Systematic heterogeneity of fractional vesicle pool sizes and release rates of hippocampal synapses

Hippocampal neurons in tissue culture develop functional synapses that exhibit considerable variation in synaptic vesicle content (20–350 vesicles). We examined absolute and fractional parameters of synaptic vesicle exocytosis of individual synapses. Their correlation to vesicle content was determined by activity-dependent discharge of FM-styryl dyes. At high frequency stimulation (30 Hz), synapses with large recycling pools released higher amounts of dye, but showed a lower fractional release compared to synapses that contained fewer vesicles. This effect gradually vanished at lower frequencies when stimulation was triggered at 20 Hz and 10 Hz, respectively. Live-cell antibody staining with anti-synaptotagmin-1-cypHer 5, and overexpression of synaptopHluorin as well as photoconversion of FM 1-43 followed by electron microscopy, consolidated the findings obtained with FM-styryl dyes. We found that the readily releasable pool grew with a power function with a coefficient of 2/3, possibly indicating a synaptic volume/surface dependency. This observation could be explained by assigning the rate-limiting factor for vesicle exocytosis at high frequency stimulation to the available active zone surface that is proportionally smaller in synapses with larger volumes.     

Do different endocytic pathways make different synaptic vesicles?

At a wide range of synapses, synaptic vesicles reside in distinct pools that respond to different stimuli. The recycling pool supplies the vesicles required for release in response to modest stimulation, whereas the reserve pool is mobilized only by strong stimulation. Multiple pathways have been proposed for the recycling of synaptic vesicles after exocytosis, but the relationship of these pathways to the different synaptic vesicle pools has remained unclear. Synaptic vesicle proteins have also been assumed to undergo recycling as a unit. However, emerging data indicate that differences in the association with distinct endocytic adaptors such as the heterotetrameric adaptor AP3 influence the trafficking of individual synaptic vesicle proteins, affecting the composition of synaptic vesicles and hence their functional characteristics. These observations might begin to account for differences in the properties of different vesicle pools.     

Synaptobrevin is essential for fast synaptic-vesicle endocytosis

Synaptobrevin-2 (VAMP-2), the major SNARE protein of synaptic vesicles, is required for fast calcium-triggered synaptic-vesicle exocytosis. Here we show that synaptobrevin-2 is also essential for fast synaptic-vesicle endocytosis. We demonstrate that after depletion of the readily releasable vesicle pool, replenishment of the pool is delayed by knockout of synaptobrevin. This delay was not from a loss of vesicles, because the total number of pre-synaptic vesicles, docked vesicles and actively recycling vesicles was unaffected. However, altered shape and size of the vesicles in synaptobrevin-deficient synapses suggests a defect in endocytosis. Consistent with such a defect, the stimulus-dependent endocytosis of horseradish peroxidase and fluorescent FM1-43 were delayed, indicating that fast vesicle endocytosis may normally be nucleated by a SNARE-dependent coat. Thus, synaptobrevin is essential for two fast synapse-specific membrane trafficking reactions: fast exocytosis for neurotransmitter release and fast endocytosis that mediates rapid reuse of synaptic vesicles.     

Mobility of synaptic vesicles in different pools in resting and stimulated frog motor nerve terminals

We used fluorescence recovery after photobleaching (FRAP) to measure the mobility of synaptic vesicles in frog motor nerve terminals. Vesicles belonging to the recycling pool or to the reserve pool were selectively labeled with FM1-43. In resting terminals, vesicles in the reserve pool were immobile, while vesicles in the recycling pool were mobile. Nerve stimulation increased the mobility of reserve pool vesicles. Treatment with latrunculin A, which destroyed actin filaments, had no significant effect on mobility, and reducing the temperature likewise had little effect, suggesting that recycling pool vesicles move by simple diffusion. Application of okadaic acid caused vesicle mobility in both pools to increase to the same level. We could model these and others' results quantitatively by taking into account the relative numbers of mobile and immobile vesicles in each pool, and vesicle packing density, which has a large effect on mobility.     

