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Synaptic vesicle tethering, priming, and neurotransmitter release require a coordinated action of multiple protein complexes. While physiological experiments, interaction data, and structural studies of purified systems were essential for our understanding of the function of the individual complexes involved, they cannot combine high structural detail with the unperturbed organization of complexes within cells to resolve how the actions of individual complexes integrate. We employed cryo-electron tomography to simultaneously image multiple presynaptic protein complexes and lipids at molecular resolution in their native composition, conformation and environment. Our results argue that tethers comprising proteins Munc13 and SNAP25 differentially and spatially confine vesicles with single nanometer precision, define vesicle tethering states, and provide molecular mechanisms that guide vesicles towards fusion, which includes molecular priming by conversion to SNARE complex-dependent tethers. These findings present an example of a cellular function performed by an extended molecular assembly comprising multiple, molecularly diverse complexes.

Active zones consist of protein scaffolds that are tightly attached to the presynaptic plasma membrane. They dock and prime synaptic vesicles, couple them to Ca2+ entry, and target neurotransmitter release to postsynaptic receptor domains. Simultaneous RIM+ELKS ablation disrupts these scaffolds, abolishes vesicle docking and removes active zone-targeted Munc13, but some vesicles remain releasable. This enduring vesicular fusogenicity may be Munc13-independent or be mediated by non-active zone-anchored Munc13. We tested its Munc13-dependence by ablating Munc13-1 and Munc13-2 on top of RIM+ELKS in cultured hippocampal neurons. The hextuple knockout synapses lacked docked vesicles, but other ultrastructural features were near-normal despite the strong genetic manipulation. Removing Munc13 in addition to RIM+ELKS further impaired action potential-evoked release by decreasing the remaining pool of releasable vesicles. We conclude that Munc13 can support some fusogenicity without RIM and ELKS, and that presynaptic recruitment of Munc13, even without active zone-anchoring, suffices to generate some fusion-competent vesicles.

Summary Compensatory endocytosis of released synaptic vesicles (SVs) relies on coordinated signaling at the lipid-protein interface. Here, we address the synaptic function of C-terminal binding protein 1 (CtBP1), a ubiquitous regulator of gene expression and membrane trafficking in cultured hippocampal neurons. In the absence of CtBP1, synapses form in greater density and show changes in SV distribution and size. The increased basal neurotransmission and enhanced synaptic depression could be attributed to a higher vesicular release probability and a smaller fraction of release-competent SVs, respectively. Rescue experiments with specifically targeted constructs indicate that, while synaptogenesis and release probability are controlled by nuclear CtBP1, the efficient recycling of SVs relies on its synaptic expression. The ability of presynaptic CtBP1 to facilitate compensatory endocytosis depends on its membrane-fission activity and the activation of the lipid-metabolizing enzyme PLD1. Thus, CtBP1 regulates SV recycling by promoting a permissive lipid environment for compensatory endocytosis., Graphical Abstract, Highlights • Nuclear CtBP1 restricts synaptogenesis and vesicular release probability • CtBP1 is required for efficient compensatory endocytosis at the presynapse • CtBP1 facilitates synaptic vesicle retrieval via activation of the lipid enzyme PLD1 • Phosphorylation by Pak1 shifts CtBP1 from the active zone to endocytic sites, Ivanova et al. demonstrate a dual role of CtBP1 in synaptic transmission. Nuclear CtBP1 restricts synaptogenesis and vesicular release probability, whereas presynaptic CtBP1 promotes compensatory endocytosis via activation of the lipid enzyme PLD1. Phosphorylation by Pak1 controls the redistribution of CtBP1 from active zones toward endocytic sites linking presynaptic exo- and endocytosis.

Serotonergic enterochromaffin (EC) cells of the gut epithelium are secretory sensory cells that communicate with vagal neurons. EC cells exhibit many features of neurons in the brain, raising the hypothesis that synapse-like contacts may mediate fast and directed signalling. To dissect functional, structural, and molecular properties underlying serotonin release from genetically identified EC cells, we employed a multidisciplinary in vitro approach combining intestinal epithelial cell and organoid cultures, electrochemistry, correlated light- and electron microscopy, and gene expression and biochemical analyses. Despite the presence of key molecules of the synaptic neurotransmitter release machinery, we found that the majority of serotonin is released with slow kinetics from large dense-core rather than small synaptic-like vesicles. While we cannot exclude synapse-like transmission between EC cells and neurons in vivo, our data support the notion that the predominant mode of serotonin secretion is similar to that of other endocrine cell types.

By performing an in vivo screen in Drosophila melanogaster, Scholz, Ehmann, et al. identify Complexin as a functional interaction partner of Bruchpilot. The two proteins mediate a physical attachment of synaptic vesicles to the active zone cytomatrix and promote rapid, sustained synaptic transmission., Information processing by the nervous system depends on neurotransmitter release from synaptic vesicles (SVs) at the presynaptic active zone. Molecular components of the cytomatrix at the active zone (CAZ) regulate the final stages of the SV cycle preceding exocytosis and thereby shape the efficacy and plasticity of synaptic transmission. Part of this regulation is reflected by a physical association of SVs with filamentous CAZ structures via largely unknown protein interactions. The very C-terminal region of Bruchpilot (Brp), a key component of the Drosophila melanogaster CAZ, participates in SV tethering. Here, we identify the conserved SNARE regulator Complexin (Cpx) in an in vivo screen for molecules that link the Brp C terminus to SVs. Brp and Cpx interact genetically and functionally. Both proteins promote SV recruitment to the Drosophila CAZ and counteract short-term synaptic depression. Analyzing SV tethering to active zone ribbons of cpx3 knockout mice supports an evolutionarily conserved role of Cpx upstream of SNARE complex assembly.

Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (2+-dependent priming as a prelude to fast and slow exocytosis triggering.

The functional consequences of the co-expression of synaptotagmin-1 and synaptotagmin-7 are unclear. We show that when present separately, synaptotagmin-1 and synaptotagmin-7 act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming. The priming effect of Synaptotagmin-7 extends to the Readily Releasable Pool, whose fusion is executed by synaptotagmin-1, indicating synergistic action of the two Ca2+-sensors, although they are only partially colocalized. Synaptotagmin-7 promotes ubMunc13-2-dependent priming and the absence of synaptotagmin-7 renders phorbolesters less effective in stimulating priming, although synaptotagmin-7 independent priming is also observed. Morphologically, synaptotagmin-7 places vesicles in close membrane apposition (< 6 nm); in its absence vesicles accumulate out of reach of the fusion complex (20-40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming and sets the stage for fast and slow exocytosis triggering.

Summary Electron microscopy can resolve synapse ultrastructure with nanometer precision, but the capture of time-resolved, activity-dependent synaptic membrane-trafficking events has remained challenging, particularly in functionally distinct synapses in a tissue context. We present a method that combines optogenetic stimulation-coupled cryofixation (“flash-and-freeze”) and electron microscopy to visualize membrane trafficking events and synapse-state-specific changes in presynaptic vesicle organization with high spatiotemporal resolution in synapses of cultured mouse brain tissue. With our experimental workflow, electrophysiological and “flash-and-freeze” electron microscopy experiments can be performed under identical conditions in artificial cerebrospinal fluid alone, without the addition of external cryoprotectants, which are otherwise needed to allow adequate tissue preservation upon freezing. Using this approach, we reveal depletion of docked vesicles and resolve compensatory membrane recycling events at individual presynaptic active zones at hippocampal mossy fiber synapses upon sustained stimulation., Graphical Abstract, Highlights • High-pressure freezing of cultured brain tissue without cryoprotectants for EM • Flash-and-freeze functional EM of identified synapses in complex neural circuits • Direct correlation of activity-induced functional states and ultrastructure • Dissection of the spatiotemporal organization of endocytosis in identified synapses, Imig et al. present an experimental workflow for electrophysiological and light-stimulation-coupled high-pressure freezing and electron microscopy to be performed under near-identical conditions in brain tissue cultures. Demonstrating proof of principle, they capture vesicle pool changes and map endocytic events in hippocampal mossy fiber boutons with high spatiotemporal resolution during defined activity states.

Dopamine controls striatal circuit function, but its transmission mechanisms are not well understood. We recently showed that dopamine secretion requires RIM, suggesting that it occurs at active zone-like sites similar to conventional synapses. Here, we establish using a systematic conditional gene knockout approach that Munc13 and Liprin-α, active zone proteins for vesicle priming and release site organization, are important for dopamine secretion. Correspondingly, RIM zinc finger and C2B domains, which bind to Munc13 and Liprin-α, respectively, are needed to restore dopamine release in RIM knockout mice. In contrast, and different from conventional synapses, the active zone scaffolds RIM-BP and ELKS, and the RIM domains that bind to them, are expendable. Hence, dopamine release necessitates priming and release site scaffolding by RIM, Munc13, and Liprin-α, but other active zone proteins are dispensable. Our work establishes that molecularly simple but efficient release site architecture mediates fast dopamine exocytosis.

Summary Axons in the adult mammalian central nervous system fail to regenerate after spinal cord injury. Neurons lose their capacity to regenerate during development, but the intracellular processes underlying this loss are unclear. We found that critical components of the presynaptic active zone prevent axon regeneration in adult mice. Transcriptomic analysis combined with live-cell imaging revealed that adult primary sensory neurons downregulate molecular constituents of the synapse as they acquire the ability to rapidly grow their axons. Pharmacogenetic reduction of neuronal excitability stimulated axon regeneration after adult spinal cord injury. Genetic gain- and loss-of-function experiments uncovered that essential synaptic vesicle priming proteins of the presynaptic active zone, but not clostridial-toxin-sensitive VAMP-family SNARE proteins, inhibit axon regeneration. Systemic administration of Baclofen reduced voltage-dependent Ca2+ influx in primary sensory neurons and promoted their regeneration after spinal cord injury. These findings indicate that functional presynaptic active zones constitute a major barrier to axon regeneration., Graphical abstract, Highlights • Axon growth-competent adult neurons downregulate critical components of the synapse • Pharmacogenetic reduction of neuronal excitability stimulates axon regeneration • The presynaptic active zone proteins Munc13 and RIM suppress axon growth • Systemic Baclofen treatment promotes regeneration after adult spinal cord injury, Combining genetic, pharmacogenetic, pharmacological, and transcriptomic approaches with live-cell, super-resolution, and 3-dimensional two-photon imaging, Hilton et al. demonstrate that critical components of the presynaptic transmitter release machinery actively inhibit axon growth. Munc13, a presynaptic active zone protein essential for synaptic transmission, suppresses axon regeneration following adult CNS injury.

© 2019 Elsevier Inc. Synapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders (“synaptopathies”). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and http://geneontology.org). The SynGO consortium presents a framework to annotate synaptic protein locations and functions and annotations for 1,112 synaptic genes based on published experimental evidence. SynGO reports exceptional features and disease associations for synaptic genes and provides an online data analysis platform.

SUMMARYDespite similarities in the composition of the molecular release machinery, synapses can exhibit strikingly different functional transmitter release properties and short- and long-term plasticity characteristics. To address the question whether ultrastructural differences could contribute to this functional synaptic heterogeneity, we employed a combination of hippocampal organotypic slice cultures, high-pressure freezing, freeze substitution, and 3D-electron tomography to resolve the spatial organization of vesicle pools at individual active zones (AZ) in two functionally distinct synapses, namely Schaffer collateral (SC) and mossy fiber (MF) synapses. We found that mature MF and SC synapses harbor equal numbers of docked vesicles at their AZs, MF synapses at rest exhibit a second pool of possibly ‘tethered’ vesicles in the AZ vicinity, and MF synapses contain at least three morphological types of docked vesicles, indicating that differences in the ultrastructural organization of MF and SC synapses may contribute to their respective functional properties and corresponding plasticity characteristics.

Neurosecretory vesicles are highly specialized trafficking organelles that store neurotransmitters that are released at presynaptic nerve endings and are, therefore, important for animal cell–cell signalling. Despite considerable anatomical and functional diversity of neurons in animals, the protein composition of neurosecretory vesicles in bilaterians appears to be similar. This similarity points towards a common evolutionary origin. Moreover, many putative homologues of key neurosecretory vesicle proteins predate the origin of the first neurons, and some even the origin of the first animals. However, little is known about the molecular toolkit of these vesicles in non-bilaterian animals and their closest unicellular relatives, making inferences about the evolutionary origin of neurosecretory vesicles extremely difficult. By comparing 28 proteins of the core neurosecretory vesicle proteome in 13 different species, we demonstrate that most of the proteins are present in unicellular organisms. Surprisingly, we find that the vesicular membrane-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein synaptobrevin is localized to the vesicle-rich apical and basal pole in the choanoflagellateSalpingoeca rosetta.Our 3D vesicle reconstructions reveal that the choanoflagellatesS. rosettaandMonosiga brevicollisexhibit a polarized and diverse vesicular landscape reminiscent of the polarized organization of chemical synapses that secrete the content of neurosecretory vesicles into the synaptic cleft. This study sheds light on the ancestral molecular machinery of neurosecretory vesicles and provides a framework to understand the origin and evolution of secretory cells, synapses and neurons.This article is part of the theme issue ‘Basal cognition: multicellularity, neurons and the cognitive lens’.

Summary: Short-term plasticity gates information transfer across neuronal synapses and is thought to be involved in fundamental brain processes, such as cortical gain control and sensory adaptation. Neurons employ synaptic vesicle priming proteins of the CAPS and Munc13 families to shape short-term plasticity in vitro, but the relevance of this phenomenon for information processing in the intact brain is unknown. By combining sensory stimulation with in vivo patch-clamp recordings in anesthetized mice, we show that genetic deletion of CAPS-1 in thalamic neurons results in more rapid adaptation of sensory-evoked subthreshold responses in layer 4 neurons of the primary visual cortex. Optogenetic experiments in acute brain slices further reveal that the enhanced adaptation is caused by more pronounced short-term synaptic depression. Our data indicate that neurons engage CAPS-family priming proteins to shape short-term plasticity for optimal sensory information transfer between thalamic and cortical neurons in the intact brain in vivo. : Nestvogel et al. perform patch-clamp recordings of layer 4 neurons in the primary visual cortex of anesthetized mice to demonstrate that CAPS-family vesicle priming proteins control synaptic short-term plasticity and adaptation of sensory-evoked thalamocortical synaptic transmission, linking the action of dedicated vesicle priming proteins to sensory processing in vivo. Keywords: Synaptic Vesicle Priming, in-vivo patch clamp technique, Acute Brain slice Patch technique, Optogenetic Stimulation, Sensory Adaptation, Visual Cortex, Ca2+- dependent Activator Protein for Secretion (CAPS)

Information processing by the nervous system depends on the release of neurotransmitter from synaptic vesicles (SVs) at the presynaptic active zone. Molecular components of the cytomatrix at the active zone (CAZ) regulate the final stages of the SV cycle preceding exocytosis and thereby shape the efficacy and plasticity of synaptic transmission. Part of this regulation is reflected by a physical association of SVs with filamentous CAZ structures. However, our understanding of the protein interactions underlying SV tethering by the CAZ is far from complete. The very C-terminal region of Bruchpilot (Brp), a key component of theDrosophilaCAZ, participates in SV tethering. Yet so far, no vesicular or cytoplasmic molecules have been reported to engage in an interaction with Brp’s C-terminus. Here, we carried out an in vivo screen for molecules that link the Brp C-terminus to SVs. This strategy identified the conserved SNARE (solubleN-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) regulator Complexin (Cpx) as a vesicular interaction partner of Brp. We show that Brp and Cpx interact genetically and functionally. Interfering with Cpx targeting to SVs mirrored distinctive features of a C-terminal Brp truncation: impaired SV recruitment to the CAZ and enhanced short-term synaptic depression. Extending the study beyondDrosophilasynapses, we interrogated active zones of mouse rod bipolar cells. Here, too, we collected evidence for an evolutionarily conserved role of Cpx upstream of SNARE complex assembly where it participates in SV tethering to the CAZ.

Summary Dendritic spines are the major transmitter reception compartments of glutamatergic synapses in most principal neurons of the mammalian brain and play a key role in the function of nerve cell circuits. The formation of functional spine synapses is thought to be critically dependent on presynaptic glutamatergic signaling. By analyzing CA1 pyramidal neurons in mutant hippocampal slice cultures that are essentially devoid of presynaptic transmitter release, we demonstrate that the formation and maintenance of dendrites and functional spines are independent of synaptic glutamate release., Highlights • Elimination of presynaptic transmitter release in hippocampal organotypic slices • Dendrite growth and maintenance are independent of presynaptic glutamate release • Spine formation and maintenance are independent of presynaptic glutamate release • Recruitment of NMDA/AMPA receptors is independent of presynaptic glutamate release, Spines are major neurotransmitter reception compartments of nerve cells. Contrary to the currently held dogma, Sigler et al. show that spine formation in the hippocampus is independent of synaptic neurotransmitter release, indicating that brain circuits are established by activity-independent cellular programs.

Transmission electron microscopy serves as a valuable tool for synaptic structure-function analyses aimed at identifying morphological features or modifications associated with specific developmental stages or dysfunctional synaptic states. By utilizing cryo-preparation techniques to minimize the introduction of structural artifacts during sample preparation, and electron tomography to reconstruct the 3D ultrastructural architecture of a synapse, the spatial organization and morphological properties of synaptic organelles and subcompartments can be quantified with unparalleled precision. In this chapter, we present an experimental approach combining organotypic slice culture, high-pressure freezing, automated freeze-substitution, and electron tomography to investigate spatial relationships between synaptic vesicles and active zone release sites in synapses from lethal mouse mutants.

Transmission electron microscopy serves as a valuable tool for synaptic structure-function analyses aimed at identifying morphological features or modifications associated with specific developmental stages or dysfunctional synaptic states. By utilizing cryo-preparation techniques to minimize the introduction of structural artifacts during sample preparation, and electron tomography to reconstruct the 3D ultrastructural architecture of a synapse, the spatial organization and morphological properties of synaptic organelles and subcompartments can be quantified with unparalleled precision. In this chapter, we present an experimental approach combining organotypic slice culture, high-pressure freezing, automated freeze-substitution, and electron tomography to investigate spatial relationships between synaptic vesicles and active zone release sites in synapses from lethal mouse mutants.

It is currently unknown whether the molecular steps of large dense-core vesicle (LDCV) docking and priming are identical to the corresponding reactions in synaptic vesicle (SV) exocytosis. Munc13s are essential for SV docking and priming, and we systematically analyzed their role in LDCV exocytosis using chromaffin cells lacking individual isoforms. We show that particularly Munc13-2 plays a fundamental role in LDCV exocytosis, but in contrast to synapses lacking Munc13s, the corresponding chromaffin cells do not exhibit a vesicle docking defect. We further demonstrate that ubMunc13-2 and Munc13-1 confer Ca2+-dependent LDCV priming with similar affinities, but distinct kinetics. Using a mathematical model, we identify an early LDCV priming step that is strongly dependent upon Munc13s. Our data demonstrate that the molecular steps of SV and LDCV priming are very similar while SV and LDCV docking mechanisms are distinct. DOI: http://dx.doi.org/10.7554/eLife.10635.001, eLife digest Mammals have adrenal glands, which secrete the stress hormone adrenaline as well as other hormones into the bloodstream. These molecules are produced in chromaffin cells, where they are packaged into compartments called large dense-core vesicles (LDCVs). To release the hormones into the bloodstream, the vesicles bind to and fuse with the membrane that surrounds the cell. This process – which is called exocytosis – is triggered by increases in the level of calcium ions inside the cells. Exocytosis also enables nerve cells to release chemical signals at junctions (known as synapses) with other nerve cells. These signals are packaged within another type of vesicle called 'synaptic' vesicles, which also release their contents by fusing with the cell membrane. However, it is not clear whether the two types of vesicle carry out exocytosis in the same way. Exocytosis requires that the vesicles physically attach to the membrane and undergo a process termed 'priming', which enables them to fuse quickly with the membrane in response to an increase in calcium ion levels. In synaptic vesicles, both of these processes – physical membrane attachment and priming – appear to occur in a single step that requires a family of proteins called the Munc13 proteins. Here, Man et al. investigate whether the Munc13 proteins are also essential for LDCV exocytosis in the chromaffin cells of mice. The experiments reveal that in contrast to synaptic vesicles, the initial binding of LDCVs to membranes does not require Munc13 proteins. However, the loss of one member of the family called Munc13-2 dramatically reduces the fusion of LDCVs with the membrane of chromaffin cells. Further experiments reveal that different Munc13 proteins differ in their ability to drive the exocytosis of LDCVs. Man et al. use a mathematical model of LDCV exocytosis, which reveals that Munc13 plays an important role in the first part of the priming step. Together, these findings show that synaptic vesicles and LDCVs use different mechanisms to bind to membranes, but are primed for fusion in a similar way. DOI: http://dx.doi.org/10.7554/eLife.10635.002

SNARE protein-driven secretion of neurotransmitters from synaptic vesicles is at the center of neuronal communication. In the absence of the cytosolic protein Munc18-1, synaptic secretion comes to a halt. Although it is believed that Munc18-1 orchestrates SNARE complexes, its mode of action is still a matter of debate. In particular, it has been challenging to clarify the role of a tight Munc18/syntaxin 1 complex, because this interaction interferes strongly with syntaxin's ability to form a SNARE complex. In this complex, two regions of syntaxin, the N -peptide and the remainder in closed conformation, bind to Munc18 simultaneously. Until now, this binary complex has been reported for neuronal tissues only, leading to the hypothesis that it might be a specialization of the neuronal secretion apparatus. Here we aimed, by comparing the core secretion machinery of the unicellular choanoflagellate Monosiga brevicollis with that of animals, to reconstruct the ancestral function of the Munc18/syntaxin1 complex. We found that the Munc18/syntaxin 1 complex from M. brevicollis is structurally and functionally highly similar to the vertebrate complex, suggesting that it constitutes a fundamental step in the reaction pathway toward SNARE assembly. We thus propose that the primordial secretion machinery of the common ancestor of choanoflagellates and animals has been co-opted for synaptic roles during the rise of animals.

SummarySynaptic vesicle docking, priming, and fusion at active zones are orchestrated by a complex molecular machinery. We employed hippocampal organotypic slice cultures from mice lacking key presynaptic proteins, cryofixation, and three-dimensional electron tomography to study the mechanism of synaptic vesicle docking in the same experimental setting, with high precision, and in a near-native state. We dissected previously indistinguishable, sequential steps in synaptic vesicle active zone recruitment (tethering) and membrane attachment (docking) and found that vesicle docking requires Munc13/CAPS family priming proteins and all three neuronal SNAREs, but not Synaptotagmin-1 or Complexins. Our data indicate that membrane-attached vesicles comprise the readily releasable pool of fusion-competent vesicles and that synaptic vesicle docking, priming, and trans-SNARE complex assembly are the respective morphological, functional, and molecular manifestations of the same process, which operates downstream of vesicle tethering by active zone components.

