Prefrontal cortical network dysfunction from acute neurotoxicant exposure.

Prefrontal cortical (PFC) dysfunction has been linked to disorders exhibiting deficits in cognitive performance, attention, motivation, and impulse control. Neurons of the PFC are susceptible to glutamatergic excitotoxicity, an effect associated with cortical degeneration in frontotemporal disorders (FTD). PFC susceptibility to environmental toxicant exposure, one possible contributor to sporadic FTD, has not been systematically studied. Here, we tested the ability of a well-known environmental neurotoxicant, methylmercury (MeHg), to induce hyperexcitability in medial prefrontal cortex (mPFC) excitatory pyramidal neurons, using whole-cell patch-clamp recording. Acute MeHg exposure (20 µM) produced significant mPFC dysfunction, with a shift in the excitatory to inhibitory (E-I) balance toward increased excitability. Both EPSC and IPSC charge were significantly increased after MeHg exposure. MeHg increased EPSC frequency, but there was no observable effect on IPSC frequency, EPSC amplitude or IPSC amplitude. Neither evoked AMPAR- nor NMDAR-mediated EPSC amplitudes were affected by MeHg. However, excitatory synapses experienced a significant reduction in paired pulse depression and probability of release. In addition, MeHg induced temporal synchrony in spontaneous IPSCs, reflecting mPFC inhibitory network dysfunction. MeHg exposure also produced increased intrinsic excitability in mPFC neurons, with an increase in action potential firing rate. The observed effects of MeHg on mPFC reflect key potential mechanisms for neuropsychological symptoms from MeHg poisoning. Therefore, MeHg has a significant effect on mPFC circuits known to contribute to cognitive and emotional function and might contribute to etiology of neurodegenerative diseases, such as FTD.

Corrigendum: An Autism-Associated de novo Mutation in GluN2B Destabilizes Growing Dendrites by Promoting Retraction and Pruning.

[This corrects the article DOI: 10.3389/fncel.2021.692232.].

GRIN2B-related neurodevelopmental disorder: current understanding of pathophysiological mechanisms.

The GRIN2B-related neurodevelopmental disorder is a rare disease caused by mutations in the GRIN2B gene, which encodes the GluN2B subunit of NMDA receptors. Most individuals with GRIN2B-related neurodevelopmental disorder present with intellectual disability and developmental delay. Motor impairments, autism spectrum disorder, and epilepsy are also common. A large number of pathogenic de novo mutations have been identified in GRIN2B. However, it is not yet known how these variants lead to the clinical symptoms of the disease. Recent research has begun to address this issue. Here, we describe key experimental approaches that have been used to better understand the pathophysiology of this disease. We discuss the impact of several distinct pathogenic GRIN2B variants on NMDA receptor properties. We then critically review pivotal studies examining the synaptic and neurodevelopmental phenotypes observed when disease-associated GluN2B variants are expressed in neurons. These data provide compelling evidence that various GluN2B mutants interfere with neuronal differentiation, dendrite morphogenesis, synaptogenesis, and synaptic plasticity. Finally, we identify important open questions and considerations for future studies aimed at understanding this complex disease. Together, the existing data provide insight into the pathophysiological mechanisms that underlie GRIN2B-related neurodevelopmental disorder and emphasize the importance of comparing the effects of individual, disease-associated variants. Understanding the molecular, cellular and circuit phenotypes produced by a wide range of GRIN2B variants should lead to the identification of core neurodevelopmental phenotypes that characterize the disease and lead to its symptoms. This information could help guide the development and application of effective therapeutic strategies for treating individuals with GRIN2B-related neurodevelopmental disorder.

An Autism-Associated de novo Mutation in GluN2B Destabilizes Growing Dendrites by Promoting Retraction and Pruning.

Mutations in GRIN2B, which encodes the GluN2B subunit of NMDA receptors, lead to autism spectrum disorders (ASD), but the pathophysiological mechanisms remain unclear. Recently, we showed that a GluN2B variant that is associated with severe ASD (GluN2B724t) impairs dendrite morphogenesis. To determine which aspects of dendrite growth are affected by GluN2B724t, we investigated the dynamics of dendrite growth and branching in rat neocortical neurons using time-lapse imaging. GluN2B724t expression shifted branch motility toward retraction and away from extension. GluN2B724t and wild-type neurons formed new branches at similar rates, but mutant neurons exhibited increased pruning of dendritic branches. The observed changes in dynamics resulted in nearly complete elimination of the net expansion of arbor size and complexity that is normally observed during this developmental period. These data demonstrate that ASD-associated mutant GluN2B interferes with dendrite morphogenesis by reducing rates of outgrowth while promoting retraction and subsequent pruning. Because mutant dendrites remain motile and capable of growth, it is possible that reducing pruning or promoting dendrite stabilization could overcome dendrite arbor defects associated with GRIN2B mutations.

