Share and share alike: trading of presynaptic elements between central synapses

Central presynaptic terminals harbour synaptic vesicles (SVs) and synapse-specific proteins necessary for neurotransmission. Classically, these elements were thought to reside more or less stably at individual mature synapses, giving rise to the idea that each terminal was essentially an independent functional unit. However, emerging evidence from fluorescence imaging studies in hippocampal cultured neurons is now challenging this view, suggesting that neighbouring synapses along axons share vesicles, and also other synaptic elements, at high levels. This raises the possibility that control of import and export might be an important regulatory target for the maintenance of release sites, modulation of synaptic efficacy and formation of new synaptic contacts. Here, temporal synaptic stability and the functional consequences for presynaptic operation will be considered.     

Sub-micromolar increase in [Ca(2+)](i) triggers delayed exocytosis of ATP in cultured astrocytes

Astrocytes release a variety of transmitter molecules, which mediate communication between glial cells in the brain and modulate synaptic transmission. ATP is a major glia-derived transmitter, but the mechanisms and kinetics of ATP release from astrocytes remain largely unknown. Here, we combined epifluorescence and total internal reflection fluorescence microscopy to monitor individual quinacrine-loaded ATP-containing vesicles undergoing exocytosis in cultured astrocytes. In resting cells, vesicles exhibited three-dimensional motility, spontaneous docking and release at low rate. Extracellular ATP application induced a Ca(2+)-dependent increase in the rate of exocytosis, which persisted for several minutes. Using UV flash photolysis of caged Ca(2+), the threshold [Ca(2+)](i) for ATP exocytosis was found to be approximately 350 nM. Subthreshold [Ca(2+)](i) transients predominantly induced vesicle docking at plasma membrane without subsequent release. ATP exocytosis triggered either by purinergic stimulation or by Ca(2+) uncaging occurred after a substantial delay ranging from tens to hundreds of seconds, with only approximately 4% of release occurring during the first 30 s. The time course of the cargo release from vesicles had two peaks centered on 相似文献   

Processing-dependent trafficking of Sonic hedgehog to the regulated secretory pathway in neurons

Neurons are an important source of the secreted morphogen Sonic hedgehog (Shh), however, little is known about neuron-specific regulation of Shh transport and secretion. To study this process, we investigated the subcellular distribution of Shh in primary neurons and differentiated cells of a neuroendocrine cell line by fluorescence microscopy and biochemical fractionation. In retinal ganglion cells, endogenous Shh was distributed as intra- and extracellular puncta at the soma, dendrites, axons and neurite terminals. Shh(+) puncta move bidirectionally and colocalize with markers of synaptic vesicles (SVs) and dense core granules. Lipid modification and proteolysis were required for Shh sorting to SVs and cell surface association. Finally, consistent with its association with regulated secretory vesicles, Shh secretion could be induced under depolarizing conditions. Taken together, these observations suggest that long-range Shh transport and signalling in neurons involves trafficking to the regulated secretory pathway and cell surface accumulation of Shh on axons and suggests a link between neuronal activity and Shh release.     

Rapid and Gentle Immunopurification of Brain Synaptic Vesicles

Current methods to isolate synaptic vesicles (SVs), the organellar quanta of synaptic transmission, require highly specialized materials and up to 24 h. These technical obstacles have thus far limited the study of SVs in models of synaptic function and pathophysiology. Here, we describe techniques for the rapid isolation of SVs by immunoprecipitation with widely available antibodies conjugated to magnetic beads. We report that the inexpensive rho1D4 monoclonal antibody binds SVs and show that elution with the 1D4 peptide yields native vesicles that are ≥ 10-fold purer than those obtained with classical techniques. These methods substantially widen the accessibility of SVs, enabling their purification in 60–90 min for downstream analyses including mass spectrometry and cryo-electron microscopy. Immunopurified SV preparations from mouse brain contained apolipoprotein E, the LDL receptor Lrp1, and enzymes involved in lipid metabolism, suggesting that SVs may play direct roles in lipid homeostasis and lipoprotein trafficking at the nerve terminal.SIGNIFICANCE STATEMENT SVs are small organelles that form and recycle at nerve terminals to enable synaptic transmission. Much remains unknown about the processes that enable the formation and function of SVs. Moreover, nerve terminals appear to be particularly vulnerable to pathophysiologic processes underlying neurodegenerative diseases and schizophrenia. Although techniques to purify synaptic vesicles thus have the potential to yield significant insights into physiology and pathophysiology of nerve terminals, current methods rely on either esoteric materials or expression of transgenes. This article addresses these problems by establishing robust, efficient methods for SV purification using widely available materials, and it highlights several promising areas of future study arising from proteomic analyses of immunopurified SVs.     

