Unraveling the molecular landscape of lead-induced cochlear synaptopathy: a quantitative proteomics analysis.

Introduction: Exposure to heavy metal lead can cause serious health effects such as developmental neurotoxicity in infants, cognitive impairment in children, and cardiovascular and nephrotoxic effects in adults. Hearing loss is one of the toxic effects induced by exposure to lead. Previous studies demonstrated that exposure to lead causes oxidative stress in the cochlea and disrupts ribbon synapses in the inner hair cells. Methods: This study investigated the underlying mechanism by evaluating the changes in the abundance of cochlear synaptosomal proteins that accompany lead-induced cochlear synaptopathy and hearing loss in mice. Young-adult CBA/J mice were given lead acetate in drinking water for 28 days. Results: Lead exposure significantly increased the hearing thresholds, particularly at the higher frequencies in both male and female mice, but it did not affect the activity of outer hair cells or induce hair cell loss. However, lead exposure decreased wave-I amplitude, suggesting lead-induced cochlear synaptopathy. In agreement, colocalization of pre- and post-synaptic markers indicated that lead exposure decreased the number of paired synapses in the basal turn of the cochlea. Proteomics analysis indicated that lead exposure increased the abundance of 352 synaptic proteins and decreased the abundance of 394 synaptic proteins in the cochlea. Bioinformatics analysis indicated that proteins that change in abundance are highly enriched in the synaptic vesicle cycle pathway. Discussion: Together, these results suggest that outer hair cells are not the primary target in lead-induced ototoxicity, that lead-induced cochlear synaptopathy is more pronounced in the basal turn of the cochlea, and that synaptic vesicle cycle signaling potentially plays a critical role in lead-induced cochlear synaptopathy.

RIM and RIM-Binding Protein Localize Synaptic CaV2 Channels to Differentially Regulate Transmission in Neuronal Circuits.

At chemical synapses, voltage-gated Ca2+ channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs to evoke rapid transmitter release. Localization is mediated by Rab3-interacting molecule (RIM) and RIM-binding proteins, which interact and bind to the C terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Although postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition (E-I) balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABAA or GABAB receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus, RIMB-1 function differentially affects excitation-inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission within circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 ß-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet it does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerge from VGCC regulation.

A Transdermal Prion-Bionics Supermolecule as a RAB3A Antagonist for Enhancing Facial Youthfulness.

The mechanism research of skin wrinkles, conducted on volunteers underwent high-intensity desk work and mice subjected to partial sleep deprivation, revealed a significant reduction in dermal thickness associated with the presence of wrinkles. This can be attributed to the activation of facial nerves in a state of hysteria due to an abnormally elevated interaction between SNAP25 and RAB3A proteins involved in the synaptic vesicle cycle (SVC). Facilitated by AI-assisted structural design, a refined peptide called RSIpep is developed to modulate this interaction and normalize SVC. Drawing inspiration from prions, which possess the ability to protect themselves against proteolysis and invade neighboring nerve cells through macropinocytosis, RSIpep is engineered to demonstrate a GSH-responsive reversible self-assembly into a prion-like supermolecule (RSIprion). RSIprion showcases protease resistance, micropinocytosis-dependent cellular internalization, and low adhesion with constituent molecules in the cuticle, thereby endowing it with the transdermic absorption and subsequent biofunction in redressing the frenzied SVC. As a facial mud mask, it effectively reduces periorbital and perinasal wrinkles in the human face. Collectively, RSIprion not only presents a clinical potential as an anti-wrinkle prion-like supermolecule, but also exemplifies a reproducible instance of bionic strategy-guided drug development that bestows transdermal ability upon the pharmaceutical molecule.

Exposure to an enriched environment modulates the synaptic vesicle cycle in a mouse spinal cord injury model.