Effect of extracellular vesicles from S. aureus-challenged human neutrophils on macrophages.

Staphylococcus aureus enhances neutrophil extracellular vesicle (EV) production. To investigate whether S. aureus viability influences EV biogenesis, EVs were isolated from human neutrophils incubated with viable bacteria (bEVs) or heat-killed bacteria (heat-killed EVs). Protein analysis, nanoparticle tracking and transmission electron microscopy showed comparable EV production between subsets, and both viable and nonviable bacteria were also detected in respective EV subsets. As anticipated, S. aureus, as well as bEVs with viable bacteria, were proinflammatory, and killing bacteria with gentamicin reduced cytokine production to baseline levels. Although heat-killed bacteria induced macrophage IL-6 production, heat-killed EVs did not. Additionally, we found that human and bacterial DNA associated with bEVs, but not heat-killed EVs, and that the DNA association could be partially decreased by disrupting electrostatic interactions. We investigated the potential for DNA isolated from EVs (EV-DNA) or EVs to cause inflammation. Although liposomal encapsulation of EV-DNA increased IL-6 production from baseline by 7.5-fold, treatment of bEVs with DNase I had no effect on IL-6 and IL-1ß production, suggesting that the DNA did not contribute to the inflammatory response. Filtered EVs, which lacked DNA and associated bacteria, exhibited less proinflammatory activity relative to bEVs, and enhanced macrophage expression of CD86 and HLA-DR. Ultimately, we show that bEVs isolated by differential centrifugation co-purify with bacteria and DNA, and studying their concerted activity and relative contribution to immune response is important to the study of host-pathogen interactions.

Acute neurotoxicant exposure induces hyperexcitability in mouse lumbar spinal motor neurons.

Spinal motor neurons (MNs) are susceptible to glutamatergic excitotoxicity, an effect associated with lumbar MN degeneration in amyotrophic lateral sclerosis (ALS). MN susceptibility to environmental toxicant exposure, one prospective contributor to sporadic ALS, has not been systematically studied. The goal of this study was to test the ability of a well-known environmental neurotoxicant to induce hyperexcitability in mouse lumbar MNs. Methylmercury (MeHg) causes neurotoxicity through mechanisms involving elevated intracellular Ca2+ concentration ([Ca2+]i), a hallmark of excitotoxicity. We tested whether acute exposure to MeHg induces hyperexcitability in MNs by altering synaptic transmission, using whole cell patch-clamp recordings of lumbar spinal MNs in vitro. Acute MeHg exposure (20 µM) led to an increase in the frequency of both spontaneous excitatory postsynaptic currents (EPSCs) and miniature EPSCs. The frequency of inhibitory postsynaptic currents (IPSCs) was also increased by MeHg. Action potential firing rates, both spontaneous and evoked, were increased by MeHg, despite increases in both EPSCs and IPSCs, indicating a shift toward hyperexcitability. Also consistent with hyperexcitability, fluo 4-AM microfluorimetry indicated that MeHg exposure induced an increase in [Ca2+]i. Spinal cord hyperexcitability is partially mediated by Ca2+-permeable AMPA receptors, as MeHg-dependent increases in EPSCs were blocked by 1-napthyl spermine. Therefore, spinal MNs appear highly susceptible to MeHg exposure, leading to significant increases in spontaneous network excitability and disruption of normal function. Prolonged hyperexcitability could lead to eventual neurodegeneration and loss of motor function as observed in spinal cord after MeHg exposure in vivo and may contribute to MeHg-induced acceleration of ALS symptoms.NEW & NOTEWORTHY Spinal motor neurons (MN) are susceptible to glutamatergic excitotoxicity, an effect associated with lumbar MN degeneration in amyotrophic lateral sclerosis (ALS). This study investigated MN susceptibility to environmental toxicant exposure, one prospective contributor to sporadic ALS. Spinal MNs appear highly susceptible to methylmercury exposure, leading to significant increases in spontaneous network excitability and disruption of normal function. Prolonged hyperexcitability could lead to neurodegeneration and loss of motor function as observed in ALS spinal cord symptoms.

Spatio-temporal dynamics of neocortical presynaptic terminal development using multi-photon imaging of the corpus callosum in vivo.

Within the developing central nervous system, the dynamics of synapse formation and elimination are insufficiently understood. It is ideal to study these processes in vivo, where neurons form synapses within appropriate behavioral and anatomical contexts. In vivo analysis is particularly important for long-range connections, since their development cannot be adequately studied in vitro. The corpus callosum (CC) represents a clinically-relevant long-range connection since several neurodevelopmental diseases involve CC defects. Here, we present a novel strategy for in vivo longitudinal and rapid time-lapse imaging of CC presynaptic terminal development. In postnatal mice, the time-course of CC presynaptic terminal formation and elimination was highly variable between axons or groups of axons. Young presynaptic terminals were remarkably dynamic - moving, dividing to generate more boutons, and merging to consolidate small terminals into large boutons. As synaptic networks matured, presynaptic mobility decreased. These rapid dynamics may be important for establishing initial synaptic contacts with postsynaptic partners, refining connectivity patterns or modifying synapse strength during development. Ultimately, this in vivo imaging approach will facilitate investigation of synapse development in other long-range connections and neurodevelopmental disease models.

An autism-associated mutation in GluN2B prevents NMDA receptor trafficking and interferes with dendrite growth.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders with multiple genetic associations. Analysis of de novo mutations identified GRIN2B, which encodes the GluN2B subunit of NMDA receptors, as a gene linked to ASDs with high probability. However, the mechanisms by which GRIN2B mutations contribute to ASD pathophysiology are not understood. Here, we investigated the cellular phenotypes induced by a human mutation that is predicted to truncate GluN2B within the extracellular loop. This mutation abolished NMDA-dependent Ca2+ influx. Mutant GluN2B co-assembled with GluN1 but was not trafficked to the cell surface or dendrites. When mutant GluN2B was expressed in developing cortical neurons, dendrites appeared underdeveloped, with shorter and fewer branches, while spine density was unaffected. Mutant dendritic arbors were often dysmorphic, displaying abnormal filopodial-like structures. Interestingly, dendrite maldevelopment appeared when mutant GluN2B was expressed on a wild-type background, reflecting the disease given that individuals are heterozygous for GRIN2B mutations. Restoring the fourth transmembrane domain and cytoplasmic tail did not rescue the phenotypes. Finally, abnormal development was not accompanied by reduced mTOR signaling. These data suggest that mutations in GluN2B contribute to ASD pathogenesis by disrupting dendrite development.

Building a Terminal: Mechanisms of Presynaptic Development in the CNS.

To create a presynaptic terminal, molecular signaling events must be orchestrated across a number of subcellular compartments. In the soma, presynaptic proteins need to be synthesized, packaged together, and attached to microtubule motors for shipment through the axon. Within the axon, transport of presynaptic packages is regulated to ensure that developing synapses receive an adequate supply of components. At individual axonal sites, extracellular interactions must be translated into intracellular signals that can incorporate mobile transport vesicles into the nascent presynaptic terminal. Even once the initial recruitment process is complete, the components and subsequent functionality of presynaptic terminals need to constantly be remodeled. Perhaps most remarkably, all of these processes need to be coordinated in space and time. In this review, we discuss how these dynamic cellular processes occur in neurons of the central nervous system in order to generate presynaptic terminals in the brain.

On the Role of Glutamate in Presynaptic Development: Possible Contributions of Presynaptic NMDA Receptors.

Proper formation and maturation of synapses during development is a crucial step in building the functional neural circuits that underlie perception and behavior. It is well established that experience modifies circuit development. Therefore, understanding how synapse formation is controlled by synaptic activity is a key question in neuroscience. In this review, we focus on the regulation of excitatory presynaptic terminal development by glutamate, the predominant excitatory neurotransmitter in the brain. We discuss the evidence that NMDA receptor activation mediates these effects of glutamate and present the hypothesis that local activation of presynaptic NMDA receptors (preNMDARs) contributes to glutamate-dependent control of presynaptic development. Abnormal glutamate signaling and aberrant synapse development are both thought to contribute to the pathogenesis of a variety of neurodevelopmental disorders, including autism spectrum disorders, intellectual disability, epilepsy, anxiety, depression, and schizophrenia. Therefore, understanding how glutamate signaling and synapse development are linked is important for understanding the etiology of these diseases.

