An antagonism between Spinophilin and Syd-1 operates upstream of memory-promoting presynaptic long-term plasticity.

We still face fundamental gaps in understanding how molecular plastic changes of synapses intersect with circuit operation to define behavioral states. Here, we show that an antagonism between two conserved regulatory proteins, Spinophilin (Spn) and Syd-1, controls presynaptic long-term plasticity and the maintenance of olfactory memories in Drosophila. While Spn mutants could not trigger nanoscopic active zone remodeling under homeostatic challenge and failed to stably potentiate neurotransmitter release, concomitant reduction of Syd-1 rescued all these deficits. The Spn/Syd-1 antagonism converged on active zone close F-actin, and genetic or acute pharmacological depolymerization of F-actin rescued the Spn deficits by allowing access to synaptic vesicle release sites. Within the intrinsic mushroom body neurons, the Spn/Syd-1 antagonism specifically controlled olfactory memory stabilization but not initial learning. Thus, this evolutionarily conserved protein complex controls behaviorally relevant presynaptic long-term plasticity, also observed in the mammalian brain but still enigmatic concerning its molecular mechanisms and behavioral relevance.

Interactive nanocluster compaction of the ELKS scaffold and Cacophony Ca2+ channels drives sustained active zone potentiation.

At presynaptic active zones (AZs), conserved scaffold protein architectures control synaptic vesicle (SV) release by defining the nanoscale distribution and density of voltage-gated Ca2+ channels (VGCCs). While AZs can potentiate SV release in the minutes range, we lack an understanding of how AZ scaffold components and VGCCs engage into potentiation. We here establish dynamic, intravital single-molecule imaging of endogenously tagged proteins at Drosophila AZs undergoing presynaptic homeostatic potentiation. During potentiation, the numbers of α1 VGCC subunit Cacophony (Cac) increased per AZ, while their mobility decreased and nanoscale distribution compacted. These dynamic Cac changes depended on the interaction between Cac channel's intracellular carboxyl terminus and the membrane-close amino-terminal region of the ELKS-family protein Bruchpilot, whose distribution compacted drastically. The Cac-ELKS/Bruchpilot interaction was also needed for sustained AZ potentiation. Our single-molecule analysis illustrates how the AZ scaffold couples to VGCC nanoscale distribution and dynamics to establish a state of sustained potentiation.

Transient active zone remodeling in the Drosophila mushroom body supports memory.

Elucidating how the distinct components of synaptic plasticity dynamically orchestrate the distinct stages of memory acquisition and maintenance within neuronal networks remains a major challenge. Specifically, plasticity processes tuning the functional and also structural state of presynaptic active zone (AZ) release sites are widely observed in vertebrates and invertebrates, but their behavioral relevance remains mostly unclear. We here provide evidence that a transient upregulation of presynaptic AZ release site proteins supports aversive olfactory mid-term memory in the Drosophila mushroom body (MB). Upon paired aversive olfactory conditioning, AZ protein levels (ELKS-family BRP/(m)unc13-family release factor Unc13A) increased for a few hours with MB-lobe-specific dynamics. Kenyon cell (KC, intrinsic MB neurons)-specific knockdown (KD) of BRP did not affect aversive olfactory short-term memory (STM) but strongly suppressed aversive mid-term memory (MTM). Different proteins crucial for the transport of AZ biosynthetic precursors (transport adaptor Aplip1/Jip-1; kinesin motor IMAC/Unc104; small GTPase Arl8) were also specifically required for the formation of aversive olfactory MTM. Consistent with the merely transitory increase of AZ proteins, BRP KD did not interfere with the formation of aversive olfactory long-term memory (LTM; i.e., 1 day). Our data suggest that the remodeling of presynaptic AZ refines the MB circuitry after paired aversive conditioning, over a time window of a few hours, to display aversive olfactory memories.

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.