Spinal cord injury (SCI) leads to motor and sensory impairment below the site of injury, thereby necessitating rehabilitation. An enriched environment (EE) increases social interaction and locomotor activity in a mouse model, similar to human rehabilitation. However, the impact of EE on presynaptic plasticity in gene expression levels remains unclear. Hence, this study aimed to investigate the therapeutic potential of EE in an SCI mouse model. Mice with spinal cord contusion were divided into two groups: those housed in standard cages (control) and those in EE conditions (EE). Each group was housed separately for either 2- or 8-weeks post-injury, after which RNA sequencing was performed and compared to a sham group (receiving only a dorsal laminectomy). The synaptic vesicle cycle (SVC) pathway and related genes showed significant downregulation after SCI at both time points. Subsequently, we investigated whether exposure to EE for 2- and 8-weeks post-SCI could modulate the SVC pathway and its related genes. Notably, exposure to EE for 8 weeks resulted in a marked reversal effect of SVC-related gene expression, along with stimulation of axon regeneration and mitigation of locomotor activity loss. Thus, prolonged exposure to EE increased presynaptic activity, fostering axon regeneration and functional improvement by modulating the SVC in the SCI mouse model. These findings suggest that EE exposure proves effective in inducing activity-dependent plasticity, offering a promising therapeutic approach akin to rehabilitation training in patients with SCI.

The active zone protein CLA-1 (Clarinet) bridges two subsynaptic domains to regulate presynaptic sorting of ATG-9.

In neuronal synapses, autophagosome biogenesis is coupled with the activity-dependent synaptic vesicle cycle via ATG-9. How vesicles containing ATG-9 are sorted at the presynapse is unknown. We performed forward genetic screens at single synapses of C. elegans neurons for mutants that disrupt ATG-9 presynaptic localization, and identified the long isoform of the active zone protein CLA-1 (Clarinet; CLA-1 L). We find that disrupting CLA-1 L results in abnormal accumulation of ATG-9-containing vesicles enriched with clathrin. The adaptor protein complexes and proteins at the periactive zone genetically interact with CLA-1 L in ATG-9 sorting. Moreover, the phenotype of the ATG-9 protein in cla-1(L) mutants was not observed for integral synaptic vesicle proteins, suggesting distinct mechanisms that regulate sorting of ATG-9-containing vesicles and synaptic vesicles. Our findings reveal novel roles for active zone proteins in the sorting of ATG-9 and in presynaptic macroautophagy/autophagy.

Mitofusin 2 Sustains the Axonal Mitochondrial Network to Support Presynaptic Ca2+ Homeostasis and the Synaptic Vesicle Cycle in Rat Hippocampal Axons.

Mitochondria exert powerful control over cellular physiology, contributing to ion homeostasis, energy production, and metabolite biosynthesis. The trafficking and function of these organelles are particularly important in neurons, with impaired mitochondrial function or altered morphology observed in every neurodegenerative disorder studied. While mitochondrial biosynthetic products play a crucial role in maintaining cellular function, their resulting byproducts can have negative consequences. Thus, organelle quality control (QC) mechanisms that maintain mitochondrial function are imperative to restrict destructive signaling cascades in the cell. Axons are particularly sensitive to damage, and there is little consensus regarding the mechanisms that mediate mitochondrial QC in this compartment. Here, we first investigated the unstressed behavior of mitochondria in rat hippocampal neurons of mixed sex, focusing on mitochondrial trafficking and fusion to better understand potential QC mechanisms. We observed size and redox asymmetry of mitochondrial traffic in axons, suggesting an active QC mechanism in this compartment. We also document biochemical complementation upon the fusion and fission of axonal mitochondria. Eliminating fusion by knocking down the neuronal mitochondrial fusion protein mitofusin 2 (MFN2) reduced the rates of axonal mitochondrial trafficking and fusion, decreased the levels of synaptic vesicle (SV) proteins, inhibited exocytosis, and impaired SV recruitment from the reserve pool during extended stimulation. MFN2 knockdown also resulted in presynaptic Ca2+ dyshomeostasis. Remarkably, upon MFN2 knockdown, presynaptic mitochondria sequestered Ca2+ more efficiently, effectively limiting presynaptic Ca2+ transients during stimulation. These results support an active mitochondrial trafficking and fusion-related QC process that supports presynaptic Ca2+ handling and the SV cycle.SIGNIFICANCE STATEMENT Decreased or altered mitochondrial function is observed in many disease states. All neurodegenerative diseases co-present with some sort of mitochondrial abnormality. Therefore, identifying quality control mechanisms that sustain the mitochondrial network in neurons, and particularly in axons, is of significant interest. The response of axonal mitochondria to acutely applied toxins or injury has been studied in detail. Although informative, the response of neurons to these insults might not be physiologically relevant, so it is crucial to also study the basal behavior of axonal mitochondria. Here, we use fluorescent biosensors to investigate the mitochondrial network in neurons and examine the role of mitofusin 2 in maintaining the axonal mitochondrial network and in supporting the synaptic vesicle cycle.