Exocytosis and endocytosis of synaptic vesicles and functional roles of vesicle pools: lessons from the Drosophila neuromuscular junction.

To maintain synaptic transmission during intense neuronal activities, the synaptic vesicle (SV) pool at release sites is effectively replenished by recruitment of SVs from the reserve pool and/or by endocytosis. The authors have studied dynamics of SVs using a fluorescence dye, FM1-43, which is incorporated into SVs during endocytosis and released by exocytosis. Drosophila is one of the most suitable preparations for genetic and pharmacological analyses, and this provides a useful model system. The authors found at the neuromuscular junctions of Drosophila that exocytosis and endocytosis of SVs are triggered by Ca(2+) influx through distinct routes and that selective inhibition of exocytosis or endocytosis resulted in depression of synaptic transmission with a distinct time course. They identified two SV pools in a single presynaptic bouton. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low-frequency stimulation and locates close to release sites in the periphery of boutons, whereas the reserve pool (RP) is loaded and unloaded only during high-frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the quantal content of evoked release, suggesting that SVs in the ECP are primarily involved in synaptic transmission. SVs in the RP are recruited to synaptic transmission by a process involving the cAMP/PKA cascade during high-frequency stimulation. Cytochalasin D blocked this recruitment process, suggesting involvement of filamentous actin. Endocytosed SVs replenish the ECP during stimulation and the RP after tetanic stimulation. Replenishment of the ECP depends on Ca(2+) influx from external solutions, and that of the RP is initiated by Ca(2+) release from internal stores. Thus, SV dynamics is closely involved in modulation of synaptic efficacy and influences synaptic plasticity.     

The synapsin cycle: a view from the synaptic endocytic zone

Although the synapsin phosphoproteins were discovered more than 30 years ago and are known to play important roles in neurotransmitter release and synaptogenesis, a complete picture of their functions within the nerve terminal is lacking. It has been shown that these proteins play an important role in the clustering of synaptic vesicles (SVs) at active zones and function as modulators of synaptic strength by acting at both pre- and postdocking levels. Recent studies have demonstrated that synapsins migrate to the endocytic zone of central synapses during neurotransmitter release, which suggests that there are additional functions for these proteins in SV recycling.     

Tissue-type plasminogen activator triggers the synaptic vesicle cycle in cerebral cortical neurons

The active zone (AZ) is a thickening of the presynaptic membrane where exocytosis takes place. Chemical synapses contain neurotransmitter-loaded synaptic vesicles (SVs) that at rest are tethered away from the synaptic release site, but after the presynaptic inflow of Ca⁺² elicited by an action potential translocate to the AZ to release their neurotransmitter load. We report that tissue-type plasminogen activator (tPA) is stored outside the AZ of cerebral cortical neurons, either intermixed with small clear-core vesicles or in direct contact with the presynaptic membrane. We found that cerebral ischemia-induced release of neuronal tPA, or treatment with recombinant tPA, recruits the cytoskeletal protein βII-spectrin to the AZ and promotes the binding of SVs to βII-spectrin, enlarging the population of SVs in proximity to the synaptic release site. This effect does not require the generation of plasmin and is followed by the recruitment of voltage gated calcium channels (VGCC) to the presynaptic terminal that leads to Ca⁺²-dependent synapsin I phosphorylation, freeing SVs to translocate to the AZ to deliver their neurotransmitter load. Our studies indicate that tPA activates the SV cycle and induces the structural and functional changes in the synapse that are required for successful neurotransmission.     

Condensate biology of synaptic vesicle clusters

Neuronal communication crucially relies on exocytosis of neurotransmitters from synaptic vesicles (SVs) which are clustered at synapses. To ensure reliable neurotransmitter release, synapses need to maintain an adequate pool of SVs at all times. Decades of research have established that SVs are clustered by synapsin 1, an abundant SV-associated phosphoprotein. The classical view postulates that SVs are crosslinked in a scaffold of protein–protein interactions between synapsins and their binding partners. Recent studies have shown that synapsins cluster SVs via liquid–liquid phase separation (LLPS), thus providing a new framework for the organization of the synapse. We discuss the evidence for phase separation of SVs, emphasizing emerging questions related to its regulation, specificity, and reversibility.     

Morphological changes in neuromuscular junctions during exercise

Long lasting exercise produces several morphological changes in teleostean neuromuscular junctions (NJs), consisting of progressive synaptic vesicles (SVs) depletion and lamellar branching of the nerve endings. Exercised fishes kept swimming during 1 hr against a 3.5 1/min flow of oxygenated water in spite of the fact that the number of SVs was reduced in about 70% after 10 min of exercise. This observation indicates that the SVs formation fails to restore their original number and consequently, under such circumstances, the transmitter release may occur by a different mechanism.     

Synapsins contribute to the dynamic spatial organization of synaptic vesicles in an activity-dependent manner

The precise subcellular organization of synaptic vesicles (SVs) at presynaptic sites allows for rapid and spatially restricted exocytotic release of neurotransmitter. The synapsins (Syns) are a family of presynaptic proteins that control the availability of SVs for exocytosis by reversibly tethering them to each other and to the actin cytoskeleton in a phosphorylation-dependent manner. Syn ablation leads to reduction in the density of SV proteins in nerve terminals and increased synaptic fatigue under high-frequency stimulation, accompanied by the development of an epileptic phenotype. We analyzed cultured neurons from wild-type and Syn I,II,III(-/-) triple knock-out (TKO) mice and found that SVs were severely dispersed in the absence of Syns. Vesicle dispersion did not affect the readily releasable pool of SVs, whereas the total number of SVs was considerably reduced at synapses of TKO mice. Interestingly, dispersion apparently involved exocytosis-competent SVs as well; it was not affected by stimulation but was reversed by chronic neuronal activity blockade. Altogether, these findings indicate that Syns are essential to maintain the dynamic structural organization of synapses and the size of the reserve pool of SVs during intense SV recycling, whereas an additional Syn-independent mechanism, whose molecular substrate remains to be clarified, targets SVs to synaptic boutons at rest and might be outpaced by activity.     

Imaging synaptic zinc release in living nervous tissue

Zinc enriched neurons have a pool of synaptic vesicles which contain free or loosely-bound zinc ions. The movement of the vesicular zinc ions into the synaptic clefts has been previously studied by microdialysis, fluorescence postmortem staining for zinc and radioactive zinc isotope. In this study the zinc fluorescence probe N-6-metoxy-p-toluensulfonamide quinoline (TSQ) has been applied as a tracer of synaptic release of zinc ions. This fluorochrome permeates cell membranes and when exposed to living brain slices gives rise to a staining pattern similar to that seen with autometallography. In the living brain slices, fluorescence emission persists after exposure to calcium saturated ethylen diamino-tetra-acetic acid (Ca-EDTA) because this chelator does not penetrate cell membranes, while sodium dethyldithiocarbamate (DEDTC), that does penetrate membranes, partially suppressed the fluorescence emission. Stimulation of slices bathed in the non-permeant chelator Ca-EDTA with 50 mM potassium chloride leads to a rapid and complete disappearance of fluorescence. In the absence of Ca-EDTA, however, potassium stimulation induces a sudden transitory increase in fluorescence. This increase is caused by a translocation of the fluorochrome (TSQ) zinc molecules from the weakly acid interior of the synaptic vesicles to the neutral extracellular space, whereby the fluorescence emission of the molecules is enhanced sufficiently to be recorded by a high sensitivity TV camera.     

Reduction of AP180 and CALM Produces Defects in Synaptic Vesicle Size and Density

Clathrin assembly proteins AP180 and CALM regulate the assembly of clathrin-coated vesicles (CCVs), which mediate diverse intracellular trafficking processes, including synaptic vesicle (SV) recycling at the synapse. Although studies using several invertebrate model systems have indicated a role for AP180 in SV recycling, less is known about AP180’s or CALM’s function in the synapse of mammalian neurons. In this study, we examined synapses of rat hippocampal neurons in which the level of AP180 or CALM had been reduced by RNA interference (RNAi). Using light microscopy, we visualized synaptic puncta in these AP180- or CALM-reduced neurons by co-expressing Synaptophysin::EGFP (Syp::EGFP). We found that neurons with reduced AP180 or reduced CALM had smaller Syp::EGFP-illuminated puncta. Using electron microscopy, we further examined the ultrastructure of the AP180- or CALM-reduced presynaptic terminals. We found that SVs became variably enlarged in both the AP180-reduced and CALM-reduced presynaptic terminals. Lower AP180 and CALM also reduced the density of SVs and the size of SV clusters. Our findings demonstrate that in the presynaptic terminals of hippocampal neurons, AP180 and CALM have a similar role in regulating synaptic vesicles. This overlapping activity may be necessary for high-precision and high-efficacy SV formation during endocytosis.     

Synapsin I senses membrane curvature by an amphipathic lipid packing sensor motif

Sustained neurotransmitter release at synapses during high-frequency synaptic activity involves the mobilization of synaptic vesicles (SVs) from the tightly clustered reserve pool (RP). Synapsin I (Syn I), a brain-specific peripheral membrane protein that undergoes activity-dependent cycles of SV association and dissociation, is implicated in RP organization via its ability to cluster SVs. Although Syn I has affinity for phospholipids, the mechanism for the reversible association of synapsin with SV membranes remains enigmatic. Here, we show that rat Syn I is able to sense membrane curvature via an evolutionary conserved amphipathic lipid packing sensor motif (ALPS). Deletion or mutational inactivation of the ALPS impairs the ability of Syn I to associate with highly curved membranes and with SVs. Furthermore, a Syn I mutant lacking ALPS displays defects in its ability to undergo activity-induced cycles of dispersion and reclustering in neurons and fails to induce vesicle clustering in vitro. Our data suggest a crucial role for ALPS-mediated sensing of membrane curvature in regulating synapsin function.     

Dynamics of Synaptic Vesicles in Cultured Spinal Cord Neurons in Relationship to Synaptogenesis

The dynamics of synaptic vesicles (SVs) during the development of presynaptic specializations in culturedXenopusspinal cord neurons was studied with the fluorescent vesicular probe FM1-43. In naive neurons that have not contacted synaptic targets, packets of SVs are distributed along the entire neurite and are quite mobile. The interaction with the synaptic target, such as a muscle cell or a latex bead coated with basic fibroblast growth factor, results in the localization and immobilization of SV packets at the contact site. Depolarization resulted in exocytosis of SVs in both naive and target-contacted neurites. Okadaic acid, a phosphatase inhibitor, caused a dispersal of SV packets in both naive and target-contacted neurites. Thus, prior to target contact, SVs are already organized into packets capable of release and recycling by a phosphorylation-dependent mechanism. Target interaction then recruits and anchors these functional SV packets into forming the presynaptic nerve terminal. With fluorescent phalloidin as a probe, F-actin was found to colocalize with SV clusters at bead-neurite contacts. Although okadaic acid caused a dispersal of SVs at the beads, F-actin localization there was relatively resistant to this drug treatment. This suggests that SVs become localized at the target by interacting with an actin-based cytoskeletal specialization in a phosphorylation-sensitive manner. The induction of this cytoskeletal specialization by the target may be an early event in presynaptic differentiation.     

RIM-Binding Protein 2 Organizes Ca2+ Channel Topography and Regulates Release Probability and Vesicle Replenishment at a Fast Central Synapse

Rab-interacting molecule (RIM)-binding protein 2 (BP2) is a multidomain protein of the presynaptic active zone (AZ). By binding to RIM, bassoon (Bsn), and voltage-gated Ca²⁺ channels (Ca_V), it is considered to be a central organizer of the topography of Ca_V and release sites of synaptic vesicles (SVs) at the AZ. Here, we used RIM-BP2 knock-out (KO) mice and their wild-type (WT) littermates of either sex to investigate the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers (ANFs) with bushy cells (BCs) of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked EPSCs. Analysis of SV pool dynamics during high-frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca²⁺-dependent fast component of SV replenishment after RRP depletion was slowed. Ultrastructural analysis by superresolution light and electron microscopy revealed an impaired topography of presynaptic Ca_V and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of Ca_V, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in BCs of RIM-BP2-deficient mice in vivo.SIGNIFICANCE STATEMENT Rab-interacting molecule (RIM)-binding proteins (BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach to the calyceal endbulb of Held synapse that transmits auditory information at rates of up to hundreds of Hertz with submillisecond precision we demonstrate a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking RIM-BP2 show a reduced release probability despite normal whole-terminal Ca²⁺ influx and abundance of the key priming protein Munc13-1, a reduced rate of SV replenishment, as well as an altered topography of voltage-gated (Ca_V)2.1 Ca²⁺ channels, and fewer docked and membrane proximal synaptic vesicles (SVs). This hampers transmission of sound onset information likely affecting downstream neural computations such as of sound localization.     

