Spontaneous neurotransmission at evocable synapses predicts their responsiveness to action potentials.

Synaptic transmission relies on presynaptic neurotransmitter (NT) release from synaptic vesicles (SVs) and on NT detection by postsynaptic receptors. Transmission exists in two principal modes: action-potential (AP) evoked and AP-independent, "spontaneous" transmission. AP-evoked neurotransmission is considered the primary mode of inter-neuronal communication, whereas spontaneous transmission is required for neuronal development, homeostasis, and plasticity. While some synapses appear dedicated to spontaneous transmission only, all AP-responsive synapses also engage spontaneously, but whether this encodes functional information regarding their excitability is unknown. Here we report on functional interdependence of both transmission modes at individual synaptic contacts of Drosophila larval neuromuscular junctions (NMJs) which were identified by the presynaptic scaffolding protein Bruchpilot (BRP) and whose activities were quantified using the genetically encoded Ca2+ indicator GCaMP. Consistent with the role of BRP in organizing the AP-dependent release machinery (voltage-dependent Ca2+ channels and SV fusion machinery), most active BRP-positive synapses (>85%) responded to APs. At these synapses, the level of spontaneous activity was a predictor for their responsiveness to AP-stimulation. AP-stimulation resulted in cross-depletion of spontaneous activity and both transmission modes were affected by the non-specific Ca2+ channel blocker cadmium and engaged overlapping postsynaptic receptors. Thus, by using overlapping machinery, spontaneous transmission is a continuous, stimulus independent predictor for the AP-responsiveness of individual synapses.

Separation of presynaptic Cav2 and Cav1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca2+ pump PMCA.

Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca2+ signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)-triggered presynaptic Ca2+ entry remain unclear. Combining Drosophila genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Drosophila Cav2 homolog, Dmca1A or cacophony, and the Cav1 homolog, Dmca1D) but with spatial and functional separation. Cav2 within active zones is required for AP-triggered neurotransmitter release. By contrast, Cav1 localizes predominantly around active zones and contributes substantially to AP-evoked Ca2+ influx but has a small impact on release. Instead, L-type calcium currents through Cav1 fine-tune short-term plasticity and facilitate SV recycling. Separate control of SV exo- and endocytosis by AP-triggered presynaptic Ca2+ influx through different channels demands efficient measures to protect the neurotransmitter release machinery against Cav1-mediated Ca2+ influx. We show that the plasma membrane Ca2+ ATPase (PMCA) resides in between active zones and isolates Cav2-triggered release from Cav1-mediated dynamic regulation of recycling and short-term plasticity, two processes which Cav2 may also contribute to. As L-type Cav1 channels also localize next to PQ-type Cav2 channels within axon terminals of some central mammalian synapses, we propose that Cav2, Cav1, and PMCA act as a conserved functional triad that enables separate control of SV release and recycling rates in presynaptic terminals.

Rab2 regulates presynaptic precursor vesicle biogenesis at the trans-Golgi.

Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals, is prerequisite for successful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. We show here that in mutants of the small GTPase Rab2, both active zone and synaptic vesicle proteins accumulated in the neuronal cell body at the trans-Golgi and were, consequently, depleted at synaptic terminals, provoking neurotransmission deficits. Ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40 × 60 nm, segregating in subfractions either positive for active zone or synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, Rab2 acts upstream of Arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we identified a Golgi-associated assembly sequence of presynaptic precursor biogenesis dependent on a Rab2-regulated protein export and sorting step at the trans-Golgi.

Antagonistic interactions between two Neuroligins coordinate pre- and postsynaptic assembly.

As a result of developmental synapse formation, the presynaptic neurotransmitter release machinery becomes accurately matched with postsynaptic neurotransmitter receptors. Trans-synaptic signaling is executed through cell adhesion proteins such as Neurexin::Neuroligin pairs but also through diffusible and cytoplasmic signals. How exactly pre-post coordination is ensured in vivo remains largely enigmatic. Here, we identified a "molecular choreography" coordinating pre- with postsynaptic assembly during the developmental formation of Drosophila neuromuscular synapses. Two presynaptic Neurexin-binding scaffold proteins, Syd-1 and Spinophilin (Spn), spatio-temporally coordinated pre-post assembly in conjunction with two postsynaptically operating, antagonistic Neuroligin species: Nlg1 and Nlg2. The Spn/Nlg2 module promoted active zone (AZ) maturation by driving the accumulation of AZ scaffold proteins critical for synaptic vesicle release. Simultaneously, these regulators restricted postsynaptic glutamate receptor incorporation. Both functions of the Spn/Nlg2 module were directly antagonized by Syd-1/Nlg1. Nlg1 and Nlg2 also had divergent effects on Nrx-1 in vivo motility. Concerning diffusible signals, Spn and Syd-1 antagonistically controlled the levels of Munc13-family protein Unc13B at nascent AZs, whose release function facilitated glutamate receptor incorporation at assembling postsynaptic specializations. As a result, we here provide direct in vivo evidence illustrating how a highly regulative and interleaved communication between cell adhesion protein signaling complexes and diffusible signals allows for a precise coordination of pre- with postsynaptic assembly. It will be interesting to analyze whether this logic also transfers to plasticity processes.

RIM-binding protein couples synaptic vesicle recruitment to release sites.

At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. We here show in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a "hinge" in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs' conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites.

Presynaptic Active Zone Plasticity Encodes Sleep Need in Drosophila.

Sleep is universal across species and essential for quality of life and health, as evidenced by the consequences of sleep loss. Sleep might homeostatically normalize synaptic gains made over wake states in order to reset information processing and storage and support learning, and sleep-associated synaptic (ultra)structural changes have been demonstrated recently. However, causal relationships between the molecular and (ultra)structural status of synapses, sleep homeostatic regulation, and learning processes have yet to be established. We show here that the status of the presynaptic active zone can directly control sleep in Drosophila. Short sleep mutants showed a brain-wide upregulation of core presynaptic scaffold proteins and release factors. Increasing the gene copy number of ELKS-family scaffold master organizer Bruchpilot (BRP) not only mimicked changes in the active zone scaffold and release proteins but importantly provoked sleep in a dosage-dependent manner, qualitatively and quantitatively reminiscent of sleep deprivation effects. Conversely, reducing the brp copy number decreased sleep in short sleep mutant backgrounds, suggesting a specific role of the active zone plasticity in homeostatic sleep regulation. Finally, elimination of BRP specifically in the sleep-promoting R2 neurons of 4xBRP animals partially restored sleep patterns and rescued learning deficits. Our results suggest that the presynaptic active zone plasticity driven by BRP operates as a sleep homeostatic actuator that also restricts periods of effective learning.

Microtubule organization in presynaptic boutons relies on the formin DAAM.

Regulation of the cytoskeleton is fundamental to the development and function of synaptic terminals, such as neuromuscular junctions. Despite the identification of numerous proteins that regulate synaptic actin and microtubule dynamics, the mechanisms of cytoskeletal control during terminal arbor formation have remained largely elusive. Here, we show that DAAM, a member of the formin family of cytoskeleton organizing factors, is an important presynaptic regulator of neuromuscular junction development in Drosophila We demonstrate that the actin filament assembly activity of DAAM plays a negligible role in terminal formation; rather, DAAM is necessary for synaptic microtubule organization. Genetic interaction studies consistently link DAAM with the Wg/Ank2/Futsch module of microtubule regulation and bouton formation. Finally, we provide evidence that DAAM is tightly associated with the synaptic active zone scaffold, and electrophysiological data point to a role in the modulation of synaptic vesicle release. Based on these results, we propose that DAAM is an important cytoskeletal effector element of the Wg/Ank2 pathway involved in the determination of basic synaptic structures, and, additionally, that DAAM may couple the active zone scaffold to the presynaptic cytoskeleton.

Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function.

Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. Here we report that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1. Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. We conclude that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis.