The molecular organization of presynaptic active zones during calcium influx-triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) were entirely lost, Ca2+ channels were reduced in density, evoked vesicle release was depressed, and short-term plasticity was altered. BRP-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity.

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, we identify BRP as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains.

Activity-dependent modifications in synapse structure play a key role in synaptic development and plasticity, but the signaling mechanisms involved are poorly understood. We demonstrate that glutamatergic Drosophila neuromuscular junctions undergo rapid changes in synaptic structure and function in response to patterned stimulation. These changes, which depend on transcription and translation, include formation of motile presynaptic filopodia, elaboration of undifferentiated varicosities, and potentiation of spontaneous release frequency. Experiments indicate that a bidirectional Wnt/Wg signaling pathway underlies these changes. Evoked activity induces Wnt1/Wg release from synaptic boutons, which stimulates both a postsynaptic DFz2 nuclear import pathway, as well as a presynaptic pathway involving GSK-3 β/Shaggy. Our findings suggest that bidirectional Wg signaling operates downstream of synaptic activity to induce modifications in synaptic structure and function. We propose that activation of the postsynaptic Wg pathway is required for the assembly of the postsynaptic apparatus, while activation of the presynaptic Wg pathway regulates cytoskeletal dynamics.

Precise apposition of presynaptic and postsynaptic domains is a fundamental property of all neuronal circuits. Experiments in vitro suggest that Neuroligins and Neurexins function as key regulatory proteins in this process. In a genetic screen, we recovered several mutant alleles of Drosophila neuroligin 1 (dnlg1) that cause a severe reduction in bouton numbers at neuromuscular junctions (NMJs). In accord with reduced synapse numbers, these NMJs show reduced synaptic transmission. Moreover, lack of postsynaptic DNlg1 leads to deficits in the accumulation of postsynaptic glutamate receptors, scaffold proteins, and subsynaptic membranes, while increased DNlg1 triggers ectopic postsynaptic differentiation via its cytoplasmic domain. DNlg1 forms discrete clusters adjacent to postsynaptic densities. Formation of these clusters depends on presynaptic Drosophila Neurexin (DNrx). However, DNrx binding is not an absolute requirement for DNlg1 function. Instead, other signaling components are likely involved in DNlg1 transsynaptic functions, with essential interactions organized by the DNlg1 extracellular domain but also by the cytoplasmic domain.

A proteomics approach identifies Drosophila Syd-1 as a Bruchpilot binding partner that controls maturation on both sides of the neuromuscular junction.

Insight into how glutamatergic synapses form in vivo is important for understanding developmental and experience-triggered changes of excitatory circuits. Here, we imaged postsynaptic densities (PSDs) expressing a functional, GFP-tagged glutamate receptor subunit (GluR-IIA(GFP)) at neuromuscular junctions of Drosophila melanogaster larvae for several days in vivo. New PSDs, associated with functional and structural presynaptic markers, formed independently of existing synapses and grew continuously until reaching a stable size within hours. Both in vivo photoactivation and photobleaching experiments showed that extrasynaptic receptors derived from diffuse, cell-wide pools preferentially entered growing PSDs. After entering PSDs, receptors were largely immobilized. In comparison, other postsynaptic proteins tested (PSD-95, NCAM and PAK homologs) exchanged faster and with no apparent preference for growing synapses. We show here that new glutamatergic synapses form de novo and not by partitioning processes from existing synapses, suggesting that the site-specific entry of particular glutamate receptor complexes directly controls the assembly of individual PSDs.

