S-SCAM is essential for synapse formation.

Synapse formation is critical for the wiring of neural circuits in the developing brain. The synaptic scaffolding protein S-SCAM/MAGI-2 has important roles in the assembly of signaling complexes at post-synaptic densities. However, the role of S-SCAM in establishing the entire synapse is not known. Here, we report significant effects of RNAi-induced S-SCAM knockdown on the number of synapses in early stages of network development in vitro. In vivo knockdown during the first three postnatal weeks reduced the number of dendritic spines in the rat brain neocortex. Knockdown of S-SCAM in cultured hippocampal neurons severely reduced the clustering of both pre- and post-synaptic components. This included synaptic vesicle proteins, pre- and post-synaptic scaffolding proteins, and cell adhesion molecules, suggesting that entire synapses fail to form. Correspondingly, functional and morphological characteristics of developing neurons were affected by reducing S-SCAM protein levels; neurons displayed severely impaired synaptic transmission and reduced dendritic arborization. A next-generation sequencing approach showed normal expression of housekeeping genes but changes in expression levels in 39 synaptic signaling molecules in cultured neurons. These results indicate that S-SCAM mediates the recruitment of all key classes of synaptic molecules during synapse assembly and is critical for the development of neural circuits in the developing brain.

The synaptic vesicle protein Mover/TPRG1L is associated with lipid droplets in astrocytes.

Crucial brain functions such as neurotransmission, myelination, and signaling pose a high demand for lipids. Lipid dysregulation is associated with neuroinflammation and neurodegeneration. Astrocytes protect neurons from lipid induced damage by accumulating and metabolizing toxic lipids in organelles called lipid droplets (LDs). LDs have long been considered as lipid storage compartments in adipocytes, but less is known about their biogenesis and composition in the brain. In particular, proteins covering the LD surface are not yet fully identified. Here, we report that the presynaptic protein Mover/TPRG1L, which regulates the probability of neurotransmitter release in neurons, is a component of the LD coat in astrocytes. Using conventional and super-resolution microscopy, we demonstrate that Mover surrounds naive and oleic acid induced astrocytic LDs. We confirm the identity of astrocytic LDs using the neutral lipid stains Bodipy and LipidTox, as well as immunofluorescence for perilipin-2, a known component of the LD coat. In astrocytes, recombinant Mover was sufficient to induce an accumulation of LDs. Furthermore, we identified point mutations that abolish targeting to LDs and show similarities in the required binding sequences for association to the presynapse and LDs. Our results show that Mover is not only a presynaptic protein but also a candidate for LD regulation. This highlights the dual role of Mover in synaptic transmission and regulation of astrocytic LDs, which may be particularly important in the context of lipid-related neurological disorders.

Ablation of the Presynaptic Protein Mover Impairs Learning Performance and Decreases Anxiety Behavior in Mice.

The presynaptic protein Mover/TPRGL/SVAP30 is absent in Drosophila and C. elegans and differentially expressed in synapses in the rodent brain, suggesting that it confers specific functions to subtypes of presynaptic terminals. In order to investigate how the absence of this protein affects behavior and learning, Mover knockout mice (KO) were subjected to a series of established learning tests. To determine possible behavioral and cognitive alterations, male and female 8-week-old KO and C57Bl/6J wildtype (WT) control mice were tested in a battery of memory and anxiety tests. Testing included the cross maze, novel object recognition test (NOR), the Morris water maze (MWM), the elevated plus maze (EPM), and the open field test (OF). Mover KO mice showed impaired recognition memory in the NOR test, and decreased anxiety behavior in the OF and the EPM. Mover KO did not lead to changes in working memory in the cross maze or spatial reference memory in the MWM. However, a detailed analysis of the swimming strategies demonstrated allocentric-specific memory deficits in male KO mice. Our data indicate that Mover appears to control synaptic properties associated with specific forms of memory formation and behavior, suggesting that it has a modulatory role in synaptic transmission.

Nanoscopical Analysis Reveals an Orderly Arrangement of the Presynaptic Scaffold Protein Bassoon at the Golgi-Apparatus.