Presynaptic NMDA receptors - dynamics and distribution in developing axons in vitro and in vivo.

During cortical development, N-methyl-D-aspartate (NMDA) receptors (NMDARs) facilitate presynaptic terminal formation, enhance neurotransmitter release and are required in presynaptic neurons for spike-timing-dependent long-term depression (tLTD). However, the extent to which NMDARs are found within cortical presynaptic terminals has remained controversial, and the sub-synaptic localization and dynamics of axonal NMDARs are unknown. Here, using live confocal imaging and biochemical purification of presynaptic membranes, we provide strong evidence that NMDARs localize to presynaptic terminals in vitro and in vivo in a developmentally regulated manner. The NR1 and NR2B subunits (also known as GRIN1 and GRIN2B, respectively) were found within the active zone membrane, where they could respond to synaptic glutamate release. Surprisingly, NR1 also appeared in glutamatergic and GABAergic synaptic vesicles. During synaptogenesis, NR1 was mobile throughout axons - including growth cones and filopodia, structures that are involved in synaptogenesis. Upon synaptogenic contact, NMDA receptors were quickly recruited to terminals by neuroligin-1 signaling. Unlike dendrites, the trafficking and distribution of axonal NR1 were insensitive to activity changes, including NMDA exposure, local glutamate uncaging or action potential blockade. These results support the idea that presynaptic NMDARs play an early role in presynaptic development.

Dynamic mechanisms of neuroligin-dependent presynaptic terminal assembly in living cortical neurons.

BACKGROUND: Synapse formation occurs when synaptogenic signals trigger coordinated development of pre and postsynaptic structures. One of the best-characterized synaptogenic signals is trans-synaptic adhesion. However, it remains unclear how synaptic proteins are recruited to sites of adhesion. In particular, it is unknown whether synaptogenic signals attract synaptic vesicle (SV) and active zone (AZ) proteins to nascent synapses or instead predominantly function to create sites that are capable of forming synapses. It is also unclear how labile synaptic proteins are at developing synapses after their initial recruitment. To address these issues, we used long-term, live confocal imaging of presynaptic terminal formation in cultured cortical neurons after contact with the synaptogenic postsynaptic adhesion proteins neuroligin-1 or SynCAM-1. RESULTS: Surprisingly, we find that trans-synaptic adhesion does not attract SV or AZ proteins nor alter their transport. In addition, although neurexin (the presynaptic partner of neuroligin) typically accumulates over the entire region of contact between axons and neuroligin-1-expressing cells, SV proteins selectively assemble at spots of enhanced neurexin clustering. The arrival and maintenance of SV proteins at these sites is highly variable over the course of minutes to hours, and this variability correlates with neurexin levels at individual synapses. CONCLUSIONS: Together, our data support a model of synaptogenesis where presynaptic proteins are trapped at specific axonal sites, where they are stabilized by trans-synaptic adhesion signaling.

Developmental up-regulation of vesicular glutamate transporter-1 promotes neocortical presynaptic terminal development.

Presynaptic terminal formation is a complex process that requires assembly of proteins responsible for synaptic transmission at sites of axo-dendritic contact. Accumulation of presynaptic proteins at developing terminals is facilitated by glutamate receptor activation. Glutamate is loaded into synaptic vesicles for release via the vesicular glutamate transporters VGLUT1 and VGLUT2. During postnatal development there is a switch from predominantly VGLUT2 expression to high VGLUT1 and low VGLUT2, raising the question of whether the developmental increase in VGLUT1 is important for presynaptic development. Here, we addressed this question using confocal microscopy and quantitative immunocytochemistry in primary cultures of rat neocortical neurons. First, in order to understand the extent to which the developmental switch from VGLUT2 to VGLUT1 occurs through an increase in VGLUT1 at individual presynaptic terminals or through addition of VGLUT1-positive presynaptic terminals, we examined the spatio-temporal dynamics of VGLUT1 and VGLUT2 expression. Between 5 and 12 days in culture, the percentage of presynaptic terminals that expressed VGLUT1 increased during synapse formation, as did expression of VGLUT1 at individual terminals. A subset of VGLUT1-positive terminals also expressed VGLUT2, which decreased at these terminals. At individual terminals, the increase in VGLUT1 correlated with greater accumulation of other synaptic vesicle proteins, such as synapsin and synaptophysin. When the developmental increase in VGLUT1 was prevented using VGLUT1-shRNA, the density of presynaptic terminals and accumulation of synapsin and synaptophysin at terminals were decreased. Since VGLUT1 knock-down was limited to a small number of neurons, the observed effects were cell-autonomous and independent of changes in overall network activity. These results demonstrate that up-regulation of VGLUT1 is important for development of presynaptic terminals in the cortex.

