Synaptic Vesicle Recycling Research Articles

Neuromodulation of Heart Functions with Infrared Light – Candidate Targets: TRP Channels

Neurostimulation regulates important physiological parameters including blood pressure, heart rate, and respiration. For this reason, neurostimulation has been considered as a therapy in many contexts (e.g., deep brain stimulation, cardiac pacemakers). Our group has discovered that using infra‐red (IR) light can be used to pace the embryonic and adult heart and modulate neuronal activity up and down. Application of a focused IR beam to small areas of the rat nodose ganglion uncovered regional functional specificity. The IR laser beam focused different regions of the ganglion elicited distinct physiological responses in blood pressure and breathing. The mechanism of IR impulse modulation is unknown but hypothesized to work through the creation of temperature gradients. In this study, we report the location of candidate target molecules in ganglia that might be triggered by IR exposure and may be responsible for the modulating effects on neuronal activity. The proposed target molecules are the transient receptor potential (TRP) channels, a calcium channel superfamily whose members are heat‐ and mechano‐sensitive. These channels are divided into several different families and are specialized for sensing different temperature ranges (noxious heat, non‐noxious heat, cold). In this study we focused on an expression of TRPV1, which is considered to be one of the main heat sensitive TRPs and is known to regulate synaptic vesicle recycling. Another important heat sensitive receptor, which is thought to work with TRPV1 is TRMP3. It has properties that make it a more promising target of the therapeutic pain control. Here we report spatial localization and distribution of various TRPs in rat and mouse nodose and superior cervical ganglion studied by immunohistology imaged using epifluorescence and confocal microscopy and in some cases tissue clearing protocols. TRPV1 and TRPM3 are found in a subset of neurons within the ganglia. These are found in clusters and scattered throughout the ganglia. TRPV1 and TRPM3 are both found in the cytoplasm evenly distributed or in some cells in a granular distribution. Both channels are expressed in small or medium size neurons. Some of the others TRPs receptors are found in neurons in rat ganglia, others are predominantly expressed in glial cells. From our preliminary data the TRP channels are present and could play an important role in heat sensing in the ganglia. There heterogeneous distribution within the ganglia could explain the region specific physiological responses. Our goal is to determine the 3‐D spatial localization of these and other IR sensitive channels in the nodose and superior cervical ganglia that modulate the function of many organ systems.Support or Funding InformationNIH grant: 1OT2OD025307‐01, Fulbright Foundation, Operational Programme Research, Development and Education (reg. n. CZ.02.2.69/0.0/0.0/16_027/0008495), Ministry of Education CZ ‐ Progres Q38.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hippocampal gene expression patterns linked to late-life physical activity oppose age and AD-related transcriptional decline

Exercise has emerged as a powerful variable that can improve cognitive function and delay age-associated cognitive decline and Alzheimer's disease (AD); however, the underlying mechanisms are poorly understood. To determine if protective mechanisms may occur at the transcriptional level, we used microarrays to investigate the relationship between physical activity levels and gene expression patterns in the cognitively intact aged human hippocampus. In parallel, hippocampal gene expression patterns associated with aging and AD were assessed using publicly available microarray data profiling hippocampus from young (20–59 years), cognitively intact aging (73–95 years) and age-matched AD cases. To identify “anti-aging/AD” transcription patterns associated with physical activity, probesets significantly associated with both physical activity and aging/AD were identified and their directions of expression change in each condition were compared. Remarkably, of the 2210 probesets significant in both data sets, nearly 95% showed opposite transcription patterns with physical activity compared with aging/AD. The majority (>70%) of these anti-aging/AD genes showed increased expression with physical activity and decreased expression in aging/AD. Enrichment analysis of the anti-aging/AD genes showing increased expression in association with physical activity revealed strong overrepresentation of mitochondrial energy production and synaptic function, along with axonal function and myelin integrity. Synaptic genes were notably enriched for synaptic vesicle priming, release and recycling, glutamate and GABA signaling, and spine plasticity. Anti-aging/AD genes showing decreased expression in association with physical activity were enriched for transcription-related function (notably negative regulation of transcription). These data reveal that physical activity is associated with a more youthful profile in the hippocampus across multiple biological processes, providing a potential molecular foundation for how physical activity can delay age- and AD-related decline of hippocampal function.

Photoconversion of FM1-43 Reveals Differences in Synaptic Vesicle Recycling and Sensitivity to Pharmacological Disruption of Actin Dynamics in Individual Synapses.

The cycling of synaptic vesicles ensures that neurons can communicate adequately through their synapses on repeated occasions when activity is sustained, and several steps in this cycle are modulated by actin. The effects of pharmacological stabilization of actin with jasplakinolide or its depolymerization with latrunculin A was assessed on the synaptic vesicle cycle at individual boutons of cerebellar granule cells, using FM1-43 imaging to track vesicle recycling and its photoconversion to specifically label recycled organelles. Remarkable differences in the recycling capacity of individual boutons are evident, and their dependence on the actin cytoskeleton for recycling is clear. Disrupting actin dynamics causes a loss of functional boutons, and while this indicates that exo/endocytotic cycling in boutons is fully dependent on such events, this dependence is only partial in other boutons. Indeed, exocytosis and vesicle trafficking are impaired significantly by stabilizing or depolymerizing actin, whereas repositioning recycled vesicles at the active zone seems to be dependent on actin polymerization alone. These findings support the hypothesis that different steps of synaptic vesicle cycling depend on actin dynamics and that such dependence varies among individual boutons.

Layer-specific involvement of endocannabinoid signaling in muscarinic-induced long-term depression in layer 2/3 pyramidal neurons of rat visual cortex

Neuromodulatory facilitation of long-term synaptic plasticity is important in learning, memory, and experience-dependent cortical plasticity. Although muscarinic-induced long-term depression (mLTD) in the visual cortex is well known, its cellular mechanisms are not fully understood yet. Since endocannabinoid signaling mediates presynaptic expression of LTD in various brain areas including the primary visual cortex of rats, we investigated the involvement of endocannabinoids in the induction of mLTD in different dendritic compartments of layer 2/3 pyramidal neurons. With an unloading experiment of FM1-43 as an indicator of synaptic vesicle recycling, we confirmed that layer 1 and layer 4 stimulations mainly activated distal apical (in layer 1) and perisomatic (in layer 2/3) dendritic compartments, respectively. Bath application of muscarine (10 min) induced LTD in synaptic inputs activated by stimulation of layers 1 (L1-mLTD) and 4 (L2/3-mLTD). Both mLTDs were blocked by intracellular Ca2+ chelator BAPTA and bath application of NMDA receptor antagonist d-AP5. However, only L2/3-mLTD exhibited an increase in paired-pulse ratio. In addition, only L2/3-mLTD was blocked by treatment with CB1 receptor antagonist AM251. Both mLTDs were blocked by intracellular NMDA receptor antagonist MK801, but not by glia-specific metabolic inhibitor fluoroacetate, implying that neither presynaptic NMDA receptors nor astrocytes are involved in mLTD. These results suggest that L2/3-mLTD is expressed presynaptically via retrograde endocannabinoid signaling while L1-mLTD is endocannabinoid independent in layer 2/3 pyramidal neurons of the visual cortex. Therefore, layer-specific involvement of endocannabinoids in the induction of mLTD might play an important role in cortical development and information processing in the neocortex.

Endophilin-A regulates presynaptic Ca2+ influx and synaptic vesicle recycling in auditory hair cells.

Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and curvature-generating properties, in mouse IHCs. Single-cell RT-PCR indicated the expression of endophilins-A1-3 in IHCs, and immunoblotting confirmed the presence of endophilin-A1 and endophilin-A2 in the cochlea. Patch-clamp recordings from endophilin-A-deficient IHCs revealed a reduction of Ca2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin-mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co-immunoprecipitated with purified endophilin-A1 protein, suggestive of a molecular interaction that might aid exocytosis-endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome-like vacuoles at IHC active zones. In summary, endophilins regulate Ca2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation.

Dysfunction of Neuromuscular Synaptic Transmission and Synaptic Vesicle Recycling in Motor Nerve Terminals of mSOD1 Transgenic Mice with Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive incurable neurodegenerative disease with selective loss of lower and upper motoneurons. Dysfunction and destruction of neuromuscular synapses leading to skeletal muscle denervation is one of the early and major events in ALS pathogenesis. Despite of the presence of studies devoted to investigation of neuromuscular transmission in ALS mouse models, no detailed information about molecular mechanisms underlying synaptic dysfunction in ALS and their temporal dynamics during ALS progression is provided. The goal of present work was to study the processes of neurotransmitter release and presynaptic vesicle recycling in neuromuscular synapses of mutated SOD1 (mSOD1) transgenic mice at different clinical stages of disease. Utilizing combination of electrophysiological recording and FM 1–43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide) fluorescent imaging, we found that mSOD1 mice at symptomatic and terminal stages of disease showed decreased baseline quantal content of end-plate potentials and prolonged synaptic vesicle recycling time comparing to wild-type mice. Despite the decrease of end-plate potential (EPP) quantal content, studied mSOD1 mice groups showed unchanged dynamics of EPP relative amplitude comparing to WT mice. We also found an increase of miniature end-plate potential amplitude in mSOD1 mice at symptomatic stage, which may reflect compensatory mechanism that alleviates reduction of EPP amplitude. Thus, we provided one of the first detailed characteristics of presynaptic dysfunction at neuromuscular junction in ALS model. Obtained data expand our understanding of the ALS pathogenesis and contribute to stage- and localization-specific description of ALS pathogenetic mechanisms.

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.

<i>Cindr</i>, the <i>Drosophila</i> Homolog of the <i>CD2AP</i> Alzheimer's Disease Susceptibility Gene, is Required for Synaptic Transmission and Proteostasis

The Alzheimer’s disease (AD) susceptibility gene, CD2-associated protein (CD2AP), encodes an actin-binding, adaptor protein, but its function in the nervous system is largely unknown. Loss of the Drosophila ortholog, cindr, enhances neurotoxicity of human Tau, which forms neurofibrillary tangle pathology in AD. Here, we show that Cindr is expressed in Drosophila neurons and present at presynaptic terminals. Flies lacking cindr show impairments in synaptic maturation, as well as synaptic vesicle recycling and release. Cindr associates and genetically interacts with 14-3-3ζ, regulates the ubiquitin-proteasome system, and affects turnover of Synapsin protein and the plasma membrane calcium ATPase (PMCA). Loss of cindr elevates PMCA levels and triggers reduction of cytosolic calcium. Studies of CD2AP mice support a conserved role in synaptic proteostasis, and CD2AP protein levels are also inversely related to Synapsin abundance in human postmortem brains. Our results reveal novel CD2AP neuronal requirements with relevance to AD susceptibility, including for proteostasis, calcium handling, and synaptic structure/function.

Differential pH Dynamics in Synaptic Vesicles From Intact Glutamatergic and GABAergic Synapses.

Synaptic transmission requires the presynaptic release of neurotransmitter from synaptic vesicles (SVs) onto the postsynaptic neuron. Vesicular neurotransmitter transporter proteins, which use a V-ATPase-generated proton gradient, play a crucial role in packaging neurotransmitter into SVs. Recent work has revealed different proton dynamics in SVs expressing the vesicular glutamate transporter (VGLUT) or the vesicular GABA transporter (VGAT) proteins. At the whole synapse level, this results in different steady-state pH and different reacidification dynamics during SV recycling (Egashira et al., 2016). In isolated SVs, the presence of VGAT causes a higher steady state pH, which is correlated with a faster proton efflux rate (Farsi et al., 2016). To address whether proton efflux from GABAergic and glutamatergic SVs in intact synapses differs, we applied a glutamatergic- or GABAergic neuron-specific expression strategy (Chang et al., 2014) to express a genetically encoded pH sensor (synaptophysin pHluorin; SypHy) and/or light-activated proton pump (pHoenix; (Rost et al., 2015). We confirm, with SypHy post-stimulation fluorescence dynamics, that the pH profile of recycling GABAergic SVs differs from that of recycling glutamatergic SVs (Egashira et al., 2016). Using light-activation of pHoenix in pH-neutral vesicles, we investigated the pH dynamics of actively filling vesicles, and could show that proton efflux from GABAergic SVs is indeed initially faster than glutamatergic SVs in intact synapses. Finally, we compared the filling rate of empty glutamatergic and GABAergic vesicles using pHoenix as a proton source, and find a slightly faster filling of glutamatergic vs. GABAergic SVs.

Presynaptic loss of dynamin-related protein 1 impairs synaptic vesicle release and recycling at the mouse calyx of Held.

This study characterizes the mechanisms underlying defects in synaptic transmission when dynamin-related protein 1 (DRP1) is genetically eliminated. Viral-mediated knockout of DRP1 from the presynaptic terminal at the mouse calyx of Held increased initial release probability, reduced the size of the synaptic vesicle recycling pool and impaired synaptic vesicle recycling. Transmission defects could be partially restored by increasing the intracellular calcium buffering capacity with EGTA-AM, implying close coupling of Ca2+ channels to synaptic vesicles was compromised. Acute restoration of ATP to physiological levels in the presynaptic terminal did not reverse the synaptic defects. Loss of DRP1 impairs mitochondrial morphology in the presynaptic terminal, which in turn seems to arrest synaptic maturation. Impaired mitochondrial biogenesis and function is implicated in many neurodegenerative diseases, and likely affects synaptic neurotransmission prior to cellular loss. Dynamin-related protein 1 (DRP1) is essential for mitochondrial fission and is disrupted in neurodegenerative disease. In this study, we used the mouse calyx of Held synapse as a model to investigate the impact of presynaptic DRP1 loss on synaptic vesicle (SV) recycling and sustained neurotransmission. In vivo viral expression of Cre recombinase in ventral cochlear neurons of floxed-DRP1 mice generated a presynaptic-specific DRP1 knockout (DRP1-preKO), where the innervated postsynaptic cell was unperturbed. Confocal reconstruction of the calyx terminal suggested SV clusters and mitochondrial content were disrupted, and presynaptic terminal volume was decreased. Using postsynaptic voltage-clamp recordings, we found that DRP1-preKO synapses had larger evoked responses at low frequency stimulation. DRP1-preKO synapses also had profoundly altered short-term plasticity, due to defects in SV recycling. Readily releasable pool size, estimated with high-frequency trains, was dramatically reduced in DRP1-preKO synapses, suggesting an important role for DRP1 in maintenance of release-competent SVs at the presynaptic terminal. Presynaptic Ca2+ accumulation in the terminal was also enhanced in DRP1-preKO synapses. Synaptic transmission defects could be partially rescued with EGTA-AM, indicating close coupling of Ca2+ channels to SV distance normally found in mature terminals may be compromised by DRP1-preKO. Using paired recordings of the presynaptic and postsynaptic compartments, recycling defects could not be reversed by acute dialysis of ATP into the calyx terminals. Taken together, our results implicate a requirement for mitochondrial fission to coordinate postnatal synapse maturation.

Time course and temperature dependence of synaptic vesicle endocytosis.

In presynaptic nerve terminals, synaptic vesicles are recycled locally via an evolutionarily conserved process that ensures maintenance of neurotransmission as well as structural integrity of synapses. Temperature is a key environmental factor that impacts critical steps involved in fusion, endocytosis and transport in different vesicle trafficking pathways. In neurons, temperature changes have been shown to impact synaptic vesicle recycling and synaptic efficacy. But contrary to non-neuronal systems, the temperature dependence of the steps involved in fusion, endocytosis and recycling of synaptic vesicles in presynaptic terminals is not completely understood, and the existing data remain highly debated. In this Review, we discuss the implications of biophysical, biochemical and functional findings on temperature dependence of membrane retrieval in multiple systems. We propose that systematic investigation of the temperature dependence of the presynaptic vesicle trafficking process can provide novel insight into poorly understood mechanisms that govern synaptic vesicle trafficking under diverse physiological conditions.

Multidimensional Dynamics of the Proteome in the Neurodegenerating and Ageing Mammalian Brain

The amount of any given protein in the brain is determined by the rates of its synthesis and destruction, which are regulated by different cellular mechanisms. Here, we combine metabolic labelling in live mice with global proteomic profiling to simultaneously quantify both the flux and amount of proteins in mouse models of neurodegeneration. In multiple models, protein turnover increases were associated with increasing pathology. This method distinguishes changes in protein expression mediated by synthesis from those mediated by degradation. In the AppNL-F knockin mouse model of Alzheimer’s disease increased turnover resulted from imbalances in both synthesis and degradation, converging on proteins associated with synaptic vesicle recycling (Dnm1, Cltc, Rims1) and mitochondria (Fis1, Ndufv1). In contrast to disease models, ageing in wildtype mice caused a widespread decrease in protein recycling associated with a decrease in autophagic flux. This simple multidimensional approach enables the comprehensive mapping of proteome dynamics and identifies affected proteins in mouse models of disease and other live animal test settings.

Riluzole attenuates the efficacy of glutamatergic transmission by interfering with the size of the readily releasable neurotransmitter pool

Riluzole is a potent neuroprotective agent which primarily inhibits excitatory neurotransmission interfering with presynaptic release, uptake and postsynaptic actions of glutamate by mechanisms that are not well understood. Riluzole and related prodrugs with improved blood brain barrier penetrance, are shown to be effective for the treatment of amyotrophic lateral sclerosis, ataxias, epilepsy and mood disorders. Our study was undertaken to decipher molecular and subcellular mechanisms of riluzole's antiglutamatergic effect, particularly focusing on presynaptic active zone structure and function. Applying multifarious live cell imaging techniques and amperometric glutamate recordings, we measured the impact of riluzole on presynaptic activity, synaptic vesicle recycling and glutamate release. Our in vitro and in vivo data revealed a unique mechanism whereby riluzole reduces the efficacy of glutamatergic transmission by selectively lowering the size of the readily releasable pool. This effect was correlated with the inhibition of protein kinase C-dependent Munc18-1 phosphorylation which is known to interfere with neurotransmitter release.

A Novel SYNJ1 Mutation in a Tunisian Family with Juvenile Parkinson's Disease Associated with Epilepsy.

Mutations in SYNJ1 gene have been described in few families with juvenile atypical Parkinson disease (PD). This gene encodes for "Synaptojanin 1," an enzyme playing a major role in the phosphorylation and the recycling of synaptic vesicles. In this study, we report two siblings, from a consanguineous Tunisian family, presenting juvenile PD. Both siblings developed mild Parkinsonism at 16 and 21years old respectively. One patient had generalized tonic-clonic seizures since the age of 7years. There was no evidence of sleep or autonomic dysfunctions and psychiatric disorders in both cases, but they developed a moderate cognitive impairment. They kept a good respond to low doses of levodopa treatment with no dyskinesia or motor fluctuations. We designed an NGS-based screening of 22 currently most prevalent parkinsonism-associated genes. Genetic study revealed a novel compound heterozygous mutation (p.Leu1406Phefs*42 and p.Lys1321Glu) in SYNJ1 gene. The p.Lys1321Glu mutation is located in the proline-rich domain and leads to a significant change in the 3D structure of the protein (RMS = 12.58Å). The p.Leu1406Phefs*42 mutation disrupt the AP2 binding sites and subsequently disable synaptic and vesicle endocytic recycling in neurons. This is the first report of mutation in the C-terminal domain of Synaptojanin 1 protein causing mild juvenile PD with generalized seizures, cognitive impairment, and good respond to levodopa treatment.

Dysfunction of Neuromuscular Synapses in the Genetic Model of Alzheimer's Disease.

The function of synaptic transmission and presynaptic vesicular cycle in the neuromuscular synapses of the diaphragm was studied in transgenic APP/PS1 mice (Alzheimer's disease model). The decrease in the quantal content of end-plate potential, intense depression of the amplitude of terminal plate potentials under conditions of lasting high frequency stimulation (50 Hz), a drastic prolongation of the synaptic vesicle recycling time in APP/PS1 mice in comparison with wild type mice were detected. Manifest dysfunction of the neuromuscular synapses, caused by disordered neurosecretion and recycling of the synaptic vesicles in the presynaptic nerve endings, was detected in the Alzheimer's disease model on transgenic APP/PS1 mice. The study supplemented the notions on the pathogenesis of Alzheimer's disease as a systemic disease, while the detected phenomena could just partially explain the development of motor disorders in this disease.

Modulation of dynamin function by small molecules.

Dynamins are essential as membrane remodelers in various cellular processes, like receptor-mediated endocytosis, synaptic vesicle recycling and spermatogenesis. Moreover, dynamin is involved in the internalization of numerous viruses and in the motility of several cancer cell lines. As tools for dissecting the underlying mechanisms of these important biological processes and as potential future therapeutics, small molecules have been developed in the last two decades that modulate the functions of dynamin. In this review we give an overview of the compound classes that are currently in use and describe how they affect dynamin function.

The Prion Protein Regulates Synaptic Transmission by Controlling the Expression of Proteins Key to Synaptic Vesicle Recycling and Exocytosis.

The cellular prion protein (PrPC), whose misfolded conformers are implicated in prion diseases, localizes to both the presynaptic membrane and postsynaptic density. To explore possible molecular contributions of PrPC to synaptic transmission, we utilized a mass spectrometry approach to quantify the release of glutamate from primary cerebellar granule neurons (CGN) expressing, or deprived of (PrP-KO), PrPC, following a depolarizing stimulus. Under the same conditions, we also tracked recycling of synaptic vesicles (SVs) in the two neuronal populations. We found that in PrP-KO CGN these processes decreased by 40 and 60%, respectively, compared to PrPC-expressing neurons. Unbiased quantitative mass spectrometry was then employed to compare the whole proteome of CGN with the two PrP genotypes. This approach allowed us to assess that, relative to the PrPC-expressing counterpart, the absence of PrPC modified the protein expression profile, including diminution of some components of SV recycling and fusion machinery. Subsequent quantitative RT-PCR closely reproduced proteomic data, indicating that PrPC is committed to ensuring optimal synaptic transmission by regulating genes involved in SV dynamics and neurotransmitter release. These novel molecular and cellular aspects of PrPC add insight into the underlying mechanisms for synaptic dysfunctions occurring in neurodegenerative disorders in which a compromised PrPC is likely to intervene.

Modulation of neurosecretion and approaches for its multistep analysis

BackgroundNeurosecretion is the multistep process occurring in separate spatial and temporal cellular boundaries which complicates its comprehensive analysis. Most of the research are focused on one distinct stage of synaptic vesicle recycling. Here, we describe approaches for complex analysis of synaptic vesicle (SV) endocytosis and separate steps of exocytosis at the level of presynaptic bouton and highly purified SVs. MethodsProposed fluorescence-based strategies and analysis of neurotransmitter transport provided the advantages in studies of exocytosis steps. We evaluated SV docking/tethering, their Ca2+-dependent fusion and release of neurotransmitters gamma-aminobutyric acid (GABA) and glutamate in two animal models. ResultsApproaches enabled us to study: 1) endocytosis/Ca2+-dependent release of fluorescent carbon nanodots (CNDs) during stimulation of nerve terminals; 2) the action of levetiracetam, modulator of SV glycoprotein SV2, on fusion competence of SVs and stimulated release of GABA and glutamate; 3) impairments of several steps of neurosecretion under vitamin D3 deficiency. ConclusionsOur algorithm enabled us to verify the method validity for multidimensional analysis of SV turnover. By increasing SV docking and the size of readily releasable pool (RRP), levetiracetam is able to selectively enhance the stimulated GABA secretion in hippocampal neurons. Findings suggest that SV2 regulates RRP through impact on the number of docked/primed SVs. General significanceMethodology can be widely applied to study the stimulated neurosecretion in presynapse, regulation of SV docking, their Ca2+-dependent fusion with target membranes, quantitative analysis of expression of neuron-specific proteins, as well as for testing the efficiency of pre-selected designed neuroactive substances.

Type II phosphatidylinositol 4-kinase regulates nerve terminal growth and synaptic vesicle recycling

Type II phosphatidylinositol 4-kinase (PI4KII) is thought to be associated with synaptic vesicles (SVs) and to be responsible for the majority of PI4K activity in the nervous system. However, the function of PI4KII at the synapse is unknown. We characterized the synaptic phenotypes of a Drosophila melanogaster PI4KII null mutant. We found increased nerve terminal growth in PI4KII null mutants indicating that PI4KII restrains nerve terminal growth. Evoked neurotransmitter release elicited in response to low frequency stimulation and spontaneous neurotransmitter release were not altered in PI4KII null mutants. However, PI4KII null mutants displayed reduced FM1-43 uptake in response to stimulation by high K+ saline, indicating impaired SV endocytosis. PI4KII null mutants did not display any defects in FM1-43 unloading, consistent with normal SV exocytosis. Thus, PI4KII is required for SV endocytosis but dispensable for SV exocytosis. Overall, our data show that PI4KII regulates both nerve terminal growth and SV recycling.

Genetic dissection of neuropeptide cell biology at high and low activity in a defined sensory neuron

Neuropeptides are ubiquitous modulators of behavior and physiology. They are packaged in specialized secretory organelles called dense core vesicles (DCVs) that are released upon neural stimulation. Unlike synaptic vesicles, which can be recycled and refilled close to release sites, DCVs must be replenished by de novo synthesis in the cell body. Here, we dissect DCV cell biology in vivo in a Caenorhabditis elegans sensory neuron whose tonic activity we can control using a natural stimulus. We express fluorescently tagged neuropeptides in the neuron and define parameters that describe their subcellular distribution. We measure these parameters at high and low neural activity in 187 mutants defective in proteins implicated in membrane traffic, neuroendocrine secretion, and neuronal or synaptic activity. Using unsupervised hierarchical clustering methods, we analyze these data and identify 62 groups of genes with similar mutant phenotypes. We explore the function of a subset of these groups. We recapitulate many previous findings, validating our paradigm. We uncover a large battery of proteins involved in recycling DCV membrane proteins, something hitherto poorly explored. We show that the unfolded protein response promotes DCV production, which may contribute to intertissue communication of stress. We also find evidence that different mechanisms of priming and exocytosis may operate at high and low neural activity. Our work provides a defined framework to study DCV biology at different neural activity levels.