The Mood Stabilizer Lithium Slows Down Synaptic Vesicle Cycling at Glutamatergic Synapses.

Lithium is a mood stabilizer broadly used to prevent and treat symptoms of mania and depression in people with bipolar disorder (BD). Little is known, however, about its mode of action. Here, we analyzed the impact of lithium on synaptic vesicle (SV) cycling at presynaptic terminals releasing glutamate, a neurotransmitter previously implicated in BD and other neuropsychiatric conditions. We used the pHluorin-based synaptic tracer vGpH and a fully automated image processing pipeline to quantify the effect of lithium on both SV exocytosis and endocytosis in hippocampal neurons. We found that lithium selectively reduces SV exocytic rates during electrical stimulation, and markedly slows down SV recycling post-stimulation. Analysis of single-bouton responses revealed the existence of functionally distinct excitatory synapses with varying sensitivity to lithium-some terminals show responses similar to untreated cells, while others are markedly impaired in their ability to recycle SVs. While the cause of this heterogeneity is unclear, these data indicate that lithium interacts with the SV machinery and influences glutamate release in a large fraction of excitatory synapses. Together, our findings show that lithium down modulates SV cycling, an effect consistent with clinical reports indicating hyperactivation of glutamate neurotransmission in BD.

Physiological Roles of ß-amyloid in Regulating Synaptic Function: Implications for AD Pathophysiology.

The physiological functions of endogenous amyloid-ß (Aß), which plays important role in the pathology of Alzheimer's disease (AD), have not been paid enough attention. Here, we review the multiple physiological effects of Aß, particularly in regulating synaptic transmission, and the possible mechanisms, in order to decipher the real characters of Aß under both physiological and pathological conditions. Some worthy studies have shown that the deprivation of endogenous Aß gives rise to synaptic dysfunction and cognitive deficiency, while the moderate elevation of this peptide enhances long term potentiation and leads to neuronal hyperexcitability. In this review, we provide a new view for understanding the role of Aß in AD pathophysiology from the perspective of physiological meaning.

Interactions between Membraneless Condensates and Membranous Organelles at the Presynapse: A Phase Separation View of Synaptic Vesicle Cycle.

Action potential-induced neurotransmitter release in presynaptic boutons involves coordinated actions of a large list of proteins that are associated directly or indirectly with membrane structures including synaptic vesicles and plasma membranes. These proteins are often highly abundant in different synaptic bouton sub-compartments, and they rarely act alone. Instead, these proteins interact with each other forming intricate and distinct molecular complexes. Many of these complexes form condensed clusters on membrane surfaces. This review summarizes findings in recent years showing that many of presynaptic protein complex assemblies are formed via phase separation. These protein condensates extensively interact with lipid membranes via distinct modes, forming various mesoscale structures by different mode of organizations between membraneless condensates and membranous organelles. We discuss that such mesoscale interactions could have deep implications on mobilization, exocytosis, and retrieval of synaptic vesicles.

Recent advance in Tau proteinopathy and circulatory disturbance of synaptic vesicles / 中华神经医学杂志

Recent studies have found that the pathological changes of Tau proteinopathy, such as Alzheimer's disease (AD) and progressive supranuclear palsy (PSP), involve transmission or circulatory disturbance of synaptic vesicles. This article reviews the relations of AD/PSP with circulatory disturbance of synaptic vesicles and explore the pathogenesis of these diseases, aiming to provide a new perspective for new therapeutic drugs.

Physiological Roles of β-amyloid in Regulating Synaptic Function: Implications for AD Pathophysiology / 神经科学通报·英文版

The physiological functions of endogenous amyloid-β (Aβ), which plays important role in the pathology of Alzheimer's disease (AD), have not been paid enough attention. Here, we review the multiple physiological effects of Aβ, particularly in regulating synaptic transmission, and the possible mechanisms, in order to decipher the real characters of Aβ under both physiological and pathological conditions. Some worthy studies have shown that the deprivation of endogenous Aβ gives rise to synaptic dysfunction and cognitive deficiency, while the moderate elevation of this peptide enhances long term potentiation and leads to neuronal hyperexcitability. In this review, we provide a new view for understanding the role of Aβ in AD pathophysiology from the perspective of physiological meaning.