Bassoon is a core scaffold protein of the presynaptic active zone. In brain synapses, the C-terminus of Bassoon is oriented toward the plasma membrane and its N-terminus is oriented toward synaptic vesicles. At the Golgi-apparatus, Bassoon is thought to assemble active zone precursor structures, but whether it is arranged in an orderly fashion is unknown. Understanding the topology of this large scaffold protein is important for models of active zone biogenesis. Using stimulated emission depletion nanoscopy in cultured hippocampal neurons, we found that an N-terminal intramolecular tag of recombinant Bassoon, but not C-terminal tag, colocalized with markers of the trans-Golgi network (TGN). The N-terminus of Bassoon was located between 48 and 69 nm away from TGN38, while its C-terminus was located between 100 and 115 nm away from TGN38. Sequences within the first 95 amino acids of Bassoon were required for this arrangement. Our results indicate that, at the Golgi-apparatus, Bassoon is oriented with its N-terminus toward and its C-terminus away from the trans Golgi network membrane. Moreover, they suggest that Bassoon is an extended molecule at the trans Golgi network with the distance between amino acids 97 and 3,938, estimated to be between 46 and 52 nm. Our data are consistent with a model, in which the N-terminus of Bassoon binds to the membranes of the trans-Golgi network, while the C-terminus associates with active zone components, thus reflecting the topographic arrangement characteristic of synapses also at the Golgi-apparatus.

Neuroligin-1 mediates presynaptic maturation through brain-derived neurotrophic factor signaling.

BACKGROUND: Maturation is a process that allows synapses to acquire full functionality, optimizing their activity to diverse neural circuits, and defects in synaptic maturation may contribute to neurodevelopmental disorders. Neuroligin-1 (NL1) is a postsynaptic cell adhesion molecule essential for synapse maturation, a role typically attributed to binding to pre-synaptic ligands, the neurexins. However, the pathways underlying the action of NL1 in synaptic maturation are incompletely understood, and some of its previously observed effects seem reminiscent of those described for the neurotrophin brain-derived neurotrophic factor (BDNF). Here, we show that maturational increases in active zone stability and synaptic vesicle recycling rely on the joint action of NL1 and brain-derived neurotrophic factor (BDNF). RESULTS: Applying BDNF to hippocampal neurons in primary cultures or organotypical slice cultures mimicked the effects of overexpressing NL1 on both structural and functional maturation. Overexpressing a NL1 mutant deficient in neurexin binding still induced presynaptic maturation. Like NL1, BDNF increased synaptic vesicle recycling and the augmentation of transmitter release by phorbol esters, both hallmarks of presynaptic maturation. Mimicking the effects of NL1, BDNF also increased the half-life of the active zone marker bassoon at synapses, reflecting increased active zone stability. Overexpressing NL1 increased the expression and synaptic accumulation of BDNF. Inhibiting BDNF signaling pharmacologically or genetically prevented the effects of NL1 on presynaptic maturation. Applying BDNF to NL1-knockout mouse cultures rescued defective presynaptic maturation, indicating that BDNF acts downstream of NL1 and can restore presynaptic maturation at late stages of network development. CONCLUSIONS: Our data introduce BDNF as a novel and essential component in a transsynaptic pathway linking NL1-mediated cell adhesion, neurotrophin action, and presynaptic maturation. Our findings connect synaptic cell adhesion and neurotrophin signaling and may provide a therapeutic approach to neurodevelopmental disorders by targeting synapse maturation.

Regulation of a subset of release-ready vesicles by the presynaptic protein Mover.

Neurotransmitter release occurs by regulated exocytosis from synaptic vesicles (SVs). Evolutionarily conserved proteins mediate the essential aspects of this process, including the membrane fusion step and priming steps that make SVs release-competent. Unlike the proteins constituting the core fusion machinery, the SV protein Mover does not occur in all species and all synapses. Its restricted expression suggests that Mover may modulate basic aspects of transmitter release and short-term plasticity. To test this hypothesis, we analyzed synaptic transmission electrophysiologically at the mouse calyx of Held synapse in slices obtained from wild-type mice and mice lacking Mover. Spontaneous transmission was unaffected, indicating that the basic release machinery works in the absence of Mover. Evoked release and vesicular release probability were slightly reduced, and the paired pulse ratio was increased in Mover knockout mice. To explore whether Mover's role is restricted to certain subpools of SVs, we analyzed our data in terms of two models of priming. A model assuming two SV pools in parallel showed a reduced release probability of so-called "superprimed vesicles" while "normally primed" ones were unaffected. For the second model, which holds that vesicles transit sequentially from a loosely docked state to a tightly docked state before exocytosis, we found that knocking out Mover selectively decreased the release probability of tight state vesicles. These results indicate that Mover regulates a subclass of primed SVs in the mouse calyx of Held.

The Calmodulin Binding Region of the Synaptic Vesicle Protein Mover Is Required for Homomeric Interaction and Presynaptic Targeting.

Neurotransmitter release is mediated by an evolutionarily conserved machinery. The synaptic vesicle (SV) associated protein Mover/TPRGL/SVAP30 does not occur in all species and all synapses. Little is known about its molecular properties and how it may interact with the conserved components of the presynaptic machinery. Here, we show by deletion analysis that regions required for homomeric interaction of Mover are distributed across the entire molecule, including N-terminal, central and C-terminal regions. The same regions are also required for the accumulation of Mover in presynaptic terminals of cultured neurons. Mutating two phosphorylation sites in N-terminal regions did not affect these properties. In contrast, a point mutation in the predicted Calmodulin (CaM) binding sequence of Mover abolished both homomeric interaction and presynaptic targeting. We show that this sequence indeed binds Calmodulin, and that recombinant Mover increases Calmodulin signaling upon heterologous expression. Our data suggest that presynaptic accumulation of Mover requires homomeric interaction mediated by regions distributed across large areas of the protein, and corroborate the hypothesis that Mover functionally interacts with Calmodulin signaling.

Differential Effect on Hippocampal Synaptic Facilitation by the Presynaptic Protein Mover.

Neurotransmitter release relies on an evolutionarily conserved presynaptic machinery. Nonetheless, some proteins occur in certain species and synapses, and are absent in others, indicating that they may have modulatory roles. How such proteins expand the power or versatility of the core release machinery is unclear. The presynaptic protein Mover/TPRGL/SVAP30 is heterogeneously expressed among synapses of the rodent brain, suggesting that it may add special functions to subtypes of presynaptic terminals. Mover is a synaptic vesicle-attached phosphoprotein that binds to Calmodulin and the active zone scaffolding protein Bassoon. Here we use a Mover knockout mouse line to investigate the role of Mover in the hippocampal mossy fiber (MF) to CA3 pyramidal cell synapse and Schaffer collateral to CA1. While Schaffer collateral synapses were unchanged by the knockout, the MFs showed strongly increased facilitation. The effect of Mover knockout in facilitation was both calcium- and age-dependent, having a stronger effect at higher calcium concentrations and in younger animals. Increasing cyclic adenosine monophosphate (cAMP) levels by forskolin equally potentiated both wildtype and knockout MF synapses, but occluded the increased facilitation observed in the knockout. These discoveries suggest that Mover has distinct roles at different synapses. At MF terminals, it acts to constrain the extent of presynaptic facilitation.

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

The Presynaptic Protein Mover Is Differentially Expressed Across Brain Areas and Synapse Types.

The assembly and function of presynaptic nerve terminals relies on evolutionarily conserved proteins. A small number of presynaptic proteins occurs only in vertebrates. These proteins may add specialized functions to certain synapses, thus increasing synaptic heterogeneity. Here, we show that the vertebrate-specific synaptic vesicle (SV) protein mover is differentially distributed in the forebrain and cerebellum of the adult mouse. Using a quantitative immunofluorescence approach, we compare the expression of mover to the expression of the general SV marker synaptophysin in 16 brain areas. We find that mover is particularly abundant in the septal nuclei (SNu), ventral pallidum (VPa), amygdala and hippocampus. Within the hippocampus, mover is predominantly associated with excitatory synapses. Its levels are low in layers that receive afferent input from the entorhinal cortex, and high in layers harboring intra-hippocampal circuits. In contrast, mover levels are high in all nuclei of the amygdala, and mover is associated with inhibitory synapses in the medioposterior amygdala. Our data reveal a striking heterogeneity in the abundance of mover on three levels, i.e., between brain areas, within individual brain areas and between synapse types. This distribution suggests a role for mover in providing specialization to subsets of synapses, thereby contributing to the functional diversity of brain areas.

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins.

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

The Kohlschütter-Tönz syndrome associated gene Rogdi encodes a novel presynaptic protein.

Mutations in the human homolog of the Drosophila gene Rogdi cause Kohlschütter-Tönz syndrome. This disorder is characterised by amelogenesis imperfecta, as well as severe neurological symptoms including epilepsy and psychomotor delay. However, little is known about the protein encoded by Rogdi, and hence the pathogenic mechanisms underlying Kohlschütter-Tönz syndrome have remained elusive. Using immunofluorescence of rat cultured hippocampal neurons and brain sections we find that Rogdi is enriched at synaptic sites. In addition, recombinant GFP-Rogdi expressed in cultured neurons was efficiently targeted to presynaptic sites, where it colocalised with the presynaptic scaffolding protein Bassoon and the synaptic vesicle markers Synaptophysin, Synapsin-1, VAMP2/Synaptobrevin and Mover. Our data indicate that GFP-Rogdi harbours efficient signals for presynaptic targeting, and that Rogdi is a presynaptic protein. Thus, the neurological symptoms associated with Kohlschütter-Tönz syndrome may arise from presynaptic dysfunction.

Modulation of Presynaptic Release Probability by the Vertebrate-Specific Protein Mover.

Mover, a member of the exquisitely small group of vertebrate-specific presynaptic proteins, has been discovered as an interaction partner of the scaffolding protein Bassoon, yet its function has not been elucidated. We used adeno-associated virus (AAV)-mediated shRNA expression to knock down Mover in the calyx of Held in vivo. Although spontaneous synaptic transmission remained unaffected, we found a strong increase of the evoked EPSC amplitude. The size of the readily releasable pool was unaltered, but short-term depression was accelerated and enhanced, consistent with an increase in release probability after Mover knockdown. This increase in release probability was not caused by alterations in Ca(2+) influx but rather by a higher Ca(2+) sensitivity of the release machinery, as demonstrated by presynaptic Ca(2+) uncaging. We therefore conclude that Mover expression in certain subsets of synapses negatively regulates synaptic release probability, constituting a novel mechanism to tune synaptic transmission.

Bassoon-disruption slows vesicle replenishment and induces homeostatic plasticity at a CNS synapse.

Endbulb of Held terminals of auditory nerve fibers (ANF) transmit auditory information at hundreds per second to bushy cells (BCs) in the anteroventral cochlear nucleus (AVCN). Here, we studied the structure and function of endbulb synapses in mice that lack the presynaptic scaffold bassoon and exhibit reduced ANF input into the AVCN. Endbulb terminals and active zones were normal in number and vesicle complement. Postsynaptic densities, quantal size and vesicular release probability were increased while vesicle replenishment and the standing pool of readily releasable vesicles were reduced. These opposing effects canceled each other out for the first evoked EPSC, which showed unaltered amplitude. We propose that ANF activity deprivation drives homeostatic plasticity in the AVCN involving synaptic upscaling and increased intrinsic BC excitability. In vivo recordings from individual mutant BCs demonstrated a slightly improved response at sound onset compared to ANF, likely reflecting the combined effects of ANF convergence and homeostatic plasticity. Further, we conclude that bassoon promotes vesicular replenishment and, consequently, a large standing pool of readily releasable synaptic vesicles at the endbulb synapse.

Mover is a homomeric phospho-protein present on synaptic vesicles.

With remarkably few exceptions, the molecules mediating synaptic vesicle exocytosis at active zones are structurally and functionally conserved between vertebrates and invertebrates. Mover was found in a yeast-2-hybrid assay using the vertebrate-specific active zone scaffolding protein bassoon as a bait. Peptides of Mover have been reported in proteomics screens for self-interacting proteins, phosphorylated proteins, and synaptic vesicle proteins, respectively. Here, we tested the predictions arising from these screens. Using flotation assays, carbonate stripping of peripheral membrane proteins, mass spectrometry, immunogold labelling of purified synaptic vesicles, and immuno-organelle isolation, we found that Mover is indeed a peripheral synaptic vesicle membrane protein. In addition, by generating an antibody against phosphorylated Mover and Western blot analysis of fractionated rat brain, we found that Mover is a bona fide phospho-protein. The localization of Mover to synaptic vesicles is phosphorylation dependent; treatment with a phosphatase caused Mover to dissociate from synaptic vesicles. A yeast-2-hybrid screen, co-immunoprecipitation and cell-based optical assays of homomerization revealed that Mover undergoes homophilic interaction, and regions within both the N- and C- terminus of the protein are required for this interaction. Deleting a region required for homomeric interaction abolished presynaptic targeting of recombinant Mover in cultured neurons. Together, these data prove that Mover is associated with synaptic vesicles, and implicate phosphorylation and multimerization in targeting of Mover to synaptic vesicles and presynaptic sites.

Neuronal profilin isoforms are addressed by different signalling pathways.

Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF), on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei.

STED nanoscopy with mass-produced laser diodes.

We show that far-field fluorescence nanoscopy by stimulated emission depletion (STED) can be realized with compact off-the-shelf laser diodes, such as those used in laser pointers and DVDs. A spatial resolution of 40-50 nm is attained by pulsing a 660 nm DVD-diode. The efficacy of these low-cost STED microscopes in biological imaging is demonstrated by differentiating between clusters of the synaptic protein bassoon and transport vesicles in hippocampal neurons, based on the feature diameter. Our results facilitate the implementation of this all-molecular-transition based superresolution method in many applications ranging from nanoscale fluorescence imaging to nanoscale fluorescence sensing.

Postsynaptic Neuroligin1 regulates presynaptic maturation.

Presynaptic nerve terminals pass through distinct stages of maturation after their initial assembly. Here we show that the postsynaptic cell adhesion molecule Neuroligin1 regulates key steps of presynaptic maturation. Presynaptic terminals from Neuroligin1-knockout mice remain structurally and functionally immature with respect to active zone stability and synaptic vesicle pool size, as analyzed in cultured hippocampal neurons. Conversely, overexpression of Neuroligin1 in immature neurons, that is within the first 5 days after plating, induced the formation of presynaptic boutons that had hallmarks of mature boutons. In particular, Neuroligin1 enhanced the size of the pool of recycling synaptic vesicles, the rate of synaptic vesicle exocytosis, the fraction of boutons responding to depolarization, as well as the responsiveness of the presynaptic release machinery to phorbol ester stimulation. Moreover, Neuroligin1 induced the formation of active zones that remained stable in the absence of F-actin, another hallmark of advanced maturation. Acquisition of F-actin independence of the active zone marker Bassoon during culture development or induced via overexpression of Neuroligin1 was activity-dependent. The extracellular domain of Neuroligin1 was sufficient to induce assembly of functional presynaptic terminals, while the intracellular domain was required for terminal maturation. These data show that induction of presynaptic terminal assembly and maturation involve mechanistically distinct actions of Neuroligins, and that Neuroligin1 is essential for presynaptic terminal maturation.

Exchange and redistribution dynamics of the cytoskeleton of the active zone molecule bassoon.

Presynaptic sites typically appear as varicosities (boutons) distributed along axons. Ultrastructurally, presynaptic boutons lack obvious physical barriers that separate them from the axon proper, yet activity-related and constitutive dynamics continuously promote the "reshuffling" of presynaptic components and even their dispersal into flanking axonal segments. How presynaptic sites manage to maintain their organization and individual characteristics over long durations is thus unclear. Conceivably, presynaptic tenacity might depend on the active zone (AZ), an electron-dense specialization of the presynaptic membrane, and particularly on the cytoskeletal matrix associated with the AZ (CAZ) that could act as a relatively stable "core scaffold" that conserves and dictates presynaptic organization. At present, however, little is known on the molecular dynamics of CAZ molecules, and thus, the factual basis for this hypothesis remains unclear. To examine the stability of the CAZ, we studied the molecular dynamics of the major CAZ molecule Bassoon in cultured hippocampal neurons. Fluorescence recovery after photobleaching and photoactivation experiments revealed that exchange rates of green fluorescent protein and photoactivatable green fluorescent protein-tagged Bassoon at individual presynaptic sites are very low (tau > 8 h). Exchange rates varied between boutons and were only slightly accelerated by stimulation. Interestingly, photoactivation experiments revealed that Bassoon lost from one synapse was occasionally assimilated into neighboring presynaptic sites. Our findings indicate that Bassoon is engaged in relatively stable associations within the CAZ and thus support the notion that the CAZ or some of its components might constitute a relatively stable presynaptic core scaffold.

Polarized targeting of neurexins to synapses is regulated by their C-terminal sequences.

Two families of cell-adhesion molecules, predominantly presynaptic neurexins and postsynaptic neuroligins, are important for the formation and functioning of synapses in the brain, and mutations in several genes encoding these transmembrane proteins have been found in autism patients. However, very little is known about how neurexins are targeted to synapses and which mechanisms regulate this process. Using various epitope-tagged neurexins in primary hippocampal neurons of wild-type and knock-out mice in vitro and in transgenic animals in vivo, we show that neurexins are trafficked throughout neurons via transport vesicles and the plasma membrane insertion of neurexins occurs preferentially in the axonal/synaptic compartment. We also observed that exit of neurexins from the ER/Golgi and correct targeting require their PDZ-binding motif at the C terminus, whereas two presumptive ER retention signals are inactive. The ubiquitous presence of neurexin-positive transport vesicles and absence of bassoon colabeling demonstrate that these carriers are not active zone precursor vesicles, but colocalization with CASK, RIM1alpha, and calcium channels suggests that they may carry additional components of the exocytotic machinery. Our data indicate that neurexins are delivered to synapses by a polarized and regulated targeting process that involves PDZ-domain mediated interactions, suggesting a novel pathway for the distribution of neurexins and other synaptic proteins.