Facilitation of neocortical presynaptic terminal development by NMDA receptor activation.

BACKGROUND: Neocortical circuits are established through the formation of synapses between cortical neurons, but the molecular mechanisms of synapse formation are only beginning to be understood. The mechanisms that control synaptic vesicle (SV) and active zone (AZ) protein assembly at developing presynaptic terminals have not yet been defined. Similarly, the role of glutamate receptor activation in control of presynaptic development remains unclear. RESULTS: Here, we use confocal imaging to demonstrate that NMDA receptor (NMDAR) activation regulates accumulation of multiple SV and AZ proteins at nascent presynaptic terminals of visual cortical neurons. NMDAR-dependent regulation of presynaptic assembly occurs even at synapses that lack postsynaptic NMDARs. We also provide evidence that this control of presynaptic terminal development is independent of glia. CONCLUSIONS: Based on these data, we propose a novel NMDAR-dependent mechanism for control of presynaptic terminal development in excitatory neocortical neurons. Control of presynaptic development by NMDARs could ultimately contribute to activity-dependent development of cortical receptive fields.

Coordinated trafficking of synaptic vesicle and active zone proteins prior to synapse formation.

BACKGROUND: The proteins required for synaptic transmission are rapidly assembled at nascent synapses, but the mechanisms through which these proteins are delivered to developing presynaptic terminals are not understood. Prior to synapse formation, active zone proteins and synaptic vesicle proteins are transported along axons in distinct organelles referred to as piccolo-bassoon transport vesicles (PTVs) and synaptic vesicle protein transport vesicles (STVs), respectively. Although both PTVs and STVs are recruited to the same site in the axon, often within minutes of axo-dendritic contact, it is not known whether or how PTV and STV trafficking is coordinated before synapse formation. RESULTS: Here, using time-lapse confocal imaging of the dynamics of PTVs and STVs in the same axon, we show that vesicle trafficking is coordinated through at least two mechanisms. First, a significant proportion of STVs and PTVs are transported together before forming a stable terminal. Second, individual PTVs and STVs share pause sites within the axon. Importantly, for both STVs and PTVs, encountering the other type of vesicle increases their propensity to pause. To determine if PTV-STV interactions are important for pausing, PTV density was reduced in axons by expression of a dominant negative construct corresponding to the syntaxin binding domain of syntabulin, which links PTVs with their KIF5B motor. This reduction in PTVs had a minimal effect on STV pausing and movement, suggesting that an interaction between STVs and PTVs is not responsible for enhancing STV pausing. CONCLUSIONS: Our results indicate that trafficking of STVs and PTVs is coordinated even prior to synapse development. This novel coordination of transport and pausing might provide mechanisms through which all of the components of a presynaptic terminal can be rapidly accumulated at sites of synapse formation.

How it's made: the synapse.

How are synapses made? This question is one of the most important issues in neurobiology today and has been the subject of intense study in recent years. This review focuses on the mechanisms of presynaptic terminal formation in the mammalian central nervous system. Building a synapse requires stabilization of contacts between axons and dendrites and formation of synaptic subcellular structures. Here, we discuss what determines where and when synapses form, how components of the nascent presynaptic terminal accumulate at the site of synapse formation, and whether assembly occurs via an ordered process dependent on a master organizer. Understanding synapse formation in the central nervous system is relevant for understanding and treating brain diseases as diverse as autism, epilepsy, anxiety disorders, brain injury, and Alzheimer's disease.

Modulation of firing rate by background synaptic noise statistics in rat visual cortical neurons.

It has been shown previously that background synaptic noise modulates the response gain of neocortical neurons. However, the role of the statistical properties of the noise in modulating firing rate is not known. Here, the dependence of firing rate on the statistical properties of the excitatory to inhibitory balance (EI) in cortical pyramidal neurons was studied. Excitatory glutamatergic and inhibitory GABAergic synaptic conductances were simulated as two stochastic processes and injected into individual neurons in vitro through use of the dynamic-clamp system. Response gain was significantly modulated as a function of the statistical interactions between excitatory and inhibitory synaptic conductances. Firing rates were compared for noisy synaptic conductance steps by varying either the EI correlation or the relative delay between correlated E and I. When inhibitory synaptic conductances exhibited a short temporal delay (5 ms) relative to correlated excitatory synaptic conductances, the response gain was increased compared with noise with no temporal delay but with an equivalent degree of correlation. The dependence of neuronal firing rate on the EI delay of the noisy background synaptic conductance suggests that individual excitatory pyramidal neurons are sensitive to the EI balance of the synaptic conductance. Therefore the statistical EI interactions encoded within the synaptic subthreshold membrane fluctuations are able to modulate neuronal firing properties.

A macromolecular complex involving the amyloid precursor protein (APP) and the cytosolic adapter FE65 is a negative regulator of axon branching.

Several studies suggest a role for the amyloid precursor protein (APP) in neurite outgrowth and synaptogenesis, but the downstream interactions that mediate the function of APP during neuron development are unknown. By introducing interaction-deficient FE65 into cultured hippocampal neurons using adenovirus, we show that a complex including APP, FE65 and an additional protein is involved in neurite outgrowth at early stages of neuronal development. Both FE65 that is unable to interact with APP (PID2 mutants) or a WW mutant increased axon branching. Although the FE65 mutants did not affect total neurite output, both mutants decreased axon segment length, consistent with an overall slowing of axonal growth cones. FE65 mutants did not alter the localization of either APP or FE65 in axonal growth cones, suggesting that the effects on neurite outgrowth are achieved by alterations in local complex formation within the axonal growth cone.

The dynamic distribution of TrkB receptors before, during, and after synapse formation between cortical neurons.

Although brain-derived neurotrophic factor (BDNF) potently regulates neuronal connectivity in the developing CNS, the mechanism by which BDNF influences the formation and/or maintenance of glutamatergic synapses remains unknown. Details about the subcellular localization of the BDNF receptor, TrkB, relative to synaptic and nonsynaptic proteins on excitatory neurons should provide insight into how BDNF might exert its effects during synapse formation. Here, we investigated the subcellular localization of tyrosine kinase receptor B (TrkB) relative to synaptic vesicle-associated proteins and NMDA receptors using immunocytochemistry, confocal microscopy, and time-lapse imaging in dissociated cultures of cortical neurons before, during, and after the peak of synapse formation. We find that TrkB is present in puncta on the surface and intracellularly in both dendrites and axons throughout development. Before synapse formation, some TrkB puncta in dendrites colocalize with NMDA receptors, and almost all TrkB puncta in axons colocalize with synaptic vesicle proteins. Clusters of TrkB fused to the enhanced green fluorescent protein (TrkB-EGFP) are highly mobile in both axons and dendrites. In axons, TrkB-EGFP dynamics are almost identical to vesicle-associated protein (VAMP2-EGFP), and these proteins are often transported together. Finally, surface TrkB is found in structures that actively participate in synapse formation: axonal growth cones and dendritic filopodia. Over time, surface TrkB becomes enriched at glutamatergic synapses, which contain both catalytic and truncated TrkB. These results suggest that TrkB is in the right place at the right time to play a direct role in the formation of glutamatergic synapses between cortical neurons.

Formation of presynaptic terminals at predefined sites along axons.

What determines where synapses will form along an axon or how proteins are deposited at nascent synapses remains unknown. Here, we show that the initial formation of presynaptic terminals occurs preferentially at predefined sites within the axons of cortical neurons. Time-lapse imaging of synaptic vesicle protein transport vesicles (STVs) indicates that STVs pause repeatedly at these sites, even in the absence of neuronal or glial contact. Contact with a neuroligin-expressing non-neuronal cell induces formation of presynaptic terminals specifically at these STV pause sites. Remarkably, formation of stable contacts with dendritic filopodia also occurs selectively at STV pause sites. Although it is not yet known which molecules comprise the predefined sites, STV pausing is regulated by cues that affect synaptogenesis. Overall, these data are consistent with the hypothesis that regulation of STV pausing might be an important mechanism for accumulation of presynaptic proteins at nascent synapses and support a new model in which many en passant synapses form specifically at predefined sites in young axons.