Synaptic inhibition is a central factor in the fine tuning of neuronal activity in the central nervous system. Symptoms consistent with reduced inhibition such as stiffness, spasms and anxiety occur in paraneoplastic stiff person syndrome with autoantibodies against the intracellular synaptic protein amphiphysin. Here we show that intrathecal application of purified anti-amphiphysin immunoglobulin G antibodies induces stiff person syndrome-like symptoms in rats, including stiffness and muscle spasms. Using in vivo recordings of Hoffmann reflexes and dorsal root potentials, we identified reduced presynaptic GABAergic inhibition as an underlying mechanism. Anti-amphiphysin immunoglobulin G was internalized into neurons by an epitope-specific mechanism and colocalized in vivo with presynaptic vesicular proteins, as shown by stimulation emission depletion microscopy. Neurons from amphiphysin deficient mice that did not internalize the immunoglobulin provided additional evidence of the specificity in antibody uptake. GABAergic synapses appeared more vulnerable than glutamatergic synapses to defective endocytosis induced by anti-amphiphysin immunoglobulin G, as shown by increased clustering of the endocytic protein AP180 and by defective loading of FM 1-43, a styryl dye used to label cell membranes. Incubation of cultured neurons with anti-amphiphysin immunoglobulin G reduced basal and stimulated release of γ-aminobutyric acid substantially more than that of glutamate. By whole-cell patch-clamp analysis of GABAergic inhibitory transmission in hippocampus granule cells we showed a faster, activity-dependent decrease of the amplitude of evoked inhibitory postsynaptic currents in brain slices treated with antibodies against amphiphysin. We suggest that these findings may explain the pathophysiology of the core signs of stiff person syndrome at the molecular level and show that autoantibodies can alter the function of inhibitory synapses in vivo upon binding to an intraneuronal key protein by disturbing vesicular endocytosis.

Understanding the process of synapse assembly in molecular and cell-biological detail is a prerequisite for understanding neural circuit development and activity-mediated remodeling, and thus is important for unraveling learning and memory processes (structural plasticity) [1][2][3] . Functional chemical synapses are characterized by two apposed compartments that must be coestablished with high spatiotemporal precision: the presynaptic active zone, where regulated and rapid fusion of neurotransmitter-filled synaptic vesicles takes place, and the postsynaptic density (PSD), which embeds neurotransmitter receptors.Glutamatergic neuromuscular junction (NMJ) terminals of Drosophila larvae grow to meet the requirements of the growing muscle fibers, a process in which new synapses are continuously added 4 (where a synapse is defined as a single active zone opposed by a single PSD 1 ). In vivo imaging has shown that presynaptic Syd-1 and Liprin-α clusters initiate active zone formation 5 . On the postsynaptic side, initial PSD growth depends on incorporation of a glutamate receptor (GluR) containing the GluRIIA subunit. Later PSD maturation is marked by the incorporation of GluRIIB-containing receptor complexes 6 . Synapse assembly is concluded at presynaptic active zones by the incorporation of the active zone scaffold component Bruchpilot (BRP) 5 .Coordinating synapse assembly requires signaling across the synaptic cleft, which separates pre-from postsynaptic membranes. Transsynaptic cell adhesion molecules are obvious candidates for coupling active zone and PSD assembly. In vitro, the Neurexin-Neuroligin (Nrx-Nlg) complex can mediate the trans-synaptic signaling required for synapse assembly 7,8 . How this signaling axis integrates with the additional assembly machinery, however, has remained largely unclear.Here, we provide evidence of a dual role for Syd-1 in early assembly of NMJ synapses. It retains Liprin-α clusters at active zones and is important for clustering of presynaptic Nrx-1, likely through a direct and PDZ-domain-dependent interaction. Consequently, Syd-1 is also needed for clustering of postsynaptic Nlg1, which organizes postsynaptic assembly. Coincident action of Syd-1 with Nrx-1-Nlg1 appears to allow active zone scaffolds to pass through an initial, still fragile assembly phase. We suggest that binding between Syd-1 and Nrx-1-Nlg1 is a means to coordinate pre-with postsynaptic assembly. Our study shows an example of how coincident action of a presynaptic active zone scaffold protein and a trans-synaptic cell adhesion protein module can spatiotemporally orchestrate synapse assembly. RESULTSInitially described in cell culture systems (for a review, see ref. 9), interaction between mammalian presynaptic Nrx proteins and postsynaptic Nlg molecules was proposed to be important for proper synapse assembly. However, genetic ablation of three Nlg (Nlgn) genes in mice 10 does not result in a substantial structural phenotype, potentially reflecting a strong capacity for compensatory processes in vivo.Nonethe...