Retrovirus-like Gag Protein Arc1 Binds RNA and Traffics across Synaptic Boutons.

Arc/Arg3.1 is required for synaptic plasticity and cognition, and mutations in this gene are linked to autism and schizophrenia. Arc bears a domain resembling retroviral/retrotransposon Gag-like proteins, which multimerize into a capsid that packages viral RNA. The significance of such a domain in a plasticity molecule is uncertain. Here, we report that the Drosophila Arc1 protein forms capsid-like structures that bind darc1 mRNA in neurons and is loaded into extracellular vesicles that are transferred from motorneurons to muscles. This loading and transfer depends on the darc1-mRNA 3' untranslated region, which contains retrotransposon-like sequences. Disrupting transfer blocks synaptic plasticity, suggesting that transfer of dArc1 complexed with its mRNA is required for this function. Notably, cultured cells also release extracellular vesicles containing the Gag region of the Copia retrotransposon complexed with its own mRNA. Taken together, our results point to a trans-synaptic mRNA transport mechanism involving retrovirus-like capsids and extracellular vesicles.

Neuron-Glia Signaling in Synapse Elimination.

Maturation of neuronal circuits requires selective elimination of synaptic connections. Although neuron-intrinsic mechanisms are important in this process, it is increasingly recognized that glial cells also play a critical role. Without proper functioning of these cells, the number, morphology, and function of synaptic contacts are profoundly altered, resulting in abnormal connectivity and behavioral abnormalities. In addition to their role in synaptic refinement, glial cells have also been implicated in pathological synapse loss and dysfunction following injury or nervous system degeneration in adults. Although mechanisms regulating glia-mediated synaptic elimination are still being uncovered, it is clear this complex process involves many cues that promote and inhibit the removal of specific synaptic connections. Gaining a greater understanding of these signals and the contribution of different cell types will not only provide insight into this critical biological event but also be instrumental in advancing knowledge of brain development and neural disease.

Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission.

Cofilin, an actin-severing protein, plays key roles in muscle sarcomere addition and maintenance. Our previous work found that Drosophila cofilin (DmCFL) knockdown in muscle causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA-sequencing analysis that unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL muscle knockdown causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown resulted in mislocalization of GluRIIA class glutamate receptors in more deteriorated muscles and strongly impaired NMJ transmission strength. These findings expand our understanding of the roles of cofilin in muscle to include NMJ structural development and suggest that NMJ defects may contribute to the pathophysiology of nemaline myopathy.

Neuronal LRP4 directs the development, maturation and cytoskeletal organization of Drosophila peripheral synapses.

Synaptic development requires multiple signaling pathways to ensure successful connections. Transmembrane receptors are optimally positioned to connect the synapse and the rest of the neuron, often acting as synaptic organizers to synchronize downstream events. One such organizer, the LDL receptor-related protein LRP4, is a cell surface receptor that has been most well-studied postsynaptically at mammalian neuromuscular junctions. Recent work, however, identified emerging roles, but how LRP4 acts as a presynaptic organizer and the downstream mechanisms of LRP4 are not well understood. Here, we show that LRP4 functions presynaptically at Drosophila neuromuscular synapses, acting in motoneurons to instruct pre- and postsynaptic development. Loss of presynaptic LRP4 results in multiple defects, impairing active zone organization, synapse growth, physiological function, microtubule organization, synaptic ultrastructure and synapse maturation. We further demonstrate that LRP4 promotes most aspects of presynaptic development via a downstream SR-protein kinase, SRPK79D. These data demonstrate a function for presynaptic LRP4 as a peripheral synaptic organizer, highlight a downstream mechanism conserved with its CNS function in Drosophila, and underscore previously unappreciated but important developmental roles for LRP4 in cytoskeletal organization, synapse maturation and active zone organization.

Nuclear envelope budding enables large ribonucleoprotein particle export during synaptic Wnt signaling.

Localized protein synthesis requires assembly and transport of translationally silenced ribonucleoprotein particles (RNPs), some of which are exceptionally large. Where in the cell such large RNP granules first assemble was heretofore unknown. We previously reported that during synapse development, a fragment of the Wnt-1 receptor, DFrizzled2, enters postsynaptic nuclei where it forms prominent foci. Here we show that these foci constitute large RNP granules harboring synaptic protein transcripts. These granules exit the nucleus by budding through the inner and the outer nuclear membranes in a nuclear egress mechanism akin to that of herpes viruses. This budding involves phosphorylation of A-type lamin, a protein linked to muscular dystrophies. Thus nuclear envelope budding is an endogenous nuclear export pathway for large RNP granules.

Wnt signaling regulates acetylcholine receptor translocation and synaptic plasticity in the adult nervous system.

The adult nervous system is plastic, allowing us to learn, remember, and forget. Experience-dependent plasticity occurs at synapses--the specialized points of contact between neurons where signaling occurs. However, the mechanisms that regulate the strength of synaptic signaling are not well understood. Here, we define a Wnt-signaling pathway that modifies synaptic strength in the adult nervous system by regulating the translocation of one class of acetylcholine receptors (AChRs) to synapses. In Caenorhabditis elegans, we show that mutations in CWN-2 (Wnt ligand), LIN-17 (Frizzled), CAM-1 (Ror receptor tyrosine kinase), or the downstream effector DSH-1 (disheveled) result in similar subsynaptic accumulations of ACR-16/α7 AChRs, a consequent reduction in synaptic current, and predictable behavioral defects. Photoconversion experiments revealed defective translocation of ACR-16/α7 to synapses in Wnt-signaling mutants. Using optogenetic nerve stimulation, we demonstrate activity-dependent synaptic plasticity and its dependence on ACR-16/α7 translocation mediated by Wnt signaling via LIN-17/CAM-1 heteromeric receptors.

Identification of secretory autophagy as a mechanism modulating activity-induced synaptic remodeling.

The ability of neurons to rapidly remodel their synaptic structure and strength in response to neuronal activity is highly conserved across species and crucial for complex brain functions. However, mechanisms required to elicit and coordinate the acute, activity-dependent structural changes across synapses are not well understood, as neurodevelopment and structural plasticity are tightly linked. Here, using an RNAi screen in Drosophila against genes affecting nervous system functions in humans, we uncouple cellular processes important for synaptic plasticity and synapse development. We find mutations associated with neurodegenerative and mental health disorders are 2-times more likely to affect activity-induced synaptic remodeling than synapse development. We report that while both synapse development and activity-induced synaptic remodeling at the fly NMJ require macroautophagy (hereafter referred to as autophagy), bifurcation in the autophagy pathway differentially impacts development and synaptic plasticity. We demonstrate that neuronal activity enhances autophagy activation but diminishes degradative autophagy, thereby driving the pathway towards autophagy-based secretion. Presynaptic knockdown of Snap29, Sec22, or Rab8, proteins implicated in the secretory autophagy pathway, is sufficient to abolish activity-induced synaptic remodeling. This study uncovers secretory autophagy as a transsynaptic signaling mechanism modulating synaptic plasticity.

Sarcopenia: Aging-Related Loss of Muscle Mass and Function.

Sarcopenia is a loss of muscle mass and function in the elderly that reduces mobility, diminishes quality of life, and can lead to fall-related injuries, which require costly hospitalization and extended rehabilitation. This review focuses on the aging-related structural changes and mechanisms at cellular and subcellular levels underlying changes in the individual motor unit: specifically, the perikaryon of the α-motoneuron, its neuromuscular junction(s), and the muscle fibers that it innervates. Loss of muscle mass with aging, which is largely due to the progressive loss of motoneurons, is associated with reduced muscle fiber number and size. Muscle function progressively declines because motoneuron loss is not adequately compensated by reinnervation of muscle fibers by the remaining motoneurons. At the intracellular level, key factors are qualitative changes in posttranslational modifications of muscle proteins and the loss of coordinated control between contractile, mitochondrial, and sarcoplasmic reticulum protein expression. Quantitative and qualitative changes in skeletal muscle during the process of aging also have been implicated in the pathogenesis of acquired and hereditary neuromuscular disorders. In experimental models, specific intervention strategies have shown encouraging results on limiting deterioration of motor unit structure and function under conditions of impaired innervation. Translated to the clinic, if these or similar interventions, by saving muscle and improving mobility, could help alleviate sarcopenia in the elderly, there would be both great humanitarian benefits and large cost savings for health care systems.

Morphological and functional alterations of neuromuscular synapses in a mouse model of ACTA1 congenital myopathy.