Properties of synaptic vesicle pools in mature central nerve terminals

Readily releasable and reserve pools of synaptic vesicles play different roles in neurotransmission, and it is important to understand their recycling and interchange in mature central synapses. Using adult rat cerebrocortical synaptosomes, we have shown that 100 mosm hypertonic sucrose caused complete exocytosis of only the readily releasable pool (RRP) of synaptic vesicles containing glutamate or gamma-aminobutyric acid. Repetitive hypertonic stimulations revealed that this pool recycled (and reloaded the neurotransmitter from the cytosol) fully in <30 s and did so independently of the reserve pool. Multiple rounds of exocytosis could occur in the constant absence of extracellular Ca(2+). However, although each vesicle cycle includes a Ca(2+)-independent exocytotic step, some other stage(s) critically require an elevation of cytosolic [Ca(2+)], and this is supplied by intracellular stores. Repetitive recycling also requires energy, but not the activity of phosphatidylinositol 4-kinase, which maintains the normal level of phosphoinositides. By varying the length of hypertonic stimulations, we found that approximately 70% of the RRP vesicles fused completely with the plasmalemma during exocytosis and could then enter silent pools, probably outside active zones. The rest of the RRP vesicles underwent very fast local recycling (possibly by kiss-and-run) and did not leave active zones. Forcing the fully fused RRP vesicles into the silent pool enabled us to measure the transfer of reserve vesicles to the RRP and to show that this process requires intact phosphatidylinositol 4-kinase and actin microfilaments. Our findings also demonstrate that respective vesicle pools have similar characteristics and requirements in excitatory and inhibitory nerve terminals.     

Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.     

Colocalization of synapsin and actin during synaptic vesicle recycling

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.     

v-SNARE composition distinguishes synaptic vesicle pools

Synaptic vesicles belong to two distinct pools, a recycling pool responsible for the evoked release of neurotransmitter and a resting pool unresponsive to stimulation. The uniform appearance of synaptic vesicles has suggested that differences in location or cytoskeletal association account for these differences in function. We now find that the v-SNARE tetanus toxin-insensitive vesicle-associated membrane protein (VAMP7) differs from other synaptic vesicle proteins in its distribution to the two pools, providing evidence that they differ in molecular composition. We also find that both resting and recycling pools undergo spontaneous release, and when activated by deletion of the longin domain, VAMP7 influences the properties of release. Further, the endocytosis that follows evoked and spontaneous release differs in mechanism, and specific sequences confer targeting to the different vesicle pools. The results suggest that different endocytic mechanisms generate synaptic vesicles with different proteins that can endow the vesicles with distinct properties.     

Polyunsaturated fatty acids influence synaptojanin localization to regulate synaptic vesicle recycling

The lipid polyunsaturated fatty acids are highly enriched in synaptic membranes, including synaptic vesicles, but their precise function there is unknown. Caenorhabditis elegans fat-3 mutants lack long-chain polyunsaturated fatty acids (LC-PUFAs); they release abnormally low levels of serotonin and acetylcholine and are depleted of synaptic vesicles, but the mechanistic basis of these defects is unclear. Here we demonstrate that synaptic vesicle endocytosis is impaired in the mutants: the synaptic vesicle protein synaptobrevin is not efficiently retrieved after synaptic vesicles fuse with the presynaptic membrane, and the presynaptic terminals contain abnormally large endosomal-like compartments and synaptic vesicles. Moreover, the mutants have abnormally low levels of the phosphoinositide phosphatase synaptojanin at release sites and accumulate the main synaptojanin substrate phosphatidylinositol 4,5-bisphosphate at these sites. Both synaptobrevin and synaptojanin mislocalization can be rescued by providing exogenous arachidonic acid, an LC-PUFA, suggesting that the endocytosis defect is caused by LC-PUFA depletion. By showing that the genes fat-3 and synaptojanin act in the same endocytic pathway at synapses, our findings suggest that LC-PUFAs are required for efficient synaptic vesicle recycling, probably by modulating synaptojanin localization at synapses.     

UNC-11, a Caenorhabditis elegans AP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.     

Modulation of Synaptic Vesicle Exocytosis in Muscle-Dependent Long-Term Depression at the Amphibian Neuromuscular Junction

We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.     