Effects of the clathrin inhibitor Pitstop-2 on synaptic vesicle recycling at a central synapse in vivo.

Four modes of endocytosis and subsequent synaptic vesicle (SV) recycling have been described at the presynapse to ensure the availability of SVs for synaptic release. However, it is unclear to what extend these modes operate under physiological activity patterns in vivo. The coat protein clathrin can regenerate SVs either directly from the plasma membrane (PM) via clathrin-mediated endocytosis (CME), or indirectly from synaptic endosomes by SV budding. Here, we examined the role of clathrin in SV recycling under physiological conditions by applying the clathrin inhibitor Pitstop-2 to the calyx of Held, a synapse optimized for high frequency synaptic transmission in the auditory brainstem, in vivo. The effects of clathrin-inhibition on SV recycling were investigated by serial sectioning scanning electron microscopy (S3EM) and 3D reconstructions of endocytic structures labeled by the endocytosis marker horseradish peroxidase (HRP). We observed large endosomal compartments as well as HRP-filled, black SVs (bSVs) that have been recently recycled. The application of Pitstop-2 led to reduced bSV but not large endosome density, increased volumes of large endosomes and shifts in the localization of both types of endocytic compartments within the synapse. These changes after perturbation of clathrin function suggest that clathrin plays a role in SV recycling from both, the PM and large endosomes, under physiological activity patterns, in vivo.

2022 Overview of Metabolic Epilepsies.

Understanding the genetic architecture of metabolic epilepsies is of paramount importance, both to current clinical practice and for the identification of further research directions. The main goals of our study were to identify the scope of metabolic epilepsies and to investigate their clinical presentation, diagnostic approaches and treatments. The International Classification of Inherited Metabolic Disorders and IEMbase were used as a basis for the identification and classification of metabolic epilepsies. Six hundred metabolic epilepsies have been identified, accounting for as much as 37% of all currently described inherited metabolic diseases (IMD). Epilepsy is a particularly common symptom in disorders of energy metabolism, congenital disorders of glycosylation, neurotransmitter disorders, disorders of the synaptic vesicle cycle and some other IMDs. Seizures in metabolic epilepsies may present variably, and most of these disorders are complex and multisystem. Abnormalities in routine laboratory tests and/or metabolic testing may be identified in 70% of all metabolic epilepsies, but in many cases they are non-specific. In total, 111 metabolic epilepsies (18% of all) have specific treatments that may significantly change health outcomes if diagnosed in time. Although metabolic epilepsies comprise an important and significant group of disorders, their real scope and frequency may have been underestimated.

Transmembrane protein ATG-9 links presynaptic autophagy with the synaptic vesicle cycle.

Macroautophagy/autophagy occurs preferentially at synapses and responds to increased neuronal activity states. How synaptic autophagy is coupled to the neuronal activity state is largely unknown. Through genetic approaches we find that ATG-9, the only transmembrane protein in the core autophagy pathway, is transported from the trans-Golgi network to synapses in C. elegans via the AP-3 complex. At synapses ATG-9 undergoes exo-endocytosis in an activity-dependent manner. Mutations that disrupt the endocytosis pathway, including a mutation associated with early onset Parkinsonism (EOP), lead to abnormal ATG-9 accumulation into subsynaptic clathrin-rich foci, and defects in activity-induced synaptic autophagy. We propose that ATG-9 exo-endocytosis links the activity-dependent synaptic vesicle cycle with autophagosome formation at synapses.

Presynaptic autophagy is coupled to the synaptic vesicle cycle via ATG-9.

Autophagy is a cellular degradation pathway essential for neuronal health and function. Autophagosome biogenesis occurs at synapses, is locally regulated, and increases in response to neuronal activity. The mechanisms that couple autophagosome biogenesis to synaptic activity remain unknown. In this study, we determine that trafficking of ATG-9, the only transmembrane protein in the core autophagy pathway, links the synaptic vesicle cycle with autophagy. ATG-9-positive vesicles in C. elegans are generated from the trans-Golgi network via AP-3-dependent budding and delivered to presynaptic sites. At presynaptic sites, ATG-9 undergoes exo-endocytosis in an activity-dependent manner. Mutations that disrupt endocytosis, including a lesion in synaptojanin 1 associated with Parkinson's disease, result in abnormal ATG-9 accumulation at clathrin-rich synaptic foci and defects in activity-induced presynaptic autophagy. Our findings uncover regulated key steps of ATG-9 trafficking at presynaptic sites and provide evidence that ATG-9 exo-endocytosis couples autophagosome biogenesis at presynaptic sites with the activity-dependent synaptic vesicle cycle.

Developmental shift to mitochondrial respiration for energetic support of sustained transmission during maturation at the calyx of Held.

A considerable amount of energy is expended following presynaptic activity to regenerate electrical polarization and maintain efficient release and recycling of neurotransmitter. Mitochondria are the major suppliers of neuronal energy, generating ATP via oxidative phosphorylation. However, the specific utilization of energy from cytosolic glycolysis rather than mitochondrial respiration at the presynaptic terminal during synaptic activity remains unclear and controversial. We use a synapse specialized for high-frequency transmission in mice, the calyx of Held, to test the sources of energy used to maintain energy during short activity bursts (<1 s) and sustained neurotransmission (30-150 s). We dissect the role of presynaptic glycolysis versus mitochondrial respiration by acutely and selectively blocking these ATP-generating pathways in a synaptic preparation where mitochondria and synaptic vesicles are prolific, under near-physiological conditions. Surprisingly, if either glycolysis or mitochondrial ATP production is intact, transmission during repetitive short bursts of activity is not affected. In slices from young animals before the onset of hearing, where the synapse is not yet fully specialized, both glycolytic and mitochondrial ATP production are required to support sustained, high-frequency neurotransmission. In mature synapses, sustained transmission relies exclusively on mitochondrial ATP production supported by bath lactate, but not glycolysis. At both ages, we observe that action potential propagation begins to fail before defects in synaptic vesicle recycling. Our data describe a specific metabolic profile to support high-frequency information transmission at the mature calyx of Held, shifting during postnatal synaptic maturation from glycolysis to rely on monocarboxylates as a fuel source.NEW & NOTEWORTHY We dissect the role of presynaptic glycolysis versus mitochondrial respiration in supporting high-frequency neurotransmission, by acutely blocking these ATP-generating pathways at a synapse tuned for high-frequency transmission. We find that massive energy expenditure is required to generate failure when only one pathway is inhibited. Action potential propagation is lost before impaired synaptic vesicle recycling. Synaptic transmission is exclusively dependent on oxidative phosphorylation in mature synapses, indicating presynaptic glycolysis may be dispensable for ATP maintenance.

(Dys)regulation of Synaptic Activity and Neurotransmitter Release by ß-Amyloid: A Look Beyond Alzheimer's Disease Pathogenesis.

Beside its widely studied role in the pathogenesis of Alzheimer's disease (AD), ß-amyloid (Aß) is a normal and soluble product of neuronal metabolism that regulates several key physiological functions, exerting neuromodulatory effects on synaptic plasticity, memory, and neurotransmitter release. Such effects have been observed to occur in a hormetic fashion, with Aß exhibiting a dual role influenced by its concentration, the different isoforms, or aggregation forms of the peptide. However, to date, our knowledge about the physiological functions of Aß and, in particular, its modulatory role on synaptic activity and neurotransmission in the normal brain is fragmentary, thus hindering a clear comprehension of the biological mechanisms underlying the derangement from function to dysfunction. In particular, according to the amyloid cascade hypothesis, the switch from physiology to pathology is linked to the abnormal increase in Aß levels, due to an imbalance in Aß production and clearance. In this regard, increased Aß levels have been hypothesized to induce early defects in synaptic function and such alterations have been suggested to account, at least in part, for the onset of neuropsychiatric symptoms (e.g., apathy, anxiety, changes in mood, depression, and agitation/aggression), frequently observed in the prodromal stage of AD. Therefore, understanding the biological mechanisms underlying early synaptic alterations in AD is a key starting point to frame the relevant time windows for AD treatment and to gain insight into AD etiopathogenesis.

The neurodevelopmental spectrum of synaptic vesicle cycling disorders.

In this review, we describe and discuss neurodevelopmental phenotypes arising from rare, high penetrance genomic variants which directly influence synaptic vesicle cycling (SVC disorders). Pathogenic variants in each SVC disorder gene lead to disturbance of at least one SVC subprocess, namely vesicle trafficking (e.g. KIF1A and GDI1), clustering (e.g. TRIO, NRXN1 and SYN1), docking and priming (e.g. STXBP1), fusion (e.g. SYT1 and PRRT2) or re-uptake (e.g. DNM1, AP1S2 and TBC1D24). We observe that SVC disorders share a common set of neurological symptoms (movement disorders, epilepsies), cognitive impairments (developmental delay, intellectual disabilities, cerebral visual impairment) and mental health difficulties (autism, ADHD, psychiatric symptoms). On the other hand, there is notable phenotypic variation between and within disorders, which may reflect selective disruption to SVC subprocesses, spatiotemporal and cell-specific gene expression profiles, mutation-specific effects, or modifying factors. Understanding the common cellular and systems mechanisms underlying neurodevelopmental phenotypes in SVC disorders, and the factors responsible for variation in clinical presentations and outcomes, may translate to personalized clinical management and improved quality of life for patients and families.

Amyloid-ß and Synaptic Vesicle Dynamics: A Cacophonic Orchestra.

It is now more than two decades since amyloid-ß (Aß), the proteolytic product of the amyloid-ß protein precursor (AßPP), was first demonstrated to be a normal and soluble product of neuronal metabolism. To date, despite a growing body of evidence suggests its regulatory role on synaptic function, the exact cellular and molecular pathways involved in Aß-driven synaptic effects remain elusive. This review provides an overview of the mounting evidence showing Aß-mediated effects on presynaptic functions and neurotransmitter release from axon terminals, focusing on its interaction with synaptic vesicle cycle. Indeed, Aß peptides have been found to interact with key presynaptic scaffold proteins and kinases affecting the consequential steps of the synaptic vesicle dynamics (e.g., synaptic vesicles exocytosis, endocytosis, and trafficking). Defects in the fine-tuning of synaptic vesicle cycle by Aß and deregulation of key molecules and kinases, which orchestrate synaptic vesicle availability, may alter synaptic homeostasis, possibly contributing to synaptic loss and cognitive decline. Elucidating the presynaptic mechanisms by which Aß regulate synaptic transmission is fundamental for a deeper comprehension of the biology of presynaptic terminals as well as of Aß-driven early synaptic defects occurring in prodromal stage of AD. Moreover, a better understating of Aß involvement in cellular signal pathways may allow to set up more effective therapeutic interventions by detecting relevant molecular mechanisms, whose imbalance might ultimately lead to synaptic impairment in AD.

Septin Polymerization Slows Synaptic Vesicle Recycling in Motor Nerve Endings.

Septins are GTP-binding proteins recognized as a component of the cytoskeleton. Despite the fact that septins are highly expressed by neurons and can interact with the proteins that participate in synaptic vesicle exocytosis and endocytosis, the role of septins in synaptic transmission and the synaptic vesicle recycling mechanisms is poorly understood. In this study, neurotransmitter release and synaptic vesicle exocytosis and endocytosis were investigated by microelectrode intracellular recording of end-plate potentials and fluorescent confocal microscopy in mouse diaphragm motor nerve endings during septin polymerization induced by forchlorfenuron application. It was shown that forchlorfenuron application reduces neurotransmission during prolonged high-frequency (20 and 50 pulses/s) stimulation. Application of pairs of short high-frequency stimulation trains showed that forchlorfenuron slows the replenishment of the readily releasable pool. Forchlorfenuron enhanced FM 1-43 fluorescent dye loading by synaptic vesicle endocytosis but decreased dye unloading from the preliminarily stained nerve endings by synaptic vesicle exocytosis. It was concluded that the septin polymerization induced by forchlorfenuron application slows the rate of synaptic vesicle recycling in motor nerve endings due to the impairment of synaptic vesicle transport.