Mutations in skeletal muscle α-actin (Acta1) cause myopathies. In a mouse model of congenital myopathy, heterozygous Acta1 (H40Y) knock-in (Acta1+/Ki) mice exhibit features of human nemaline myopathy, including premature lethality, severe muscle weakness, reduced mobility, and the presence of nemaline rods in muscle fibers. In this study, we investigated the impact of Acta1 (H40Y) mutation on the neuromuscular junction (NMJ). We found that the NMJs were markedly fragmented in Acta1+/Ki mice. Electrophysiological analysis revealed a decrease in amplitude but increase in frequency of miniature end-plate potential (mEPP) at the NMJs in Acta1+/Ki mice, compared with those in wild type (Acta1+/+) mice. Evoked end-plate potential (EPP) remained similar at the NMJs in Acta1+/Ki and Acta1+/+ mice, but quantal content was increased at the NMJs in Acta1+/Ki, compared with Acta1+/+ mice, suggesting a homeostatic compensation at the NMJs in Acta1+/Ki mice to maintain normal levels of neurotransmitter release. Furthermore, short-term synaptic plasticity of the NMJs was compromised in Acta1+/Ki mice. Together, these results demonstrate that skeletal Acta1 H40Y mutation, albeit muscle-origin, leads to both morphological and functional defects at the NMJ.

Vcp overexpression and leucine supplementation extend lifespan and ameliorate neuromuscular junction phenotypes of a SOD1G93A-ALS mouse model.

Many genes with distinct molecular functions have been linked to genetically heterogeneous amyotrophic lateral sclerosis (ALS), including SuperOxide Dismutase 1 (SOD1) and Valosin-Containing Protein (VCP). SOD1 converts superoxide to oxygen and hydrogen peroxide. VCP acts as a chaperon to regulate protein degradation and synthesis and various other cellular responses. Although the functions of these two genes differ, in the current report we show that overexpression of wild-type VCP in mice enhances lifespan and maintains the size of neuromuscular junctions (NMJs) of both male and female SOD1G93A mice, a well-known ALS mouse model. Although VCP exerts multiple functions, its regulation of ER formation and consequent protein synthesis has been shown to play the most important role in controlling dendritic spine formation and social and memory behaviors. Given that SOD1 mutation results in protein accumulation and aggregation, it may direct VCP to the protein degradation pathway, thereby impairing protein synthesis. Since we previously showed that the protein synthesis defects caused by Vcp deficiency can be improved by leucine supplementation, to confirm the role of the VCP-protein synthesis pathway in SOD1-linked ALS, we applied leucine supplementation to SOD1G93A mice and, similar to Vcp overexpression, we found that it extends SOD1G93A mouse lifespan. In addition, the phenotypes of reduced muscle strength and fewer NMJs of SOD1G93A mice are also improved by leucine supplementation. These results support the existence of crosstalk between SOD1 and VCP and suggest a critical role for protein synthesis in ASL. Our study also implies a potential therapeutic treatment for ALS.

Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins.

Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. In this work, we present evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release.

The YAP1/TAZ-TEAD transcriptional network regulates gene expression at neuromuscular junctions in skeletal muscle fibers.

We examined YAP1/TAZ-TEAD signaling pathway activity at neuromuscular junctions (NMJs) of skeletal muscle fibers in adult mice. Our investigations revealed that muscle-specific knockouts of Yap1 or Taz, or both, demonstrate that these transcriptional coactivators regulate synaptic gene expression, the number and morphology of NMJs, and synaptic nuclei. Yap1 or Taz single knockout mice display reduced grip strength, fragmentation of NMJs, and accumulation of synaptic nuclei. Yap1/Taz muscle-specific double knockout mice do not survive beyond birth and possess almost no NMJs, the few detectable show severely impaired morphology and are organized in widened endplate bands; and with motor nerve endings being mostly absent. Myogenic gene expression is significantly impaired in the denervated muscles of knockout mice. We found that Tead1 and Tead4 transcription rates were increased upon incubation of control primary myotubes with AGRN-conditioned medium. Reduced AGRN-dependent acetylcholine receptor clustering and synaptic gene transcription were observed in differentiated primary Tead1 and Tead4 knockout myotubes. In silico analysis of previously reported genomic occupancy sites of TEAD1/4 revealed evolutionary conserved regions of potential TEAD binding motifs in key synaptic genes, the relevance of which was functionally confirmed by reporter assays. Collectively, our data suggest a role for YAP1/TAZ-TEAD1/TEAD4 signaling, particularly through TAZ-TEAD4, in regulating synaptic gene expression and acetylcholine receptor clustering at NMJs.

Structural insights into the assembly of the agrin/LRP4/MuSK signaling complex.

MuSK is a receptor tyrosine kinase (RTK) that plays essential roles in the formation and maintenance of the neuromuscular junction. Distinct from most members of RTK family, MuSK activation requires not only its cognate ligand agrin but also its coreceptors LRP4. However, how agrin and LRP4 coactivate MuSK remains unclear. Here, we report the cryo-EM structure of the extracellular ternary complex of agrin/LRP4/MuSK in a stoichiometry of 1:1:1. This structure reveals that arc-shaped LRP4 simultaneously recruits both agrin and MuSK to its central cavity, thereby promoting a direct interaction between agrin and MuSK. Our cryo-EM analyses therefore uncover the assembly mechanism of agrin/LRP4/MuSK signaling complex and reveal how MuSK receptor is activated by concurrent binding of agrin and LRP4.

PTPN11/Corkscrew Activates Local Presynaptic Mapk Signaling to Regulate Synapsin, Synaptic Vesicle Pools, and Neurotransmission Strength, with a Dual Requirement in Neurons and Glia.

Cytoplasmic protein tyrosine phosphatase nonreceptor type 11 (PTPN11) and Drosophila homolog Corkscrew (Csw) regulate the mitogen-activated protein kinase (MAPK) pathway via a conserved autoinhibitory mechanism. Disease-causing loss-of-function (LoF) and gain-of-function (GoF) mutations both disrupt this autoinhibition to potentiate MAPK signaling. At the Drosophila neuromuscular junction glutamatergic synapse, LoF/GoF mutations elevate transmission strength and reduce activity-dependent synaptic depression. In both sexes of LoF/GoF mutations, the synaptic vesicles (SV)-colocalized synapsin phosphoprotein tether is highly elevated at rest, but quickly reduced with stimulation, suggesting a larger SV reserve pool with greatly heightened activity-dependent recruitment. Transmission electron microscopy of mutants reveals an elevated number of SVs clustered at the presynaptic active zones, suggesting that the increased vesicle availability is causative for the elevated neurotransmission. Direct neuron-targeted extracellular signal-regulated kinase (ERK) GoF phenocopies both increased local presynaptic MAPK/ERK signaling and synaptic transmission strength in mutants, confirming the presynaptic regulatory mechanism. Synapsin loss blocks this elevation in both presynaptic PTPN11 and ERK mutants. However, csw null mutants cannot be rescued by wild-type Csw in neurons: neurotransmission is only rescued by expressing Csw in both neurons and glia simultaneously. Nevertheless, targeted LoF/GoF mutations in either neurons or glia alone recapitulate the elevated neurotransmission. Thus, PTPN11/Csw mutations in either cell type are sufficient to upregulate presynaptic function, but a dual requirement in neurons and glia is necessary for neurotransmission. Taken together, we conclude that PTPN11/Csw acts in both neurons and glia, with LoF and GoF similarly upregulating MAPK/ERK signaling to enhance presynaptic Synapsin-mediated SV trafficking.

Cholesterol is required for activity-dependent synaptic growth.

Changes in cholesterol content of neuronal membranes occur during development and brain aging. Little is known about whether synaptic activity regulates cholesterol levels in neuronal membranes and whether these changes affect neuronal development and function. We generated transgenic flies that express the cholesterol-binding D4H domain of perfringolysin O toxin and found increased levels of cholesterol in presynaptic terminals of Drosophila larval neuromuscular junctions following increased synaptic activity. Reduced cholesterol impaired synaptic growth and largely prevented activity-dependent synaptic growth. Presynaptic knockdown of adenylyl cyclase phenocopied the impaired synaptic growth caused by reducing cholesterol. Furthermore, the effects of knocking down adenylyl cyclase and reducing cholesterol were not additive, suggesting that they function in the same pathway. Increasing cAMP levels using a dunce mutant with reduced phosphodiesterase activity failed to rescue this impaired synaptic growth, suggesting that cholesterol functions downstream of cAMP. We used a protein kinase A (PKA) sensor to show that reducing cholesterol levels reduced presynaptic PKA activity. Collectively, our results demonstrate that enhanced synaptic activity increased cholesterol levels in presynaptic terminals and that these changes likely activate the cAMP-PKA pathway during activity-dependent growth.

Clinical and genetic characterisation of a large Indian congenital myasthenic syndrome cohort.

Congenital myasthenic syndromes (CMS) are a rare group of inherited disorders caused by gene defects associated with the neuromuscular junction and potentially treatable with commonly available medications such as acetylcholinesterase inhibitors and ß2 adrenergic receptor agonists. In this study, we identified and genetically characterized the largest cohort of CMS patients from India to date. Genetic testing of clinically suspected patients evaluated in a South Indian hospital during the period 2014-19 was carried out by standard diagnostic gene panel testing or using a two-step method that included hotspot screening followed by whole-exome sequencing. In total, 156 genetically diagnosed patients (141 families) were characterized and the mutational spectrum and genotype-phenotype correlation described. Overall, 87 males and 69 females were evaluated, with the age of onset ranging from congenital to fourth decade (mean 6.6 ± 9.8 years). The mean age at diagnosis was 19 ± 12.8 (1-56 years), with a mean diagnostic delay of 12.5 ± 9.9 (0-49 years). Disease-causing variants in 17 CMS-associated genes were identified in 132 families (93.6%), while in nine families (6.4%), variants in genes not associated with CMS were found. Overall, postsynaptic defects were most common (62.4%), followed by glycosylation defects (21.3%), synaptic basal lamina genes (4.3%) and presynaptic defects (2.8%). Other genes found to cause neuromuscular junction defects (DES, TEFM) in our cohort accounted for 2.8%. Among the individual CMS genes, the most commonly affected gene was CHRNE (39.4%), followed by DOK7 (14.4%), DPAGT1 (9.8%), GFPT1 (7.6%), MUSK (6.1%), GMPPB (5.3%) and COLQ (4.5%). We identified 22 recurrent variants in this study, out of which eight were found to be geographically specific to the Indian subcontinent. Apart from the known common CHRNE variants p.E443Kfs*64 (11.4%) and DOK7 p.A378Sfs*30 (9.3%), we identified seven novel recurrent variants specific to this cohort, including DPAGT1 p.T380I and DES c.1023+5G>A, for which founder haplotypes are suspected. This study highlights the geographic differences in the frequencies of various causative CMS genes and underlines the increasing significance of glycosylation genes (DPAGT1, GFPT1 and GMPPB) as a cause of neuromuscular junction defects. Myopathy and muscular dystrophy genes such as GMPPB and DES, presenting as gradually progressive limb girdle CMS, expand the phenotypic spectrum. The novel genes MACF1 and TEFM identified in this cohort add to the expanding list of genes with new mechanisms causing neuromuscular junction defects.

The MuSK agonist antibody protects the neuromuscular junction and extends the lifespan in C9orf72-ALS mice.

The disassembly of the neuromuscular junction (NMJ) is an early event in amyotrophic lateral sclerosis (ALS), ultimately leading to motor dysfunction and lethal respiratory paralysis. The hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most common genetic mutation, and the dipeptide repeat (DPR) proteins have been shown to cause neurodegeneration. While no drugs can treat ALS patients efficiently, new treatment strategies are urgently needed. Here, we report that a MuSK agonist antibody alleviates poly-PR-induced NMJ deficits in C9orf72-ALS mice. The HB9-PRF/F mice, which express poly-PR proteins in motor neurons, exhibited impaired motor behavior and NMJ deficits. Mechanistically, poly-PR proteins interacted with Agrin to disrupt the interaction between Agrin and Lrp4, leading to attenuated activation of MuSK. Treatment with a MuSK agonist antibody rescued NMJ deficits, and extended the lifespan of C9orf72-ALS mice. Moreover, impaired NMJ transmission was observed in C9orf72-ALS patients. These findings identify the mechanism by which poly-PR proteins attenuate MuSK activation and NMJ transmission, highlighting the potential of promoting MuSK activation with an agonist antibody as a therapeutic strategy to protect NMJ function and prolong the lifespan of ALS patients.

Rapid homeostatic modulation of transsynaptic nanocolumn rings.

Robust neural information transfer relies on a delicate molecular nano-architecture of chemical synapses. Neurotransmitter release is controlled by a specific arrangement of proteins within presynaptic active zones. How the specific presynaptic molecular architecture relates to postsynaptic organization and how synaptic nano-architecture is transsynaptically regulated to enable stable synaptic transmission remain enigmatic. Using time-gated stimulated emission-depletion microscopy at the Drosophila neuromuscular junction, we found that presynaptic nanorings formed by the active-zone scaffold Bruchpilot (Brp) align with postsynaptic glutamate receptor (GluR) rings. Individual rings harbor approximately four transsynaptically aligned Brp-GluR nanocolumns. Similar nanocolumn rings are formed by the presynaptic protein Unc13A and GluRs. Intriguingly, acute GluR impairment triggers transsynaptic nanocolumn formation on the minute timescale during homeostatic plasticity. We reveal distinct phases of structural transsynaptic homeostatic plasticity, with postsynaptic GluR reorganization preceding presynaptic Brp modulation. Finally, homeostatic control of transsynaptic nano-architecture and neurotransmitter release requires the auxiliary GluR subunit Neto. Thus, transsynaptic nanocolumn rings provide a substrate for rapid homeostatic stabilization of synaptic efficacy.

CASK and FARP localize two classes of post-synaptic ACh receptors thereby promoting cholinergic transmission.

Changes in neurotransmitter receptor abundance at post-synaptic elements play a pivotal role in regulating synaptic strength. For this reason, there is significant interest in identifying and characterizing the scaffolds required for receptor localization at different synapses. Here we analyze the role of two C. elegans post-synaptic scaffolding proteins (LIN-2/CASK and FRM-3/FARP) at cholinergic neuromuscular junctions. Constitutive knockouts or muscle specific inactivation of lin-2 and frm-3 dramatically reduced spontaneous and evoked post-synaptic currents. These synaptic defects resulted from the decreased abundance of two classes of post-synaptic ionotropic acetylcholine receptors (ACR-16/CHRNA7 and levamisole-activated AChRs). LIN-2's AChR scaffolding function is mediated by its SH3 and PDZ domains, which interact with AChRs and FRM-3/FARP, respectively. Thus, our findings show that post-synaptic LIN-2/FRM-3 complexes promote cholinergic synaptic transmission by recruiting AChRs to post-synaptic elements.

Physiologic and Nanoscale Distinctions Define Glutamatergic Synapses in Tonic vs Phasic Neurons.

Neurons exhibit a striking degree of functional diversity, each one tuned to the needs of the circuitry in which it is embedded. A fundamental functional dichotomy occurs in activity patterns, with some neurons firing at a relatively constant "tonic" rate, while others fire in bursts, a "phasic" pattern. Synapses formed by tonic versus phasic neurons are also functionally differentiated, yet the bases of their distinctive properties remain enigmatic. A major challenge toward illuminating the synaptic differences between tonic and phasic neurons is the difficulty in isolating their physiological properties. At the Drosophila neuromuscular junction, most muscle fibers are coinnervated by two motor neurons: the tonic "MN-Ib" and phasic "MN-Is." Here, we used selective expression of a newly developed botulinum neurotoxin transgene to silence tonic or phasic motor neurons in Drosophila larvae of either sex. This approach highlighted major differences in their neurotransmitter release properties, including probability, short-term plasticity, and vesicle pools. Furthermore, Ca2+ imaging demonstrated ∼2-fold greater Ca2+ influx at phasic neuron release sites relative to tonic, along with an enhanced synaptic vesicle coupling. Finally, confocal and super-resolution imaging revealed that phasic neuron release sites are organized in a more compact arrangement, with enhanced stoichiometry of voltage-gated Ca2+ channels relative to other active zone scaffolds. These data suggest that distinctions in active zone nano-architecture and Ca2+ influx collaborate to differentially tune glutamate release at tonic versus phasic synaptic subtypes.SIGNIFICANCE STATEMENT "Tonic" and "phasic" neuronal subtypes, based on differential firing properties, are common across many nervous systems. Using a recently developed approach to selectively silence transmission from one of these two neurons, we reveal specialized synaptic functional and structural properties that distinguish these specialized neurons. This study provides important insights into how input-specific synaptic diversity is achieved, which could have implications for neurologic disorders that involve changes in synaptic function.