Vesicle Pool Size Research Articles

Direct evidence for ultrastructures of the α-synuclein-associated synaptic vesicle pool in presynaptic terminals

SNCA/PARK1 encodes α-synuclein, which is associated with familial Parkinson's disease. Despite its abundance in presynaptic terminals, the aggregation mechanism of α-synuclein and its relationship with Parkinson's disease have not yet been elucidated. Moreover, the ultrastructures of α-synuclein localization sites in neuronal presynaptic terminals remain unclear. Therefore, we herein generated transgenic mice expressing human α-synuclein tagged with mKate2 (hSNCA-mKate2 mice). These mice exhibited normal growth and fertility and had no motor dysfunction relative to their wild-type littermates, even at one year old. α-Synuclein-mKate2 accumulated in presynaptic terminals, particularly between Purkinje cells in the cerebellum and neurons in cerebellar nuclei. α-Synuclein-mKate2 was associated with the presynaptic marker, synaptophysin. In-resin CLEM and immunoelectron or electron microscopy revealed that α-synuclein-mKate2 localized on the surface of synaptic vesicles that were tightly arranged and assembled to form large synaptic pools in the cerebellum with negligible effects on the active zone. These results suggest that α-synuclein-associated ultrastructures in the presynaptic terminals of hSNCA-mKate2 mice reflect the structures of α-synuclein-assembled synaptic vesicle pools, and the size of vesicle pools increased. This transgenic mouse model will be a valuable tool for studying α-synuclein-associated synaptic vesicle pools.

Presynaptic quantal size enhancement counteracts post-tetanic release depression.

Repetitive synaptic stimulation can induce different forms of synaptic plasticity but may also limit the robustness of synaptic transmission by exhausting key resources. Little is known about how synaptic transmission is stabilized after high-frequency stimulation.In the present study, we observed that tetanic stimulation of the Drosophila neuromuscular junction (NMJ) decreases quantal content, release-ready vesicle pool size and synaptic vesicle density for minutes after stimulation. This was accompanied by a pronounced increase in quantal size. Interestingly, action potential-evoked synaptic transmission remained largely unchanged. EPSC amplitude fluctuation analysis confirmed the post-tetanic increase in quantal size and the decrease in quantal content, suggesting that the quantal size increase counteracts release depression to maintain evoked transmission. The magnitude of the post-tetanic quantal size increase and release depression correlated with stimulation frequency and duration, indicating activity-dependent stabilization of synaptic transmission. The post-tetanic quantal size increase persisted after genetic ablation of the glutamate receptor subunits GluRIIA or GluRIIB, and glutamate receptor calcium permeability, as well as blockade of postsynaptic calcium channels. By contrast, it was strongly attenuated by pharmacological or presynaptic genetic perturbation of the GTPase dynamin. Similar observations were made after inhibition of the H+-ATPase, suggesting that the quantal size increase is presynaptically driven. Additionally, dynamin and H+-ATPase perturbation resulted in a post-tetanic decrease in evoked amplitudes. Finally, we observed an increase in synaptic vesicle diameter after tetanic stimulation. Thus, a presynaptically-driven quantal size increase, likely mediated by larger synaptic vesicles, counterbalances post-tetanic release depression, thereby conferring robustness to synaptic transmission on the minute time scale. KEY POINTS: Many synapses transmit robustly aftersustained activity despite the limitation of key resources, such as release-ready synaptic vesicles. We report robust synaptic transmission after sustained high-frequency stimulation of the Drosophila neuromuscular junction despite a reduction in release-ready vesicle number. An increased postsynaptic response to individual vesicles, likely driven by an increase in vesicle size due to endocytosis defects, stabilizes synaptic efficacy for minutes after sustained activity. Our study provides novel insights into the mechanisms governing synaptic stability after sustained neural activity.

Glial Cx43 hemichannels and neuronal Panx1 hemichannels and P2X7 receptors orchestrate presynaptic homeostatic plasticity

The emerging role of glial cells in modulating neuronal excitability and synaptic strength is a growing field in neuroscience. In recent years, a pivotal role of gliotransmission in homeostatic presynaptic plasticity has been highlighted and glial-derived ATP arises as a key contributor. However, very little is known about the glial non-vesicular ATP-release pathway and how ATP participates in the modulation of synaptic strength. Here, we investigated the functional changes occurring in neurons upon chronic inactivity and the role of the purinergic signaling, connexin43 and pannexin1 hemichannels in this process. By using hippocampal dissociated cultures, we showed that blocking connexin43 and pannexin1 hemichannels decreases the amount of extracellular ATP. Moreover, Ca2+ imaging assays using Fluo-4/AM revealed that blocking connexin43, neuronal P2X7Rs and pannexin1 hemichannels decreases the amount of basal Ca2+ in neurons. A significant impairment in synaptic vesicle pool size was also evidenced under these conditions. Interestingly, rescue experiments where Panx1HCs are blocked showed that the compensatory adjustment of cytosolic Ca2+ was recovered after P2X7Rs activation, suggesting that Panx1 acts downstream P2X7Rs. These changes were accompanied by a modulation of neuronal permeability, as revealed by ethidium bromide uptake experiments. In particular, the permeability of neuronal P2X7Rs and pannexin1 hemichannels is increased upon 24 h of inactivity. Taken together, we have uncovered a role for connexin43-dependent ATP release and neuronal P2X7Rs and pannexin1 hemichannels in the adjustment of presynaptic strength by modulating neuronal permeability, the entrance of Ca2+ into neurons and the size of the recycling pool of synaptic vesicles.

GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the "Flash and Freeze-fracture" method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.

Synapse-specific burst coding sustained by local axonal translation

Neurotransmission in the brain is unreliable, suggesting that high-frequency spike bursts rather than individual spikes carry the neural code. For instance, cortical pyramidal neurons rely on bursts in memory formation. Protein synthesis is another key factor in long-term synaptic plasticity and learning but is widely considered unnecessary for synaptic transmission. Here, however, we show that burst neurotransmission at synapses between neocortical layer 5 pyramidal cells depends on axonal protein synthesis linked to presynaptic NMDA receptors and mTOR. We localized protein synthesis to axons with laser axotomy and puromycylation live imaging. We whole-cell recorded connected neurons to reveal how translation sustained readily releasable vesicle pool size and replenishment rate. We live imaged axons and found sparsely docked RNA granules, suggesting synapse-specific regulation. In agreement, translation boosted neurotransmission onto excitatory but not inhibitory basket or Martinotti cells. Local axonal mRNA translation is thus a hitherto unappreciated principle for sustaining burst coding at specific synapse types.

The synaptic basis of activity-dependent eye-specific competition.

Binocular vision requires proper developmental wiring of eye-specific inputs to the brain. In the thalamus, axons from the two eyes initially overlap in the dorsal lateral geniculate nucleus and undergo activity-dependent competition to segregate into target domains. Here, we combine eye-specific tract tracing with volumetric super-resolution imaging to measure the nanoscale molecular reorganization of developing retinogeniculate eye-specific synapses in the mouse brain. We show there are eye-specific differences in presynaptic vesicle pool size and vesicle association with the active zone at the earliest stages of retinogeniculate refinement but find no evidence of eye-specific differences in subsynaptic domain number, size, or transsynaptic alignment across development. Genetic disruption of spontaneous retinal activity decreases retinogeniculate synapse density, delays the emergence eye-specific differences in vesicle organization, and disrupts subsynaptic domain maturation. These results suggest that activity-dependent eye-specific presynaptic maturation underlies synaptic competition in the mammalian visual system.

Signal transmission in mature mammalian vestibular hair cells.

The maintenance of balance and gaze relies on the faithful and rapid signaling of head movements to the brain. In mammals, vestibular organs contain two types of sensory hair cells, type-I and type-II, which convert the head motion-induced movement of their hair bundles into a graded receptor potential that drives action potential activity in their afferent fibers. While signal transmission in both hair cell types involves Ca2+-dependent quantal release of glutamate at ribbon synapses, type-I cells appear to also exhibit a non-quantal mechanism that is believed to increase transmission speed. However, the reliance of mature type-I hair cells on non-quantal transmission remains unknown. Here we investigated synaptic transmission in mammalian utricular hair cells using patch-clamp recording of Ca2+ currents and changes in membrane capacitance (ΔCm). We found that mature type-II hair cells showed robust exocytosis with a high-order dependence on Ca2+ entry. By contrast, exocytosis was approximately 10 times smaller in type-I hair cells. Synaptic vesicle exocytosis was largely absent in mature vestibular hair cells of CaV1.3 (CaV1.3−/−) and otoferlin (Otof−/−) knockout mice. Even though Ca2+-dependent exocytosis was small in type-I hair cells of wild-type mice, or absent in CaV1.3−/− and Otof−/−mice, these cells were able to drive action potential activity in the postsynaptic calyces. This supports a functional role for non-quantal synaptic transmission in type-I cells. The large vesicle pools in type-II cells would facilitate sustained transmission of tonic or low-frequency signals. In type-I cells, the restricted vesicle pool size, together with a rapid non-quantal mechanism, could allow them to sustain high-frequency phasic signal transmission at their specialized large calyceal synapses.

Use of soft X-ray tomography to capture the dynamic biochemical nature of intact pancreatic beta cells

Characterizing relationships between cell structures and functions requires quantitative mesoscale mapping of intact cells showing subcellular rearrangements following stimulation; however, current approaches are limited in this regard. Thus, we investigated the application of soft X-ray tomography to generate three-dimensional reconstructions of whole pancreatic β-cells at different timepoints following glucose-stimulated insulin secretion. Reconstructions following stimulation show distinct insulin vesicle distribution patterns reflective of altered vesicle pool sizes as they mature and travel through the secretory pathway.

Synaptic Vesicle Pool Monitoring with Synapto-pHluorin.

Synaptic vesicle exocytosis can be monitored with genetically encoded pH sensors in an in vitro fluorescence microscopy setup. Here, we describe a workflow starting with preparation of a primary cell culture to eventually estimate synaptic vesicle pool sizes based on electrical current-evoked vesicle release, which is reported by the synaptobrevin 2-EGFP fusion protein synapto-pHluorin (spH) that is expressed inside the synaptic vesicle membrane. The readily releasable pool and the recycling pool of synaptic vesicles are released separately in response to electrical stimulation. As vesicle reacidification is blocked in this experimental design, every released vesicle is counted only once. This spH-based approach offers different information than styryl-dye (FM dyes)-based approaches because the total synaptic pool size is measured by an alkalinization step. This provides a normalization constant for quantifying and comparing the synaptic vesicle pool sizes. In addition to investigation of basic research questions, spH-reported vesicle release is valuable to determine presynaptic effects of, e.g., pharmacological drug treatments.

Postnatal maturation of glutamate clearance and release kinetics at the rat and mouse calyx of Held synapses.

Although calyx of Held synapses undergo dramatic changes around the hearing onset, previous in vivo studies suggest that the calyx synapses undergo further post-hearing maturation process. While developmental changes over the hearing onset have been extensively studied, this post-hearing maturation process remained relatively little investigated. Because of post-hearing maturation, previous results from studies around hearing onset and studies of post-hearing calyx synapses are somewhat inconsistent. Here, we characterized the post-hearing maturation of calyx synapses with regard to in vitro electrophysiological properties in rats and mice. We found that parameters for residual glutamate in the cleft during a train, EPSC kinetics, and vesicle pool size became close to a full mature level by P14, but they further matured until P16 in the rats. Consistently, the phasic and slow EPSCs evoked by action potential trains at P16 calyx synapses were not different from those at P18 or P25 under physiological extracellular [Ca2+ ]o (1.2mM). In contrast, the parameters for residual current and EPSC kinetics displayed drastic changes until P16 in mice, and slow EPSCs during the train further decreased between P16 and P18, suggesting that maturation of calyx synapses progresses at least up to P16 in rats and P18 in mice.

Distinct synaptic transfer functions in same-type photoreceptors.

Many sensory systems use ribbon-type synapses to transmit their signals to downstream circuits. The properties of this synaptic transfer fundamentally dictate which aspects in the original stimulus will be accentuated or suppressed, thereby partially defining the detection limits of the circuit. Accordingly, sensory neurons have evolved a wide variety of ribbon geometries and vesicle pool properties to best support their diverse functional requirements. However, the need for diverse synaptic functions does not only arise across neuron types, but also within. Here we show that UV-cones, a single type of photoreceptor of the larval zebrafish eye, exhibit striking differences in their synaptic ultrastructure and consequent calcium to glutamate transfer function depending on their location in the eye. We arrive at this conclusion by combining serial section electron microscopy and simultaneous 'dual-colour' two-photon imaging of calcium and glutamate signals from the same synapse in vivo. We further use the functional dataset to fit a cascade-like model of the ribbon synapse with different vesicle pool sizes, transfer rates, and other synaptic properties. Exploiting recent developments in simulation-based inference, we obtain full posterior estimates for the parameters and compare these across different retinal regions. The model enables us to extrapolate to new stimuli and to systematically investigate different response behaviours of various ribbon configurations. We also provide an interactive, easy-to-use version of this model as an online tool. Overall, we show that already on the synaptic level of single-neuron types there exist highly specialised mechanisms which are advantageous for the encoding of different visual features.

Loss of miR-183/96 Alters Synaptic Strength via Presynaptic and Postsynaptic Mechanisms at a Central Synapse.

A point mutation in miR-96 causes non-syndromic progressive peripheral hearing loss and alters structure and physiology of the central auditory system. To gain further insight into the functions of microRNAs (miRNAs) within the central auditory system, we investigated constitutive Mir-183/96dko mice of both sexes. In this mouse model, the genomically clustered miR-183 and miR-96 are constitutively deleted. It shows significantly and specifically reduced volumes of auditory hindbrain nuclei, because of decreases in cell number and soma size. Electrophysiological analysis of the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) demonstrated strongly altered synaptic transmission in young-adult mice. We observed an increase in quantal content and readily releasable vesicle pool size in the presynapse while the overall morphology of the calyx was unchanged. Detailed analysis of the active zones (AZs) revealed differences in its molecular composition and synaptic vesicle (SV) distribution. Postsynaptically, altered clustering and increased synaptic abundancy of the AMPA receptor subunit GluA1 was observed resulting in an increase in quantal amplitude. Together, these presynaptic and postsynaptic alterations led to a 2-fold increase of the evoked excitatory postsynaptic currents in MNTB neurons. None of these changes were observed in deaf Cldn14ko mice, confirming an on-site role of miR-183 and miR-96 in the auditory hindbrain. Our data suggest that the Mir-183/96 cluster plays a key role for proper synaptic transmission at the calyx of Held and for the development of the auditory hindbrain.SIGNIFICANCE STATEMENT The calyx of Held is the outstanding model system to study basic synaptic physiology. Yet, genetic factors driving its morphologic and functional maturation are largely unknown. Here, we identify the Mir-183/96 cluster as an important factor to regulate its synaptic strength. Presynaptically, Mir-183/96dko calyces show an increase in release-ready synaptic vesicles (SVs), quantal content and abundance of the proteins Bassoon and Piccolo. Postsynaptically, the quantal size as well as number and size of GluA1 puncta were increased. The two microRNAs (miRNAs) are thus attractive candidates for regulation of synaptic maturation and long-term adaptations to sound levels. Moreover, the different phenotypic outcomes of different types of mutations in the Mir-183 cluster corroborate the requirement of mutation-tailored therapies in patients with hearing loss.

Ultrastructural maturation of the endbulb of Held active zones comparing wild-type and otoferlin-deficient mice

SummaryEndbulbs of Held are located in the anteroventral cochlear nucleus and present the first central synapses of the auditory pathway. During development, endbulbs mature functionally to enable rapid and powerful synaptic transmission with high temporal precision. This process is accompanied by morphological changes of endbulb terminals. Loss of the hair cell-specific protein otoferlin (Otof) abolishes neurotransmission in the cochlea and results in the smaller endbulb of Held terminals. Thus, peripheral hearing impairment likely also leads to alterations in the morphological synaptic vesicle (SV) pool size at individual endbulb of Held active zones (AZs). Here, we investigated endbulb AZs in pre-hearing, young, and adult wild-type and Otof−/− mice. During maturation, SV numbers at endbulb AZs increased in wild-type mice but were found to be reduced in Otof−/− mice. The SV population at a distance of 0–15 nm was most strongly affected. Finally, overall SV diameters decreased in Otof−/− animals during maturation.

The BAD-BAX-Caspase-3 Cascade Modulates Synaptic Vesicle Pools via Autophagy.

The BAD-BAX-caspase-3 cascade is a canonical apoptosis pathway. Macroautophagy ("autophagy" hereinafter) is a process by which organelles and aggregated proteins are delivered to lysosomes for degradation. Here, we report a new function of the BAD-BAX-caspase-3 cascade and autophagy in the control of synaptic vesicle pools. We found that, in hippocampal neurons of male mice, the BAD-BAX-caspase-3 pathway regulates autophagy, which in turn limits the size of synaptic vesicle pools and influences the kinetics of activity-induced depletion and recovery of synaptic vesicle pools. Moreover, the caspase-autophagy pathway is engaged by fear conditioning to facilitate associative fear learning and memory. This work identifies a new mechanism for controlling synaptic vesicle pools, and a novel, nonapoptotic, presynaptic function of the BAD-BAX-caspase-3 cascade.SIGNIFICANCE STATEMENT Despite the importance of synaptic vesicles for neurons, little is known about how the size of synaptic vesicle pools is maintained under basal conditions and regulated by neural activity. This study identifies a new mechanism for the control of synaptic vesicle pools, and a new, nonapoptotic function of the BAD-BAX-caspase-3 pathway in presynaptic terminals. Additionally, it indicates that autophagy is not only a homeostatic mechanism to maintain the integrity of cells and tissues, but also a process engaged by neural activity to regulate synaptic vesicle pools for optimal synaptic responses, learning, and memory.

Visualizing subcellular rearrangements in intact β cells using soft x-ray tomography.

Characterizing relationships between cell structures and functions requires mesoscale mapping of intact cells showing subcellular rearrangements following stimulation; however, current approaches are limited in this regard. Here, we report a unique application of soft x-ray tomography to generate three-dimensional reconstructions of whole pancreatic β cells at different time points following glucose-stimulated insulin secretion. Reconstructions following stimulation showed distinct insulin vesicle distribution patterns reflective of altered vesicle pool sizes as they travel through the secretory pathway. Our results show that glucose stimulation caused rapid changes in biochemical composition and/or density of insulin packing, increased mitochondrial volume, and closer proximity of insulin vesicles to mitochondria. Costimulation with exendin-4 (a glucagon-like peptide-1 receptor agonist) prolonged these effects and increased insulin packaging efficiency and vesicle maturation. This study provides unique perspectives on the coordinated structural reorganization and interactions of organelles that dictate cell responses.

Measurements of Exocytosis by Capacitance Recordings and Calcium Uncaging in Mouse Adrenal Chromaffin Cells.

Fusion of vesicles with the plasma membrane and liberation of their contents is a multistep process involving several proteins. Correctly assigning the role of specific proteins and reactions in this cascade requires a measurement method with high temporal resolution. Patch-clamp recordings of cell membrane capacitance in combination with calcium measurements, calcium uncaging, and carbon-fiber amperometry allow for the accurate determination of vesicle pool sizes, their fusion kinetics, and their secreted oxidizable content. Here, we will describe this method in a model system for neurosecretion, the adrenal chromaffin cells, which secrete adrenaline.

The potential LXRβ agonist stigmasterol protects against hypoxia/reoxygenation injury by modulating mitophagy in primary hippocampal neurons

BackgroundNeuronal excitotoxicity induces a plethora of downstream signaling pathways, resulting in the calcium overload-induced excitotoxic cell death, a well-known phenomenon in cerebrovascular and neurodegenerative disorders. The naturally occurring phytosterol, stigmasterol (ST) is known for its potential role in cholesterol homeostasis and neuronal development. However, the ability of ST to protect against the induced excitotoxicity in hippocampal neurons has not been investigated yet. PurposeThe present study aimed to investigate whether ST could protect against hypoxia/reoxygenation (H/R)-induced excitotoxicity in hippocampal neurons. MethodsAfter H/R, neurons were initially subjected to trypan blue exclusion assay for the assessment of cell viability. Live staining using fluorescence dyes namely JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine iodide), DCFDA (2′,7′–dichlorofluorescein diacetate) and FM1-43 (N-(3-triethylammoniumpropyl)−4-(4-(dibutylamino)styryl) were used to measure MMP, ROS and synaptic vesicle pool size. Immunostaining was performed to analyze the expression levels of vesicular glutamate transporter 1 (VGLUT1), N-methyl-D-acetate receptor subunit 2B (GluN2B), LC3BII, p62, and PTEN induced protein kinase 1 (PINK1) in neuron after H/R. Western blotting was carried out to measure the protein expression of GluN2B. The molecular dynamics simulation was employed to elucidate the LXRβ agonistic conformation of ST. ResultPre-incubation of neuronal cultures with ST (20 μM) protected against excitotoxicity, and attenuated reactive oxygen species (ROS) generation, double-stranded DNA break, and mitochondrial membrane potential (MMP) loss. ST treatment also resulted in the downregulation of the expressions of VGLUT1 and GluN2B and the reduction of the size of recyclable synaptic vesicle (SV) pool. Like LXRβ agonist GW3695, ST suppressed the expression of GluN2B. Furthermore, ST induced mitophagy through upregulating the expressions of LC3BII, p62, and PINK1. The molecular simulation study showed that ST interacted with the ligand binding domain of liver X receptor β (LXRβ), a known binding receptor of ST, through multiple hydrogen bonding. ConclusionCollectively, these findings revealed that ST exhibited a promising neuroprotective effect by regulating both pre- and post-synaptic events following H/R, particularly, attenuation of GluN2B-mediated excitotoxicity and oxidative stress, and induction of mitophagy, and suggested that ST might be a therapeutic promise against ischemic stroke and its associated neurological disorders.

Disentangling the Roles of RIM and Munc13 in Synaptic Vesicle Localization and Neurotransmission

Efficient neurotransmitter release at the presynaptic terminal requires docking of synaptic vesicles to the active zone membrane and formation of fusion-competent synaptic vesicles near voltage-gated Ca2+ channels. Rab3-interacting molecule (RIM) is a critical active zone organizer, as it recruits Ca2+ channels and activates synaptic vesicle docking and priming via Munc13-1. However, our knowledge about Munc13-independent contributions of RIM to active zone functions is limited. To identify the functions that are solely mediated by RIM, we used genetic manipulations to control RIM and Munc13-1 activity in cultured hippocampal neurons from mice of either sex and compared synaptic ultrastructure and neurotransmission. We found that RIM modulates synaptic vesicle localization in the proximity of the active zone membrane independent of Munc13-1. In another step, both RIM and Munc13 mediate synaptic vesicle docking and priming. In addition, while the activity of both RIM and Munc13-1 is required for Ca2+-evoked release, RIM uniquely controls neurotransmitter release efficiency. However, activity-dependent augmentation of synaptic vesicle pool size relies exclusively on the action of Munc13s. Based on our results, we extend previous findings and propose a refined model in which RIM and Munc13-1 act in overlapping and independent stages of synaptic vesicle localization and release.SIGNIFICANCE STATEMENT The presynaptic active zone is composed of scaffolding proteins that functionally interact to localize synaptic vesicles to release sites, ensuring neurotransmission. Our current knowledge of the presynaptic active zone function relies on structure-function analysis, which has provided detailed information on the network of interactions and the impact of active zone proteins. Yet, the hierarchical, redundant, or independent cooperation of each active zone protein to synapse functions is not fully understood. Rab3-interacting molecule and Munc13 are the two key functionally interacting active zone proteins. Here, we dissected the distinct actions of Rab3-interacting molecule and Munc13-1 from both ultrastructural and physiological aspects. Our findings provide a more detailed view of how these two presynaptic proteins orchestrate their functions to achieve synaptic transmission.

Vesicle Pools of Memory at Mossy Fiber Synapses

In this issue of Neuron, Vandael etal. (2020) reveal that post-tetanic potentiation at dentate gyrus mossy fiber synapses is induced by natural activity patterns. This plasticity is mediated by an increase in readily releasable vesicle pool size and is extended in the absence of activity, forming a "pool engram."

Spike Activity Regulates Vesicle Filling at a Glutamatergic Synapse.

Synaptic vesicles need to be recycled and refilled rapidly to maintain high-frequency synaptic transmission. However, little is known about the control of neurotransmitter transport into synaptic vesicles, which determines the contents of synaptic vesicles and the strength of synaptic transmission. Here, we report that Na+ substantially accumulated in the calyx of Held terminals of juvenile mice of either sex during high-frequency spiking. The activity-induced elevation of cytosolic Na+ activated vesicular Na+/H+ exchanger, facilitated glutamate loading into synaptic vesicles, and increased quantal size of asynchronous released vesicles but did not affect the vesicle pool size or release probability. Consequently, presynaptic Na+ increased the EPSCs and was required to maintain the reliable high-frequency signal transmission from the presynaptic calyces to the postsynaptic medial nucleus of the trapezoid body (MNTB) neurons. Blocking Na+/H+ exchange activity decreased the postsynaptic current and caused failures in postsynaptic firing. Therefore, during high-frequency synaptic transmission, when large amounts of glutamate are released, Na+ accumulated in the terminals, activated vesicular Na+/H+ exchanger, and regulated glutamate loading as a function of the level of vesicle release.SIGNIFICANCE STATEMENT Auditory information is encoded by action potentials (APs) phase-locked to sound frequency at high rates. A large number of synaptic vesicles are released during high-frequency synaptic transmission; accordingly, synaptic vesicles need to be recycled and refilled rapidly. We have recently found that a Na+/H+ exchanger expressed on synaptic vesicles promotes vesicle filling with glutamate. Here, we showed that when a large number of synaptic vesicles are released during high-frequency synaptic transmission, Na+ accumulates in axon terminals and facilitates glutamate uptake into synaptic vesicles. Na+ thus accelerates vesicle replenishment and sustains reliable synaptic transmission.