Munc13 proteins are essential regulators of exocytosis. In hippocampal glutamatergic neurons, the genetic deletion of Munc13s results in the complete loss of primed synaptic vesicles (SVs) in direct contact with the presynaptic active zone membrane, and in a total block of neurotransmitter release. Similarly drastic consequences of Munc13 loss are detectable in hippocampal and striatal GABAergic neurons. We show here that, in the adult mouse retina, the two Munc13-2 splice variants bMunc13-2 and ubMunc13-2 are selectively localized to conventional and ribbon synapses, respectively, and that ubMunc13-2 is the only Munc13 isoform in mature photoreceptor ribbon synapses. Strikingly, the genetic deletion of ubMunc13-2 has little effect on synaptic signaling by photoreceptor ribbon synapses and does not prevent membrane attachment of synaptic vesicles at the photoreceptor ribbon synaptic site. Thus, photoreceptor ribbon synapses and conventional synapses differ fundamentally with regard to their dependence on SV priming proteins of the Munc13 family. Their function is only moderately affected by Munc13 loss, which leads to slight perturbations of signal integration in the retina.

The high degree of similarity between mouse and human physiology and genomes makes mice the animal model of choice to study the functions and dysfunctions of the central nervous system (CNS). The considerable knowledge accumulated in the past decades and the steadily growing number of genetically modified mouse lines allow for the increasingly accurate understanding of biological circuits. Electron microscopy (EM) contributes to unravel the biology of neuronal networks and the myelinating glia by allowing a fine morphological scrutiny of the nervous tissue. We provide detailed descriptions of the conventional processing as well as cryopreparation methods such as high-pressure freezing (HPF), freeze-substitution (FS), and SDS-digested freeze-fracture replica labeling (SDS-FRL) on selected CNS regions such as the retina, optic nerve, and cerebellum. By taking example of the ribbon synapse in the retina and myelinated retinal ganglion cell axons of the optic nerve, we discuss the advantages and drawbacks of these methods in a comparative way.

The high degree of similarity between mouse and human physiology and genomes makes mice the animal model of choice to study the functions and dysfunctions of the central nervous system (CNS). The considerable knowledge accumulated in the past decades and the steadily growing number of genetically modified mouse lines allow for the increasingly accurate understanding of biological circuits. Electron microscopy (EM) contributes to unravel the biology of neuronal networks and the myelinating glia by allowing a fine morphological scrutiny of the nervous tissue. We provide detailed descriptions of the conventional processing as well as cryopreparation methods such as high-pressure freezing (HPF), freeze-substitution (FS), and SDS-digested freeze-fracture replica labeling (SDS-FRL) on selected CNS regions such as the retina, optic nerve, and cerebellum. By taking example of the ribbon synapse in the retina and myelinated retinal ganglion cell axons of the optic nerve, we discuss the advantages and drawbacks of these methods in a comparative way.

Summary Although similar in molecular composition, synapses can exhibit strikingly distinct functional transmitter release and plasticity characteristics. To determine whether ultrastructural differences co-define this functional heterogeneity, we combine hippocampal organotypic slice cultures, high-pressure freezing, freeze substitution, and 3D-electron tomography to compare two functionally distinct synapses: hippocampal Schaffer collateral and mossy fiber synapses. We find that mossy fiber synapses, which exhibit a lower release probability and stronger short-term facilitation than Schaffer collateral synapses, harbor lower numbers of docked synaptic vesicles at active zones and a second pool of possibly tethered vesicles in their vicinity. Our data indicate that differences in the ratio of docked versus tethered vesicles at active zones contribute to distinct functional characteristics of synapses., Graphical Abstract, Highlights • Electron tomography enables the dissection of vesicle pools at synaptic active zones • Docked and primed vesicle availability contributes to initial release probability • The ratio of docked and tethered vesicles may co-determine short-term plasticity • Hippocampal mossy fibers contain three morphological types of docked vesicles, Distinct synapse types exhibit strikingly different morphological and functional properties. To investigate how the ultrastructural architecture of synaptic release sites contributes to such diversity, Maus et al. exploit electron tomography to correlate the nanoscale organization of synaptic vesicle pools with fundamental functional properties such as short-term synaptic plasticity characteristics.