Rabphilin regulates SNARE-dependent re-priming of synaptic vesicles for fusion

Synaptic vesicle fusion is catalyzed by assembly of synaptic SNARE complexes, and is regulated by the synaptic vesicle GTP-binding protein Rab3 that binds to RIM and to rabphilin. RIM is a known physiological regulator of fusion, but the role of rabphilin remains obscure. We now show that rabphilin regulates recovery of synaptic vesicles from use-dependent depression, probably by a direct interaction with the SNARE protein SNAP-25. Deletion of rabphilin dramatically accelerates recovery of depressed synaptic responses; this phenotype is rescued by viral expression of wild-type rabphilin, but not of mutant rabphilin lacking the second rabphilin C2 domain that binds to SNAP-25. Moreover, deletion of rabphilin also increases the size of synaptic responses in synapses lacking the vesicular SNARE protein synaptobrevin in which synaptic responses are severely depressed. Our data suggest that binding of rabphilin to SNAP-25 regulates exocytosis of synaptic vesicles after the readily releasable pool has either been physiologically exhausted by use-dependent depression, or has been artificially depleted by deletion of synaptobrevin.     

Synaptotagmin-7 controls the size of the reserve and resting pools of synaptic vesicles in hippocampal neurons

Continuous neurotransmitter release is subjected to synaptic vesicle availability, which in turn depends on vesicle recycling and the traffic of vesicles between pools. We studied the role of Synaptotagmin-7 (Syt-7) in synaptic vesicle accessibility for release in hippocampal neurons in culture. Synaptic boutons from Syt-7 knockout (KO) mice displayed normal basal secretion with no alteration in the RRP size or the probability of release. However, stronger stimuli revealed an increase in the size of the reserve and resting vesicle pools in Syt-7 KO boutons compared with WT. These data suggest that Syt-7 plays a significant role in the vesicle pool homeostasis and, consequently, in the availability of vesicles for synaptic transmission during strong stimulation, probably, by facilitating advancing synaptic vesicles to the readily releasable pool.     

Peculiarities of synaptic vesicle recycling in frog and mouse motor nerve terminals

Using electrophysiology and fluorescence microscopy with dye FM 1-43, a comparative study of peculiarities of neurotransmitter secretion, synaptic vesicle exo-endocytosis and recycling has been carried out in nerve terminals (NT) of the skin-sternal muscle of the frog Rana ridibunda and of the white mouse diaphragm muscle during a long-term high-frequency stimulation (20 imp/s). The obtained data have allowed identifying three synaptic vesicle pools and two recycling ways in the motor NT. In the frog NT, the long-term high-frequency stimulation induced consecutive expenditure of the pool ready to release, the mobilizational, and reserve vesicle pools. The exocytosis rate exceeded markedly the endocytosis rate; the slow synaptic vesicle recycling with replenishment of the reserve pool was predominant. In the mouse NT, only the vesicles of the ready to release and the mobilizational pools, which are replenished predominantly by fast recycling, were exocytosed. The exo- and endocytosis occurred practically in parallel, while vesicles of the reserve pool did not participate in the neurotransmitter secretion. It is suggested that evolution of the motor NT from the poikilothermal to homoiothermal animals went by the way of a decrease of the vesicle pool size, the more economic expenditure and the more effective reuse of synaptic vesicles owing to the high rates of endocytosis and recycling. These peculiarities can provide in NT of homoiothermal animals a long maintenance of neurotransmitter secretion at the steady and sufficiently high level to preserve reliability of synaptic transmission in the process of the high-frequency activity.     

Vesicle cycle in the presynaptic nerve terminal

Presynaptic nerve terminals contain a great number ofsynaptic vesicles filled with neurotransmitter. The transmission of information in synapses is mediated by release of transmitter from vesicles: exocytosis, after their fusion with presynaptic membrane. At the functioning synapses, the continuous recycling of synaptic vesicles occurs (vesicle cycle), which provides multiple reuse of vesicular membrane material during synaptic activity. Vesicle cycle consists of large number of steps, including vesicle fusion--exocytosis, formation of new vesicles--endocytosis, vesicle sorting, filling of vesicles with transmitter, intraterminal vesicle transport driving the vesicles to different vesicle pools and preparing to next exocytic event. At this paper, I presented the latest literature and our data regarding the steps and mechanisms of vesicle cycle at synapses. Special attention was paid to neuromuscular synapse as the most thoroughly investigated and as my favorite preparation.     

Vesicle cycle in mouse diaphragm motor nerve terminals

In our research on mouse diaphragm muscles the dynamic of neurotransmitter secretion and synaptic vesicles recycling (exo-endocytosis cycle) at the long-term rhythmic stimulation (20Hz) are explored using an intracellular microelectrode registration and a fluorescent microscopy. It have been shown, thate change of end plant potentials (EPP) amplitude at the rhythmic training occurs in three phases: initial transient decrease, long amplitude stabilization (1-2 min)--the plateau and secondary slow decrease. After 3 minute stimulations the EPP amplitude recovery observed during several seconds. Loading the synaptic vesicle by fluorescent endocytic dye FM 1-43 had shown that the rhythmic stimulation results to gradual (during 5-6 mines) fluorescence decrease in NT, indicating the synaptic vesicle exocytosis. The quantum analysis of the electrophysiological data and their comparison to the fluorescent researches date has allowed to assume, that mouse motor nerve terminals are characterized by high rate of endocytosis and fast synaptic vesicle reuse (average recycling time about 50 sec) that can provide effective maintenance of synaptic transmission at long high-frequency activity. Sizes of ready releasable and recycling synaptic vesicle pools are quantitatively determined. It is assumed, that vesicle recycling occurs on a short fast way to inclusion in recycling pool. So, in the stimulation protocol that were used the synaptic vesicles from reserve pool remain unused. Thus in our conditions recycling pool vesicles cycle repeatedly without reserve pool release.     

Motorneurons require cysteine string protein-α to maintain the readily releasable vesicular pool and synaptic vesicle recycling

Cysteine string protein-α (CSP-α) is a synaptic vesicle protein that prevents activity-dependent neurodegeneration by poorly understood mechanisms. We have studied the synaptic vesicle cycle at the motor nerve terminals of CSP-α knock-out mice expressing the synaptopHluorin transgene. Mutant nerve terminals fail to sustain prolonged release and the number of vesicles available to be released decreases. Strikingly, the SNARE protein SNAP-25 is dramatically reduced. In addition, endocytosis during the stimulus fails to maintain the size of the recycling synaptic vesicle pool during prolonged stimulation. Upon depolarization, the styryl dye FM?2-10 becomes trapped and poorly releasable. Consistently with the functional results, electron microscopy analysis revealed characteristic features of impaired synaptic vesicle recycling. The unexpected defect in vesicle recycling in CSP-α knock-out mice provides insights into understanding molecular mechanisms of degeneration in motor nerve terminals.     

Mixing and matching during synaptic vesicle endocytosis

The question of how synapses maintain an active recycling pool of synaptic vesicles to support high-frequency synaptic transmission has been a perplexing and often controversial problem. In this issue of Neuron, Fernandez-Alfonso et al. present data indicating that at least two synaptic vesicle proteins, synaptotagmin 1 and VAMP-2, are present in a large pool on the synaptic and axonal plasma membrane and can interchange with recently exocytosed proteins. These findings suggest that a plasma membrane pool of synaptic vesicle proteins provides a reservoir that can facilitate rapid endocytosis.     

Probing vesicle dynamics in single hippocampal synapses

We use fluorescence correlation spectroscopy and fluorescence recovery after photobleaching to study vesicle dynamics inside the synapses of cultured hippocampal neurons labeled with the fluorescent vesicle marker FM 1-43. These studies show that when the cell is electrically at rest, only a small population of vesicles is mobile, taking seconds to traverse the synapse. Applying the phosphatase inhibitor okadaic acid causes vesicles to diffuse freely, moving 30 times faster than vesicles in control synapses. These results suggest that vesicles move sluggishly due to binding to elements of the synaptic cytomatrix and that this binding is altered by phosphorylation. Motivated by these results, a model is constructed consisting of diffusing vesicles that bind reversibly to the cytomatrix. This stick-and-diffuse model accounts for the fluorescence correlation spectroscopy and fluorescence recovery after photobleaching data, and also predicts the well-known exponential refilling of the readily releasable pool. Our measurements suggest that the movement of vesicles to the active zone is the rate-limiting step in this process.     

Evidence for a primary endocytic vesicle involved in synaptic vesicle biogenesis

The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from "classical" synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles.