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Summary The accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed., Highlights • VAMP4 is the first identified ADBE cargo • VAMP4 is essential for ADBE • Most synaptic vesicle cargoes are not selectively recovered by ADBE, Nicholson-Fish et al. show that VAMP4 is selectively retrieved by activity-dependent bulk endocytosis (ADBE) and is essential for ADBE to proceed. Thus, synaptic vesicles generated by ADBE will have a specific molecular composition that defines their physiological function.

Summary Age-related neurodegenerative disorders are characterized by a slow, persistent accumulation of aggregated proteins. Although cells can elicit physiological responses to enhance cellular clearance and counteract accumulation, it is unclear how pathogenic proteins evade this process in disease. We find that Parkinson’s disease α-synuclein perturbs the physiological response to lysosomal stress by impeding the SNARE protein ykt6. Cytosolic ykt6 is normally autoinhibited by a unique farnesyl-mediated regulatory mechanism; however, during lysosomal stress, it activates and redistributes into membranes to preferentially promote hydrolase trafficking and enhance cellular clearance. α-Synuclein aberrantly binds and deactivates ykt6 in patient-derived neurons, thereby disabling the lysosomal stress response and facilitating protein accumulation. Activating ykt6 by small-molecule farnesyltransferase inhibitors restores lysosomal activity and reduces α-synuclein in patient-derived neurons and mice. Our findings indicate that α-synuclein creates a permissive environment for aggregate persistence by inhibiting regulated cellular clearance and provide a therapeutic strategy to restore protein homeostasis by harnessing SNARE activity.

SummarySpontaneous synaptic vesicle fusion is a common property of all synapses. To trace the origin of spontaneously fused vesicles in hippocampal synapses, we tagged vesicles with fluorescent styryl dyes, antibodies against synaptotagmin-1, or horseradish peroxidase. We could show that synaptic vesicles recycle at rest, and after spontaneous exo-endocytosis, they populate a reluctantly releasable pool of limited size. Interestingly, vesicles in this spontaneously labeled pool were more likely to re-fuse spontaneously compared to vesicles labeled with activity. We found that blocking vesicle refilling at rest selectively depleted neurotransmitter from spontaneously fusing vesicles without significantly altering evoked transmission. Furthermore, in the absence of the vesicle SNARE protein synaptobrevin (VAMP), activity-dependent and spontaneously recycling vesicles could mix, suggesting a role for synaptobrevin in the separation of the two pools. Taken together these results suggest that spontaneously recycling vesicles and activity-dependent recycling vesicles originate from distinct pools with limited cross-talk with each other.

Drosophila olfactory receptor neurons (ORNs) elaborate a precise internal representation of the external olfactory world in the antennal lobe (AL), a structure analagous to the vertebrate olfactory bulb. ORNs expressing the same odorant receptor innervate common targets in a highly organized neuropilar structure inside the AL, the glomerulus. During normal development, ORNs target to specific regions of the AL and segregate into subclass-specific aggregates called protoglomeruli prior to extensive intermingling with target dendrites to form mature glomeruli. Using a panel of ORN subclass-specific markers, we demonstrate that in the adult AL, N-cadherin (N-cad) mutant ORN terminals remain segregated from dendrites of target neurons. N-cad plays a crucial role in protoglomerulus formation but is largely dispensible for targeting to the appropriate region of the AL. We propose that N-cad, a homophilic cell adhesion molecule, acts in a permissive fashion to promote subclass-specific sorting of ORN axon terminals into protoglomeruli.

During neurotransmitter release, the neuronal SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 form a four-helix bundle, the SNARE complex, that pulls the synaptic vesicle and plasma membranes together possibly causing membrane fusion. Complexin binds tightly to the SNARE complex and is essential for efficient Ca2+-evoked neurotransmitter release. A combined X-ray and TROSY-based NMR study now reveals the atomic structure of the complexin/SNARE complex. Complexin binds in an antiparallel α-helical conformation to the groove between the synaptobrevin and syntaxin helices. This interaction stabilizes the interface between these two helices, which bears the repulsive forces between the apposed membranes. These results suggest that complexin stabilizes the fully assembled SNARE complex as a key step that enables the exquisitely high speed of Ca2+-evoked neurotransmitter release.

Syntaxin, vesicle-associated membrane protein (VAMP), and synaptosome-associated protein of 25 kDa (SNAP-25) form a ternary "core complex" central to the process of synaptic vesicle docking and fusion. Several lines of evidence support the hypothesis that the proteins assemble in a coiled-coil structure, but the alignment of α helices in this coil and the overall conformation of the coil are unknown. We employ the technique of fluorescence resonance energy transfer (FRET) to investigate the alignment between syntaxin and VAMP. With the acceptor probe coupled to the amino-terminal end of the VAMP coiled-coil domain, the donor probe fluorescence is quenched to a greater extent when it is on the amino-terminal end of the syntaxin H3 domain than when it is on the carboxy-terminal end. The data indicate that syntaxin and VAMP bind primarily in a parallel arrangement and suggest a coiled-coil structure that is bent rather than fully extended. We propose a model in which binding of SNAP receptor (SNARE) protein coiled-coil domains helps drive vesicle fusion.

N-type Ca 2+ channels bind directly to the synaptic core complex of VAMP/synaptobrevin, syntaxin, and SNAP-25. Peptides containing the synaptic protein interaction ("synprint") site caused dissociation of N-type Ca 2+ channels from the synaptic core complex. Introduction of synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibited synaptic transmission. Fast EPSPs due to synchronous transmitter release were inhibited, while late EPSPs arising from asynchronous release following a train of action potentials were increased and paired-pulse facilitation was increased. The corresponding peptides from L-type Ca 2+ channels had no effect, and the N-type peptides had no effect on Ca 2+ currents through N-type Ca 2+ channels. These results are consistent with the hypothesis that binding of the synaptic core complex to presynaptic N-type Ca 2+ channels is required for Ca 2+ influx to elicit rapid, synchronous neurotransmitter release.

Synaptic vesicles are proposed to dock at the presynaptic plasma membrane through the interaction of two integral membrane proteins of synaptic vesicles, VAMP and synaptotagmin, and two plasma membrane proteins, syntaxin and SNAP-25. We have characterized the binding properties of these proteins and observed SNAP-25 potentiation of VAMP 2 binding to syntaxins la and 4 but not syntaxins 2 or 3. n-sec1, a neuron-specific syntaxin-binding protein, bound syntaxin with nanomolar affinity, forming a complex that is distinct from the previously identified 7S and 20S syntaxin-containing complexes. This suggests that syntaxin exists in at least three states: bound to n-sec1, in a 7S particle, and in a 20S particle. Recombinant n-secl inhibited VAMP or SNAP-25 binding to syntaxin. We propose that the specific associations of VAMP, SNAP-25, and syntaxin mediate vesicle docking and that a syntaxin/n-sec1 complex precedes and/or regulates formation of these complexes.

SummaryRecent studies suggest that synaptic vesicles (SVs) giving rise to spontaneous neurotransmission are distinct from those that carry out evoked release. However, the molecular basis of this dichotomy remains unclear. Here, we focused on two noncanonical SNARE molecules, Vps10p-tail-interactor-1a (vti1a) and VAMP7, previously shown to reside on SVs. Using simultaneous multicolor imaging at individual synapses, we could show that compared to the more abundant vesicular SNARE synaptobrevin2, both vti1a and VAMP7 were reluctantly mobilized during activity. Vti1a, but not VAMP7, showed robust trafficking under resting conditions that could be partly matched by synaptobrevin2. Furthermore, loss of vti1a function selectively reduced high-frequency spontaneous neurotransmitter release detected postsynaptically. Expression of a truncated version of vti1a augmented spontaneous release more than full-length vti1a, suggesting that an autoinhibitory process regulates vti1a function. Taken together, these results support the premise that in its native form vti1a selectively maintains spontaneous neurotransmitter release.

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 the first 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 which 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 which can endow the vesicles with distinct properties.

SummaryThe mechanism coupling exocytosis and endocytosis remains to be elucidated at central synapses. Here, we show that the mechanism linking these two processes is dependent on microdomain-[Ca2+]i similar to that which triggers exocytosis, as well as the exocytotic protein synaptobrevin/VAMP. Furthermore, block of endocytosis has a limited, retrograde action on exocytosis, delaying recruitment of release-ready vesicles and enhancing short-term depression. This effect sets in so rapidly that it cannot be explained by the nonavailability of recycled vesicles. Rather, we postulate that perturbation of a step linking exocytosis and endocytosis temporarily prevents new vesicles from docking at specialized sites for exocytosis.

SummaryIn order to release neurotransmitter synchronously in response to a presynaptic action potential, synaptic vesicles must be both release competent and located close to presynaptic Ca2+ channels. It has not been shown, however, which of the two is the more decisive factor. We tested this issue at the calyx of Held synapse by combining Ca2+ uncaging and electrophysiological measurements of postsynaptic responses. After depletion of the synaptic vesicles that are responsible for synchronous release during action potentials, uniform elevation of intracellular Ca2+ by Ca2+ uncaging could still elicit rapid release. The Ca2+ sensitivity of remaining vesicles was reduced no more than 2-fold, which is insufficient to explain the slow-down of the kinetics of release (10-fold) observed during a depolarizing pulse. We conclude that recruitment of synaptic vesicles to sites where Ca2+ channels cluster, rather than fusion competence, is a limiting step for rapid neurotransmitter release in response to presynaptic action potentials.

We have developed a genetic approach to examine the role of spontaneous activity and synaptic release in the establishment and maintenance of an olfactory sensory map. Conditional expression of tetanus toxin light chain, a molecule that inhibits synaptic release, does not perturb targeting during development, but neurons that express this molecule in a competitive environment fail to maintain appropriate synaptic connections and disappear. Overexpression of the inward rectifying potassium channel, Kir2.1, diminishes the excitability of sensory neurons and more severely disrupts the formation of an olfactory map. These studies suggest that spontaneous neural activity is required for the establishment and maintenance of the precise connectivity inherent in an olfactory sensory map.

We have investigated the trafficking of two endogenous axonal membrane proteins, VAMP2 and NgCAM, in order to elucidate the cellular events that underlie their polarization. We found that VAMP2 is delivered to the surface of both axons and dendrites, but preferentially endocytosed from the dendritic membrane. A mutation in the cytoplasmic domain of VAMP2 that inhibits endocytosis abolished its axonal polarization. In contrast, the targeting of NgCAM depends on sequences in its ectodomain, which mediate its sorting into carriers that preferentially deliver their cargo proteins to the axonal membrane. These observations show that neurons use two distinct mechanisms to polarize proteins to the axonal domain: selective retention in the case of VAMP2, selective delivery in the case of NgCAM.

We have investigated mechanisms in postendocytic processing of synaptic vesicles at hippocampal synapses, using synaptobrevin/vesicle-associated membrane protein (VAMP) tagged with variants of the green fluorescent protein. Following exocytosis, VAMP is retrieved at synaptic and adjoining axonal regions. Retrieved VAMP-containing vesicles return to synaptic vesicle clusters at a rate slower than endocytosis. Vesicles containing a different protein, synaptophysin, recluster at a similar rate, suggesting common vesicular intermediates for the two proteins. Activity prolongs the time taken by endocytosed vesicles to return to synapses. Exogenous calcium buffers slow endocytosis but have no additional effect on the time course of reclustering. In contrast, the protein kinase inhibitor staurosporine does not affect endocytosis but slows reclustering. Finally, since VAMP can move freely on surface membranes, sustained synaptic activity leads to mixing of this vesicular component between adjacent synapses.

Intracellular membrane fusion is mediated by the formation of a four-helix bundle comprised of SNARE proteins. Every cell expresses a large number of SNARE proteins that are localized to particular membrane compartments, suggesting that the fidelity of vesicle trafficking might in part be determined by specific SNARE pairing. However, the promiscuity of SNARE pairing in vitro suggests that the information for membrane compartment organization is not encoded in the inherent ability of SNAREs to form complexes. Here, we show that exocytosis of norepinephrine from PC12 cells is only inhibited or rescued by specific SNAREs. The data suggest that SNARE pairing does underlie vesicle trafficking fidelity, and that specific SNARE interactions with other proteins may facilitate the correct pairing.

Piccolo is a novel component of the presynaptic cytoskeletal matrix (PCM) assembled at the active zone of neurotransmitter release. Analysis of its primary structure reveals that Piccolo is a multidomain zinc finger protein structurally related to Bassoon, another PCM protein. Both proteins were found to be shared components of glutamatergic and GABAergic CNS synapses but not of the cholinergic neuromuscular junction. The Piccolo zinc fingers were found to interact with the dual prenylated rab3A and VAMP2/Synaptobrevin II receptor PRA1. We show that PRA1 is a synaptic vesicle–associated protein that is colocalized with Piccolo in nerve terminals of hippocampal primary neurons. These data suggest that Piccolo plays a role in the trafficking of synaptic vesicles (SVs) at the active zone.

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that forms the SNARE complex with VAMP/synaptobrevin and SNAP-25. Identifying proteins that modulate SNARE complex formation is critical for understanding the molecular mechanisms underlying neurotransmitter release and its modulation. We have cloned and characterized a protein called syntaphilin that is selectively expressed in brain. Syntaphilin competes with SNAP-25 for binding to syntaxin-1 and inhibits SNARE complex formation by absorbing free syntaxin-1. Transient overexpression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls free syntaxin-1 availability for the assembly of the SNARE complex, and thereby regulates synaptic vesicle exocytosis.

Exocytosis requires the fusion of vesicular and plasma membranes and results in the release of vesicle contents into the extracellular space. This membrane fusion event is mediated in large part by specific interactions of proteins on the vesicle membrane (v-SNAREs) with proteins on the target membrane (t-SNAREs) (6xRothman, J.E. Nature. 1994; 372: 55–63CrossRef | PubMed | Scopus (1801)See all References, 1xBajjalieh, S.M. and Scheller, R.H. J. Biol. Chem. 1995; 270: 1971–1974CrossRef | PubMed | Scopus (165)See all References, 7xSudhof, T.C. Nature. 1995; 375: 645–653CrossRef | PubMedSee all References). In neurotransmitter release, for example, the vesicle protein VAMP (also called synaptobrevin) forms a tight SDS-resistant protein complex with two target membrane proteins: syntaxin and SNAP-25. This trimeric protein complex has been proposed to constitute part of the minimal machinery required for membrane fusion (Jahn and Hanson 1998xJahn, R. and Hanson, P.I. Nature. 1998; 393: 14–15CrossRef | PubMed | Scopus (71)See all ReferencesJahn and Hanson 1998).SNARE complexes assemble into a four-helix bundle formed by the 70 membrane-proximal residues of SNARE proteins. The minimal core consists of two helical domains from SNAP-25 and one from syntaxin arranged in parallel with one helical domain from VAMP (Fasshauer et al. 1998xFasshauer, D., Eliason, W.K., Brunger, A.T., and Jahn, R. Biochemistry. 1998; 37: 10354–10362CrossRef | PubMed | Scopus (175)See all ReferencesFasshauer et al. 1998). Importantly, the generation of the SNARE core complexes is highly regulated. Despite their ability to form SDS-resistant complexes, most VAMP, syntaxin, and SNAP-25 proteins are not bound to each other under steady state conditions. Instead, it appears that the availability of syntaxin and VAMP proteins to form SNARE complexes is limited by their interactions with other proteins, including Munc18 (also called rSec1) and synaptophysin, respectively. Munc18 in vitro competitively inhibits the binding of SNAP-25 or VAMP to syntaxin-1a (Pevsner et al. 1994xPevsner, J., Hsu, S.C., Braun, J.E., Calakos, N., Ting, A.E., Bennett, M.K., and Scheller, R.H. Neuron. 1994; 13: 353–361Abstract | Full Text PDF | PubMed | Scopus (473)See all ReferencesPevsner et al. 1994); yet, how these complexes are rearranged during the priming and triggering of exocytosis remains to be determined.Recently, a new syntaxin-1–binding protein, tomosyn, was shown to have the ability to displace Munc18 from syntaxin-1 and subsequently to form a new complex with syntaxin, SNAP-25 and synaptotagmin (a syntaxin-binding protein) (Fujita et al. 1998xFujita, Y., Shirataki, H., Sakisaka, T., Asakura, T., Ohya, T., Kotani, H., Yokoyama, S., Nishioka, H., Matsuura, Y., Mizoguchi, A. et al. Neuron. 1998; 20: 905–915Abstract | Full Text | Full Text PDF | PubMed | Scopus (188)See all ReferencesFujita et al. 1998). Although the exact nature of the interaction was not determined, tomosyn was found to interact with the coiled coil domain of syntaxin-1a. Furthermore, it was also suggested that tomosyn would be later replaced by VAMP to form the SNARE complex required for membrane fusion, since the characterization of the SNARE proteins present in different high molecular complexes showed that the presence of tomosyn and VAMP was mutually exclusive (Fujita et al. 1998xFujita, Y., Shirataki, H., Sakisaka, T., Asakura, T., Ohya, T., Kotani, H., Yokoyama, S., Nishioka, H., Matsuura, Y., Mizoguchi, A. et al. Neuron. 1998; 20: 905–915Abstract | Full Text | Full Text PDF | PubMed | Scopus (188)See all ReferencesFujita et al. 1998). In this way, tomosyn may play a key role in regulating the generation of fusion-competent SNARE complexes.We have started, among several approaches, to search for proteins that interact with SNARE proteins from mast cells using a yeast two-hybrid system in order to identify and map regulatory components of the exocytic process in these cells. Interestingly, using SNAP-23 (the counterpart for SNAP-25 in mast cells) as a bait, we found 3 different interacting clones among 3 × 106 clones screened which encoded the carboxyl terminus of tomosyn. Similarly, 6 different clones encoding the carboxyl terminus of tomosyn were recovered among 3 × 107 clones screened using syntaxin-4 as a bait. These results indicate that tomosyn, like VAMP, has the capacity to bind not only syntaxin-1a but also syntaxin-4 and SNAP-23. The results also show that tomosyn is expressed in mast cells, specifically the murine mast cell line MC-9, and suggest that, as in neurons and PC12 cells, tomosyn plays a role in the regulation of mast cell exocytosis.Altogether, the 9 cDNA clones encoded peptides containing the carboxy-terminal 82–142 amino acid residues of tomosyn. This defines the carboxyl terminus as the domain of tomosyn that interacts with the coiled coil domains of syntaxin and SNAP-23. Analysis of this carboxy-terminal interacting domain of tomosyn with the COILS program (21 amino acid window size) predicted a coiled coil centered on arginine 1188 with a probability of 0.9. In a blast search of the nonredundant database, two VAMP homologs are the fourth and fifth highest scores behind tomosyn itself and previously discussed tumor suppressor–related genes. In addition, the VAMP1 homology is 10 and the VAMP4 homology is 8 standard deviations above the mean randomized homology found between the sequences (Figure 1Figure 1). Comparison to several other known coiled coil sequences results in insignificant scores ranging from 0–4 standard deviations above the mean randomized homology. Interestingly, sensitive profile searches based on the coiled coil domains of the SNARE proteins have shown that all SNARE coiled coils are distantly related (Weimbs et al. 1998xWeimbs, T., Mostov, K., and Hui Low, S. Trends Cell Biol. 1998; 8: 260–262Abstract | Full Text | Full Text PDF | PubMed | Scopus (100)See all ReferencesWeimbs et al. 1998). Moreover, alignment of the coiled coil domains of the SNARE superfamily reveal the conservation of an arginine residue in the d position of a central heptad repeat in all VAMP-like SNAREs. A glutamine residue replaces the arginine in all other SNARE coiled coils (Weimbs et al. 1998xWeimbs, T., Mostov, K., and Hui Low, S. Trends Cell Biol. 1998; 8: 260–262Abstract | Full Text | Full Text PDF | PubMed | Scopus (100)See all ReferencesWeimbs et al. 1998). The tomosyn predicted coiled coil includes the conserved arginine at position d in the central heptad repeat of the α helix (Figure 1Figure 1). This finding thus argues that tomosyn, like VAMP proteins, is capable of forming trimeric complexes with t-SNAREs and is consistent with the proposed replacement of tomosyn by VAMP during the exocytic process. We propose that tomosyn is the first of a class of membrane trafficking regulators that utilizes a SNARE coil domain to substitute for one of the components of the four-helix bundle that makes up the core complex. Further characterization of the tomosyn molecule should shed light on the regulation of dissociation and association of SNARE complexes during the exocytic membrane fusion events.Figure 1Schematic Representation of Tomosyn and Sequence Alignment of Its Carboxy-Terminal Domain Residues with the Coiled Coil Sequences of Representative VAMP Family ProteinsThe amino acid sequence of the 82 residues from the smallest interacting domain of murine tomosyn was identical to the sequence from rat tomosyn (black box). Numbers indicating the position of tomosyn residues correspond to the rat sequence (ra Tomosyn, U92072). GenBank accession numbers for the other sequences are as follows: mu VAMP1, U61751; mu VAMP4, AF061516; and sc Snc1, U12980 (mu, murine; sc, S. cerevisiae). Black boxes indicate identical residues present in all sequences. White boxes indicate positions that show 75% match (3 out of 4) to similar amino acid residues. The black triangle marks the arginine (R) residue conserved in all VAMP family members. The a and d positions in the heptad repeats are marked by black dots below the alignment.View Large Image | View Hi-Res Image | Download PowerPoint Slide

In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.

We have used the squid giant synapse to determine the role of synaptobrevin, integral membrane proteins of small synaptic vesicles, in neurotransmitter release. The sequence of squid synaptobrevin, deduced by cDNA cloning, is 65%–68% identical to mammalian isoforms and includes the conserved cleavage site for tetanus and botulinum B toxins. Injection of either toxin into squid nerve terminals caused a slow, irreversible inhibition of release without affecting the Ca 2+ signal which triggers release. Microinjection of a recombinant protein corresponding to the cytoplasmic domain of synaptobrevin produced a more rapid and reversible inhibition of release, whereas two smaller peptide fragments were without effect. Electron microscopy of tetanus-injected terminals revealed an increased number of both docked and undocked synaptic vesicles. These data indicate that synaptobrevin participates in neurotransmitter release at a step between vesicle docking and fusion.

Regulated delivery and removal of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) from postsynaptic elements has been proposed as a mechanism for regulating synaptic strength. Here we test the role of ubiquitin in regulating synapses that contain a C. elegans GluR, GLR-1. GLR-1 receptors were ubiquitinated in vivo. Mutations that decreased ubiquitination of GLR-1 increased the abundance of GLR-1 at synapses and altered locomotion behavior in a manner that is consistent with increased synaptic strength. By contrast, overexpression of ubiquitin decreased the abundance of GLR-1 at synapses and decreased the density of GLR-1-containing synapses, and these effects were prevented by mutations in the unc-11 gene, which encodes a clathrin adaptin protein (AP180). These results suggest that ubiquitination of GLR-1 receptors regulates synaptic strength and the formation or stability of GLR-1-containing synapses.

Summary Reelin is a glycoprotein that is critical for proper layering of neocortex during development as well as dynamic regulation of glutamatergic postsynaptic signaling in mature synapses. Here, we show that Reelin also acts presynaptically, resulting in robust rapid enhancement of spontaneous neurotransmitter release without affecting properties of evoked neurotransmission. This effect of Reelin requires a modest but significant increase in presynaptic Ca 2+ initiated via ApoER2 signaling. The specificity of Reelin action on spontaneous neurotransmitter release is encoded at the level of vesicular SNARE machinery as it requires VAMP7 and SNAP-25 but not synaptobrevin2, VAMP4, or vti1a. These results uncover a presynaptic regulatory pathway that utilizes the heterogeneity of synaptic vesicle-associated SNAREs and selectively augments action potential-independent neurotransmission.

Summary Cerebellar basket cells inhibit postsynaptic Purkinje cells in a rapid and precise manner. To investigate the mechanisms of transmitter release underlying this rapid inhibition, Ca 2+ uncaging was employed to measure the intracellular Ca 2+ dependence of transmitter release and the kinetics of synaptic vesicle pool transitions in immature basket cell synapses at room temperature. Vesicle release properties distinct from those previously observed at excitatory synapses were seen, including a relatively high intracellular Ca 2+ sensitivity of vesicle fusion, rapid vesicle pool mobilization with few reluctant vesicles, and vesicle replenishment driven by unusually high Ca 2+ levels from both local and residual Ca 2+ sources during action potential trains. These results suggest that inhibitory basket cell synapses are optimized for rapid and precise temporal and spatial Ca 2+ coordination of synaptic vesicle fusion and replenishment, which may contribute to the unique physiology of inhibitory synaptic transmission, including phasic release during action potential trains and tonic release by residual intracellular Ca 2+ .

The structure of synaptobrevin, an intrinsic membrane protein of small synaptic vesicles from mammalian brain, was studied by purification and molecular cloning. Its message in bovine brain encodes a 116 amino acid protein whose sequence reveals it to be the mammalian homolog of Torpedo VAMP-1. Antibody probing demonstrates that the protein is also present in Drosophila, and its Drosophila homolog was cloned. Alignment of the sequences of synaptobrevin/VAMP-1 from the three species shows it to contain four domains, including a highly conserved central region of 63 amino acids that contains 75% invariant residues. The finding that a membrane protein from vertebrate synaptic vesicles is conserved in Drosophila points toward a central role of this protein in neurotransmission and should allow a genetic approach to neurotransmitter release.