at the electric organ of T. Californica. In addition, this approach can be applied to other synapses in order to address the prevalence of neurotransmitter synergy and co-release at chemical synapses.

Lasers et techniques épilatoires : vérités et nouveautés

Synaptic vesicle exo- and endocytosis are usually driven by neuronal activity but can also occur spontaneously. The identity and differences between vesicles supporting evoked and spontaneous neurotransmission remain highly debated. Here we combined nanometer-resolution imaging with a transient motion analysis approach to examine the dynamics of individual synaptic vesicles in hippocampal terminals under physiological conditions. We found that vesicles undergoing spontaneous and stimulated endocytosis differ in their dynamic behavior, particularly in the ability to engage in directed motion. Our data indicate that such motional differences depend on the myosin family of motor proteins, particularly myosin II. Analysis of synaptic transmission in the presence of myosin II inhibitor confirmed a specific role for myosin II in evoked, but not spontaneous, neurotransmission and also suggested a functional role of myosin II-mediated vesicle motion in supporting vesicle mobilization during neural activity.

Dynamics of Multiple Trafficking Behaviors of Individual Synaptic Vesicles Revealed by Quantum-Dot Based Presynaptic Probe

Although quantum dots (QDs) have provided invaluable information regarding the diffusive behaviors of postsynaptic receptors, their application in presynaptic terminals has been rather limited. In addition, the diffraction-limited nature of the presynaptic bouton has hampered detailed analyses of the behaviors of synaptic vesicles (SVs) at synapses. Here, we created a quantum-dot based presynaptic probe and characterized the dynamic behaviors of individual SVs. As previously reported, the SVs exhibited multiple exchanges between neighboring boutons. Actin disruption induced a dramatic decrease in the diffusive behaviors of SVs at synapses while microtubule disruption only reduced extrasynaptic mobility. Glycine-induced synaptic potentiation produced significant increases in synaptic and inter-boutonal trafficking of SVs, which were NMDA receptor- and actin-dependent while NMDA-induced synaptic depression decreased the mobility of the SVs at synapses. Together, our results show that sPH-AP-QD revealed previously unobserved trafficking properties of SVs around synapses, and the dynamic modulation of SV mobility could regulate presynaptic efficacy during synaptic activity.

A Synaptic Vesicle in Time

Tracking of individual synaptic vesicles reveals that kiss-and-run fusion is concentrated near the center of the synapse.