Caudal neurosecretory system synaptic morphology following deafferentation: An electron microscopic degeneration study

The caudal neurosecretory system of Poecilia sphenops (molly) is an isolated population of neurosecretory cells located in the caudal most aspect of the teleost spinal cord. The structure of this neuroendocrine system is favorable for studies on the synaptic control of neurosecretory mechanisms. Little is known about the detailed synaptology of the system. Morphological and electrophysiological reports have shown that the caudal neurosecretory system is linked to higher brain centers by descending spinal projections. To examine the synaptology of the descending synaptic input, surgical deafferentation was performed by microsuction removal of a segment of spinal cord rostral to the caudal system. The degeneration of axon terminals was studied at various times following deafferentation and compared to control synaptology. Based on vesicle content and morphology, three axon terminal types were found in the caudal neurosecretory system. These terminals formed axosomatic, axodendritic, and axoaxonic synaptic contacts. Following deafferentation, axon terminals with dense-cored vesicles and boutons with round clear vesicles degenerated as evidenced by the electron dense dark reaction and the electron lucent reaction respectively. This suggested that at least two different types of axon terminals arise from the descending projection to the caudal neurosecretory system and that two different neurotransmitters may be influencing the neurosecretory activity of these cells.     

Munc13-independent vesicle priming at mouse photoreceptor ribbon synapses

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.     

L-type calcium channels are involved in fast endocytosis at the mouse neuromuscular junction

We used fluorescence microscopy of FM dyes-labeled synaptic vesicles and electrophysiological recordings to examine the functional characteristics of vesicle recycling and study how different types of voltage-dependent Ca²⁺ channels (VDCCs) regulate the coupling of exocytosis and endocytosis at mouse neuromuscular junction.
Our results demonstrate the presence of at least two different pools of recycling vesicles: a high-probability release pool (i.e. a fast destaining vesicle pool), which is preferentially loaded during the first 5 s (250 action potentials) at 50 Hz; and a low-probability release pool (i.e. a slow destaining vesicle pool), which is loaded during prolonged stimulation and keeps on refilling after end of stimulation.
Our results suggest that a fast recycling pool mediates neurotransmitter release when vesicle use is minimal (i.e. during brief high-frequency stimulation), while vesicle mobilization from a reserve pool is the prevailing mechanism when the level of synaptic activity increases.
We observed that specific N- and L -type VDCC blockers had no effect on evoked transmitter release upon low-frequency stimulation (5 Hz). However, at high-frequency stimulation (50 Hz), L -type Ca²⁺ channel blocker increased FM2-10 destaining and at the same time diminished quantal release. Furthermore, when L -type channels were blocked, FM2-10 loading during stimulation was diminished, while the amount of endocytosis after stimulation was increased.
Our experiments suggest that L -type VDCCs promote endocytosis of synaptic vesicles, directing the newly formed vesicles to a high-probability release pool where they compete against unused vesicles.     

Kiss and run exocytosis of dense core secretory vesicles

The mechanisms by which large, dense core, peptide-containing vesicles release their cargo from neuroendocrine cells has been the subject of intense debate during the past few years. Thanks to recent studies of the vesicle membrane post fusion, the view that exocytosis is obligatorily linked to the complete collapse of these vesicles at the cell surface is gradually being revised. We discuss here the evidence supporting a model in which (1) exocytosis and endocytosis of dense core vesicles are closely coupled, and (2) the release of peptides occurs from largely intact vesicles via the formation of a large, but transient, fusion pore.     

Membrane recycling due to low and high rates of nerve stimulation at release sites in the amphibian (Bufo marinus) neuromuscular junction

The activity-dependent labelling of motor nerve terminals with the dye FM1-43 has been used to estimate the relative levels of membrane recycling (due to synaptic vesicle exocytosis and recovery) at release sites in response to 1,200 nerve stimulations delivered at either low (0.5 Hz) or high (30 Hz) frequency. Dye in terminals appears as fluorescent spots distributed along the terminal branches; each spot is thought to be a cluster of labelled vesicles associated with a release site. Relative fluorescence in spots was quantified from images obtained with a confocal microscope. Spot intensities varied widely within branches following labelling at both frequencies, but the distribution was highly skewed towards lower intensities at low frequency stimulation; at high frequency, more spots had stronger fluorescence. Both weak and strongly stained spots were uniformly distributed along the length of terminal branches after low frequency stimulation; however, there was a gradual decline in all spot intensities towards the distal end of branches loaded with dye at high frequency stimulation. Antibody staining for synaptic vesicles was, on average, uniformly distributed along the branches. The increase in number of more strongly FM1-43-labelled spots in terminal branches stimulated at high compared with low frequency suggests that more release sites are active at high rates of nerve stimulation. This "recruitment" of release sites at high frequency stimulation occurs mostly in the proximal half of terminal branches and is not related to the abundance of synaptic vesicles in the terminal.