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Sample records for activates synaptic vesicle

  1. Synaptic Vesicle Proteins and Active Zone Plasticity

    Kittel, Robert J.; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention.

  2. Synaptic Vesicle Exocytosis

    Südhof, Thomas C; Rizo, Josep

    2011-01-01

    Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for “soluble NSF-attachment protein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap aro...

  3. Signaling for Vesicle Mobilization and Synaptic Plasticity

    Levitan, Edwin S.

    2008-01-01

    The hypothesis that release of classical neurotransmitters and neuropeptides is facilitated by increasing the mobility of small synaptic vesicles (SSVs) and dense core vesicles (DCVs) could not be tested until the advent of methods for visualizing these secretory vesicles in living nerve terminals. In fact, fluorescence imaging studies have only since 2005 established that activity increases secretory vesicle mobility in motoneuron terminals and chromaffin cells. Mobilization of DCVs and SSVs...

  4. The molecular physiology of activity-dependent bulk endocytosis of synaptic vesicles.

    Clayton, E. L.; Cousin, M. A.

    2009-01-01

    Central nerve terminals release neurotransmitter in response to a wide variety of stimuli. Because maintenance of neurotransmitter release is dependent on the continual supply of synaptic vesicles (SVs), nerve terminals possess an array of endocytosis modes to retrieve and recycle SV membrane and proteins. During mild stimulation conditions, single SV retrieval modes such as clathrin-mediated endocytosis predominate. However, during increased neuronal activity, additional SV retrieval capacit...

  5. The Molecular Physiology of Activity-Dependent Bulk Endocytosis of Synaptic Vesicles

    Clayton, Emma L.; Cousin, Michael A

    2009-01-01

    Central nerve terminals release neurotransmitter in response to a wide variety of stimuli. Since maintenance of neurotransmitter release is dependent on the continual supply of synaptic vesicles (SVs), nerve terminals possess an array of endocytosis modes to retrieve and recycle SV membrane and proteins. During mild stimulation conditions single SV retrieval modes such as clathrin-mediated endocytosis (CME) predominate. However during increased neuronal activity additional SV retrieval capaci...

  6. Coordinated trafficking of synaptic vesicle and active zone proteins prior to synapse formation

    Sabo Shasta L

    2011-05-01

    Full Text Available Abstract Background The proteins required for synaptic transmission are rapidly assembled at nascent synapses, but the mechanisms through which these proteins are delivered to developing presynaptic terminals are not understood. Prior to synapse formation, active zone proteins and synaptic vesicle proteins are transported along axons in distinct organelles referred to as piccolo-bassoon transport vesicles (PTVs and synaptic vesicle protein transport vesicles (STVs, respectively. Although both PTVs and STVs are recruited to the same site in the axon, often within minutes of axo-dendritic contact, it is not known whether or how PTV and STV trafficking is coordinated before synapse formation. Results Here, using time-lapse confocal imaging of the dynamics of PTVs and STVs in the same axon, we show that vesicle trafficking is coordinated through at least two mechanisms. First, a significant proportion of STVs and PTVs are transported together before forming a stable terminal. Second, individual PTVs and STVs share pause sites within the axon. Importantly, for both STVs and PTVs, encountering the other type of vesicle increases their propensity to pause. To determine if PTV-STV interactions are important for pausing, PTV density was reduced in axons by expression of a dominant negative construct corresponding to the syntaxin binding domain of syntabulin, which links PTVs with their KIF5B motor. This reduction in PTVs had a minimal effect on STV pausing and movement, suggesting that an interaction between STVs and PTVs is not responsible for enhancing STV pausing. Conclusions Our results indicate that trafficking of STVs and PTVs is coordinated even prior to synapse development. This novel coordination of transport and pausing might provide mechanisms through which all of the components of a presynaptic terminal can be rapidly accumulated at sites of synapse formation.

  7. Open Syntaxin Docks Synaptic Vesicles

    Marc Hammarlund; Mark T Palfreyman; Shigeki Watanabe; Shawn Olsen; Erik M. Jorgensen

    2007-01-01

    Author Summary Like Olympic swimmers crouched on their starting blocks, synaptic vesicles prepare for fusion with the neuronal plasma membrane long before the starting gun fires. This preparation enables vesicles to fuse rapidly, synchronously, and in the correct place when the signal finally arrives. A well-known but poorly understood part of vesicle preparation is docking, in which vesicles prepare for release by attaching to the plasma membrane at the eventual site of release. Here, we out...

  8. LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex.

    Cirnaru, Maria D; Marte, Antonella; Belluzzi, Elisa; Russo, Isabella; Gabrielli, Martina; Longo, Francesco; Arcuri, Ludovico; Murru, Luca; Bubacco, Luigi; Matteoli, Michela; Fedele, Ernesto; Sala, Carlo; Passafaro, Maria; Morari, Michele; Greggio, Elisa; Onofri, Franco; Piccoli, Giovanni

    2014-01-01

    Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex. PMID:24904275

  9. Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons.

    Yepes, M; Wu, F; Torre, E; Cuellar-Giraldo, D; Jia, D; Cheng, L

    2016-04-01

    The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca(2+)-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis. PMID:26820595

  10. Cannabinoid agonists rearrange synaptic vesicles at excitatory synapses and depress motoneuron activity in vivo.

    García-Morales, Victoria; Montero, Fernando; Moreno-López, Bernardo

    2015-05-01

    Impairment of motor skills is one of the most common acute adverse effects of cannabis. Related studies have focused mainly on psychomotor alterations, and little is known about the direct impact of cannabinoids (CBs) on motoneuron physiology. As key modulators of synaptic function, CBs regulate multiple neuronal functions and behaviors. Presynaptic CB1 mediates synaptic strength depression by inhibiting neurotransmitter release, via a poorly understood mechanism. The present study examined the effect of CB agonists on excitatory synaptic inputs incoming to hypoglossal motoneurons (HMNs) in vitro and in vivo. The endocannabinoid anandamide (AEA) and the synthetic CB agonist WIN 55,212-2 rapidly and reversibly induced short-term depression (STD) of glutamatergic synapses on motoneurons by a presynaptic mechanism. Presynaptic effects were fully reversed by the CB1-selective antagonist AM281. Electrophysiological and electron microscopy analysis showed that WIN 55,212-2 reduced the number of synaptic vesicles (SVs) docked to active zones in excitatory boutons. Given that AM281 fully abolished depolarization-induced depression of excitation, motoneurons can be feasible sources of CBs, which in turn act as retrograde messengers regulating synaptic function. Finally, microiontophoretic application of the CB agonist O-2545 reversibly depressed, presumably via CB1, glutamatergic inspiratory-related activity of HMNs in vivo. Therefore, evidence support that CBs, via presynaptic CB1, induce excitatory STD by reducing the readily releasable pool of SVs at excitatory synapses, then attenuating motoneuron activity. These outcomes contribute a possible mechanistic basis for cannabis-associated motor performance disturbances such as ataxia, dysarthria and dyscoordination. PMID:25595101

  11. The Molecular Physiology of Activity-Dependent Bulk Endocytosis of Synaptic Vesicles

    Clayton, Emma L.; Cousin, Michael A.

    2010-01-01

    Central nerve terminals release neurotransmitter in response to a wide variety of stimuli. Since maintenance of neurotransmitter release is dependent on the continual supply of synaptic vesicles (SVs), nerve terminals possess an array of endocytosis modes to retrieve and recycle SV membrane and proteins. During mild stimulation conditions single SV retrieval modes such as clathrin-mediated endocytosis (CME) predominate. However during increased neuronal activity additional SV retrieval capacity is required, which is provided by activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mechanism during elevated neuronal activity. It is a high capacity SV retrieval mode that is immediately triggered during such stimulation conditions. This review will summarise the current knowledge regarding the molecular mechanism of ADBE, including molecules required for its triggering and subsequent steps, including SV budding from bulk endosomes. The molecular relationship between ADBE and the SV reserve pool will also be discussed. It is becoming clear that an understanding of the molecular physiology of ADBE will be of critical importance in attempts to modulate both normal and abnormal synaptic function during intense neuronal activity. PMID:19765184

  12. Concurrent Imaging of Synaptic Vesicle Recycling and Calcium Dynamics

    Li, Haiyan; Foss, Sarah M.; Dobryy, Yuriy L.; Park, C. Kevin; Hires, Samuel Andrew; Shaner, Nathan C.; Tsien, Roger Y.; Osborne, Leslie C.; Voglmaier, Susan M.

    2011-01-01

    Synaptic transmission involves the calcium dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-sh...

  13. Concurrent imaging of synaptic vesicle recycling and calcium dynamics.

    Haiyan eLi; Foss, Sarah M.; Yuriy eDobryy; C. Kevin ePark; Samuel Andrew Hires; Shaner, Nathan C.; Tsien, Roger Y.; Osborne, Leslie C.; Voglmaier, Susan M.

    2011-01-01

    Synaptic transmission involves the calcium-dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-...

  14. Protein tyrosine phosphorylation in synaptic vesicles.

    Pang, D T; Wang, J K; Valtorta, F; Benfenati, F; Greengard, P.

    1988-01-01

    Protein tyrosine phosphorylation in purified synaptic vesicles from rat forebrain has been studied in the presence of Mn2+ and orthovanadate. High levels of endogenous protein tyrosine phosphorylation were observed. Four major phosphoproteins, with apparent molecular masses of 105, 94, 38, and 30 kDa, were shown to contain phosphotyrosine. The 38-kDa phosphoprotein was identified as synaptophysin (p38), a well-characterized integral membrane protein of synaptic vesicles. The three other phosp...

  15. Concurrent imaging of synaptic vesicle recycling and calcium dynamics.

    Haiyan eLi

    2011-11-01

    Full Text Available Synaptic transmission involves the calcium-dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2, and a presynaptically-localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3 with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Re-acidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real-time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released.

  16. Concurrent Imaging of Synaptic Vesicle Recycling and Calcium Dynamics

    Li, Haiyan; Foss, Sarah M.; Dobryy, Yuriy L.; Park, C. Kevin; Hires, Samuel Andrew; Shaner, Nathan C.; Tsien, Roger Y.; Osborne, Leslie C.; Voglmaier, Susan M.

    2011-01-01

    Synaptic transmission involves the calcium dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2), and a presynaptically localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3) with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Reacidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released. PMID:22065946

  17. Colocalization of synapsin and actin during synaptic vesicle recycling

    Bloom, Ona; Evergren, Emma; Tomilin, Nikolay;

    2003-01-01

    activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin......-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known...

  18. Okadaic acid disrupts clusters of synaptic vesicles in frog motor nerve terminals

    1994-01-01

    The fluorophore FM1-43 appears to stain membranes of recycled synaptic vesicles. We used FM1-43 to study mechanisms of synaptic vesicle clustering and mobilization in living frog motor nerve terminals. FM1- 43 staining of these terminals produces a linear series of fluorescent spots, each spot marking the cluster of several hundred synaptic vesicles at an active zone. Most agents we tested did not affect staining, but the phosphatase inhibitor okadaic acid (OA) disrupted the fluorescent spots...

  19. Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate

    Schotten, Sebastiaan; Meijer, Marieke; Walter, Alexander Matthias;

    2015-01-01

    supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced by......-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca2+-dependent release....

  20. Mobility and Turnover of Vesicles at the Synaptic Ribbon

    LoGiudice, Lisamarie; Sterling, Peter; Matthews, Gary

    2008-01-01

    Ribbon synapses release neurotransmitter continuously at high rates, and the ribbons tether a large pool of synaptic vesicles. To determine if the tethered vesicles are actually released, we tracked vesicles labeled with FM4-64 dye in mouse retinal bipolar cell terminals whose ribbons had been labeled with a fluorescent peptide. We photobleached vesicles in regions with ribbons and without them and then followed recovery of fluorescence as bleached regions were repopulated by labeled vesicles...

  1. Overexpression of synapsin Ia in the rat calyx of Held accelerates short-term plasticity and decreases synaptic vesicle volume and active zone area

    Mariya Vasileva

    2013-12-01

    Full Text Available Synapsins are synaptic vesicle (SV proteins organizing a component of the reserve pool of vesicles at most central nervous system synapses. Alternative splicing of the three mammalian genes results in multiple isoforms that may differentially contribute to the organization and maintenance of the synaptic vesicle-pools. To address this, we first characterized the expression pattern of synapsin isoforms in the rat calyx of Held. At postnatal day 16, synapsins Ia, Ib, IIb and IIIa were present, while IIa – known to sustain repetitive transmission in glutamatergic terminals – was not detectable. To test if the synapsin I isoforms could mediate IIa-like effect, and if this depends on the presence of the E-domain, we overexpressed either synapsin Ia or synapsin Ib in the rat calyx of Held via recombinant adeno-associated virus-mediated gene transfer. Although the size and overall structure of the perturbed calyces remained unchanged, short-term depression and recovery from depression were accelerated upon overexpression of synapsin I isoforms. Thus, at the calyx of Held, synapsin Ia may not substitute for the synapsin IIa-function reported for hippocampal synapses. Using electron microscopic three-dimensional reconstructions we found a redistribution of SV clusters proximal to the active zones (AZ alongside with a decrease of both AZ area and SV volume. The number of SVs at individual AZs was strongly reduced. Hence, our data indicate that the amount of synapsin Ia expressed in the calyx regulates the rate and extent of short-term synaptic plasticity by affecting vesicle recruitment to the AZ. Finally, our study reveals a novel contribution of synapsin Ia to define the surface area of AZs.

  2. Vesicular glutamate transporter 1 orchestrates recruitment of other synaptic vesicle cargo proteins during synaptic vesicle recycling.

    Pan, Ping-Yue; Marrs, Julia; Ryan, Timothy A

    2015-09-11

    A long standing question in synaptic physiology is how neurotransmitter-filled vesicles are rebuilt after exocytosis. Among the first steps in this process is the endocytic retrieval of the transmembrane proteins that are enriched in synaptic vesicles (SVs). At least six types of transmembrane proteins must be recovered, but the rules for how this multiple cargo selection is accomplished are poorly understood. Among these SV cargos is the vesicular glutamate transporter (vGlut). We show here that vGlut1 has a strong influence on the kinetics of retrieval of half of the known SV cargos and that specifically impairing the endocytosis of vGlut1 in turn slows down other SV cargos, demonstrating that cargo retrieval is a collective cargo-driven process. Finally, we demonstrate that different cargos can be retrieved in the same synapse with different kinetics, suggesting that additional post-endocytic sorting steps likely occur in the nerve terminal. PMID:26224632

  3. Analysing the distribution of synaptic vesicles using a spatial point process model

    Khanmohammadi, Mahdieh; Waagepetersen, Rasmus Plenge; Nava, Nicoletta; Nyengaard, Jens; Sporring, Jon

    2015-01-01

    Stress can affect the brain functionality in many ways. As the synaptic vesicles have a major role in nervous signal transportation in synapses, their distribution in relationship to the active zone is very important in studying the neuron responses. We study the effect of stress on brain...... functionality by statistically modelling the distribution of the synaptic vesicles in two groups of rats: a control group subjected to sham stress and a stressed group subjected to a single acute foot-shock (FS)-stress episode. We hypothesize that the synaptic vesicles have different spatial distributions in...

  4. Analysing the distribution of synaptic vesicles using a spatial point process model

    Khanmohammadi, Mahdieh; Waagepetersen, Rasmus; Nava, Nicoletta; Nyengaard, Jens; Sporring, Jon

    Stress can affect the brain functionality in many ways. As the synaptic vesicles have a major role in nervous signal transportation in synapses, their distribution in relationship to the active zone is very important in studying the neuron responses. We study the effect of stress on brain...... functionality by statistically modelling the distribution of the synaptic vesicles in two groups of rats: a control group subjected to sham stress and a stressed group subjected to a single acute foot-shock (FS)-stress episode. We hypothesize that the synaptic vesicles have different spatial distributions in...

  5. Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling

    1992-01-01

    The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater tha...

  6. Amplification of neuromuscular transmission by methylprednisolone involves activation of presynaptic facilitatory adenosine A2A receptors and redistribution of synaptic vesicles.

    Oliveira, L; Costa, A C; Noronha-Matos, J B; Silva, I; Cavalcante, W L G; Timóteo, M A; Corrado, A P; Dal Belo, C A; Ambiel, C R; Alves-do-Prado, W; Correia-de-Sá, P

    2015-02-01

    The mechanisms underlying improvement of neuromuscular transmission deficits by glucocorticoids are still a matter of debate despite these compounds have been used for decades in the treatment of autoimmune myasthenic syndromes. Besides their immunosuppressive action, corticosteroids may directly facilitate transmitter release during high-frequency motor nerve activity. This effect coincides with the predominant adenosine A2A receptor tonus, which coordinates the interplay with other receptors (e.g. muscarinic) on motor nerve endings to sustain acetylcholine (ACh) release that is required to overcome tetanic neuromuscular depression in myasthenics. Using myographic recordings, measurements of evoked [(3)H]ACh release and real-time video microscopy with the FM4-64 fluorescent dye, results show that tonic activation of facilitatory A2A receptors by endogenous adenosine accumulated during 50 Hz bursts delivered to the rat phrenic nerve is essential for methylprednisolone (0.3 mM)-induced transmitter release facilitation, because its effect was prevented by the A2A receptor antagonist, ZM 241385 (10 nM). Concurrent activation of the positive feedback loop operated by pirenzepine-sensitive muscarinic M1 autoreceptors may also play a role, whereas the corticosteroid action is restrained by the activation of co-expressed inhibitory M2 and A1 receptors blocked by methoctramine (0.1 μM) and DPCPX (2.5 nM), respectively. Inhibition of FM4-64 loading (endocytosis) by methylprednisolone following a brief tetanic stimulus (50 Hz for 5 s) suggests that it may negatively modulate synaptic vesicle turnover, thus increasing the release probability of newly recycled vesicles. Interestingly, bulk endocytosis was rehabilitated when methylprednisolone was co-applied with ZM241385. Data suggest that amplification of neuromuscular transmission by methylprednisolone may involve activation of presynaptic facilitatory adenosine A2A receptors by endogenous adenosine leading to synaptic

  7. Imaging Exocytosis of Single Synaptic Vesicles at a Fast CNS Presynaptic Terminal.

    Midorikawa, Mitsuharu; Sakaba, Takeshi

    2015-11-01

    Synaptic vesicles are tethered to the active zone where they are docked/primed so that they can fuse rapidly upon Ca(2+) influx. To directly study these steps at a CNS presynaptic terminal, we used total internal reflection fluorescence (TIRF) microscopy at the live isolated calyx of Held terminal and measured the movements of single synaptic vesicle just beneath the plasma membrane. Only a subset of vesicles within the TIRF field underwent exocytosis. Following exocytosis, new vesicles (newcomers) approached the membrane and refilled the release sites slowly with a time constant of several seconds. Uniform elevation of the intracellular Ca(2+) using flash photolysis elicited an exocytotic burst followed by the sustained component, representing release of the readily releasable vesicles and vesicle replenishment, respectively. Surprisingly, newcomers were not released within a second of high Ca(2+). Instead, already-tethered vesicles became release-ready and mediated the replenishment. Our results reveal an important feature of conventional synapses. PMID:26539890

  8. 3D ESTIMATION OF SYNAPTIC VESICLE DISTRIBUTIONS IN SERIAL SECTION TRANSMISSION ELECTRON MICROSCOPY

    Khanmohammadi, Mahdieh; Darkner, Sune; Nava, Nicoletta;

    directly. It is hypothesized that in a rat model of behavioral stress the vesicles distribution varies. We propose methods for estimating the 3-dimensional distribution of synaptic vesicles from the active zone through serial section transmission electron microscope images (ssTEM) from Sprague-Dawley rat...

  9. Synapsin IIa controls the reserve pool of glutamatergic synaptic vesicles

    Gitler, Daniel; Cheng, Qing; Greengard, Paul; Augustine, George J.

    2008-01-01

    Synapsins regulate synaptic transmission by controlling the reserve pool of synaptic vesicles. Each of the three mammalian synapsin genes is subject to alternative splicing, yielding several isoforms whose roles are unknown. To investigate the function of these isoforms, we examined the synaptic effects of introducing each isoform into glutamatergic cultured hippocampal neurons from synapsin triple knock-out mice. Remarkably, we found that synapsin IIa was the only isoform that could rescue t...

  10. Endosome-mediated endocytic mechanism replenishes the majority of synaptic vesicles at mature CNS synapses in an activity-dependent manner.

    Park, Joohyun; Cho, Oh Yeon; Kim, Jung Ah; Chang, Sunghoe

    2016-01-01

    Whether synaptic vesicles (SVs) are recovered via endosome-mediated pathways is a matter of debate; however, recent evidence suggests that clathrin-independent bulk endocytosis (CIE) via endosomes is functional and preferentially replenishes SV pools during strong stimulation. Here, using brefeldin-A (BFA) to block CIE, we found that CIE retrieved a minority of SVs at developing CNS synapses during strong stimulation, but its contribution increased up to 61% at mature CNS synapses. Contrary to previous views, BFA not only blocked SV formation from the endosome but also blocked the endosome formation at the plasma membrane. Adaptor protein 1 and 3 (AP-1/3) have key roles in SV reformation from endosomes during CIE, and AP-1 also affects bulk endosome formation from the plasma membrane. Finally, temporary blocking of chronic or acute neuronal activity with tetrodotoxin in mature neurons redirected most SV retrieval to endosome-independent pathways. These results show that during high neuronal activity, CIE becomes the major endocytic pathway at mature CNS synapses. Moreover, mature neurons use clathrin-mediated endocytosis and the CIE pathway to different extents depending on their previous activity; this may result in activity-dependent alterations of the SV composition which ultimately influence transmitter release and contribute to synaptic plasticity. PMID:27534442

  11. Amyloid precursor protein is trafficked and secreted via synaptic vesicles.

    Teja W Groemer

    Full Text Available A large body of evidence has implicated amyloid precursor protein (APP and its proteolytic derivatives as key players in the physiological context of neuronal synaptogenesis and synapse maintenance, as well as in the pathology of Alzheimer's Disease (AD. Although APP processing and release are known to occur in response to neuronal stimulation, the exact mechanism by which APP reaches the neuronal surface is unclear. We now demonstrate that a small but relevant number of synaptic vesicles contain APP, which can be released during neuronal activity, and most likely represent the major exocytic pathway of APP. This novel finding leads us to propose a revised model of presynaptic APP trafficking that reconciles existing knowledge on APP with our present understanding of vesicular release and recycling.

  12. Cdk5 is essential for synaptic vesicle endocytosis

    Tan, Timothy C; Valova, Valentina A; Malladi, Chandra S;

    2003-01-01

    Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynami...

  13. Dysregulations of Synaptic Vesicle Trafficking in Schizophrenia.

    Egbujo, Chijioke N; Sinclair, Duncan; Hahn, Chang-Gyu

    2016-08-01

    Schizophrenia is a serious psychiatric illness which is experienced by about 1 % of individuals worldwide and has a debilitating impact on perception, cognition, and social function. Over the years, several models/hypotheses have been developed which link schizophrenia to dysregulations of the dopamine, glutamate, and serotonin receptor pathways. An important segment of these pathways that have been extensively studied for the pathophysiology of schizophrenia is the presynaptic neurotransmitter release mechanism. This set of molecular events is an evolutionarily well-conserved process that involves vesicle recruitment, docking, membrane fusion, and recycling, leading to efficient neurotransmitter delivery at the synapse. Accumulated evidence indicate dysregulation of this mechanism impacting postsynaptic signal transduction via different neurotransmitters in key brain regions implicated in schizophrenia. In recent years, after ground-breaking work that elucidated the operations of this mechanism, research efforts have focused on the alterations in the messenger RNA (mRNA) and protein expression of presynaptic neurotransmitter release molecules in schizophrenia and other neuropsychiatric conditions. In this review article, we present recent evidence from schizophrenia human postmortem studies that key proteins involved in the presynaptic release mechanism are dysregulated in the disorder. We also discuss the potential impact of dysfunctional presynaptic neurotransmitter release on the various neurotransmitter systems implicated in schizophrenia. PMID:27371030

  14. Designing the lipid raft marker protein for synaptic vesicles

    Lv Jihua; Sui Senfang

    2009-01-01

    Lipid rafts are cholesterol-enriched microdomains and implicated in many essential physiological activities such as the neurotransmitter release. Many studies have been carried out on the function of rafts in the plasma membranes, whereas little is known about the information of such microdomains in subcellular compartments especially synaptic vesicles (SVs). In the well-studied plasma membranes, several proteins have been recognized as raft markers, which are used to label or trace rafts. But the raft marker protein on SVs has not been identified yet. Although some SV proteins, including VAMP and CPE, have been found in raft fractions, they cannot be used as markers due to their low abundance in rafts. In this work, we designed several chimera proteins and tested their characteristics for using as SV raft makers. First, we detected whether they located in SVs, and then the chimeras exhibiting the better localization in SVs were further examined for their enrichment in raft using detergent treatment and gradient density floatation analysis. Our results indicate that one of the chimeric proteins is primarily located in SVs and distributed in raft microdomains, which strongly suggests that it could be served as a raft marker for SVs.

  15. β-Hydroxybutyrate supports synaptic vesicle cycling but reduces endocytosis and exocytosis in rat brain synaptosomes.

    Hrynevich, Sviatlana V; Waseem, Tatyana V; Hébert, Audrey; Pellerin, Luc; Fedorovich, Sergei V

    2016-02-01

    The ketogenic diet is used as a prophylactic treatment for different types of brain diseases, such as epilepsy or Alzheimer's disease. In such a diet, carbohydrates are replaced by fats in everyday food, resulting in an elevation of blood-borne ketone bodies levels. Despite clinical applications of this treatment, the molecular mechanisms by which the ketogenic diet exerts its beneficial effects are still uncertain. In this study, we investigated the effect of replacing glucose by the ketone body β-hydroxybutyrate as the main energy substrate on synaptic vesicle recycling in rat brain synaptosomes. First, we observed that exposing presynaptic terminals to nonglycolytic energy substrates instead of glucose did not alter the plasma membrane potential. Next, we found that synaptosomes were able to maintain the synaptic vesicle cycle monitored with the fluorescent dye acridine orange when glucose was replaced by β-hydroxybutyrate. However, in presence of β-hydroxybutyrate, synaptic vesicle recycling was modified with reduced endocytosis. Replacing glucose by pyruvate also led to a reduced endocytosis. Addition of β-hydroxybutyrate to glucose-containing incubation medium was without effect. Reduced endocytosis in presence of β-hydroxybutyrate as sole energy substrate was confirmed using the fluorescent dye FM2-10. Also we found that replacement of glucose by ketone bodies leads to inhibition of exocytosis, monitored by FM2-10. However this reduction was smaller than the effect on endocytosis under the same conditions. Using both acridine orange in synaptosomes and the genetically encoded sensor synaptopHluorin in cortical neurons, we observed that replacing glucose by β-hydroxybutyrate did not modify the pH gradient of synaptic vesicles. In conclusion, the nonglycolytic energy substrates β-hydroxybutyrate and pyruvate are able to support synaptic vesicle recycling. However, they both reduce endocytosis. Reduction of both endocytosis and exocytosis together with

  16. Kinetics of synaptic depression and vesicle recycling after tetanic stimulation of frog motor nerve terminals.

    Wu, L G; Betz, W J

    1998-01-01

    We measured the time courses of two key components of the synaptic vesicle cycle during recovery from synaptic depression under different conditions, and used this and other information to create a kinetic model of the vesicle cycle. End plate potential (EPP) amplitudes were used to follow recovery from synaptic depression after different amounts of tetanic stimulation. This provided an estimate of the time course of vesicle mobilization from the reserve pool to the docked (readily releasable...

  17. Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

    Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua

    2016-07-01

    At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as

  18. Working Memory Impairment in Calcineurin Knock-out Mice Is Associated with Alterations in Synaptic Vesicle Cycling and Disruption of High-Frequency Synaptic and Network Activity in Prefrontal Cortex

    Cottrell, Jeffrey R.; Levenson, Jonathan M.; Kim, Sung Hyun; Gibson, Helen E.; Richardson, Kristen A.; Sivula, Michael; Li, Bing; Ashford, Crystle J.; Heindl, Karen A.; Babcock, Ryan J.; Rose, David M.; Hempel, Chris M; Wiig, Kjesten A.; Laeng, Pascal; Levin, Margaret E.

    2013-01-01

    Working memory is an essential component of higher cognitive function, and its impairment is a core symptom of multiple CNS disorders, including schizophrenia. Neuronal mechanisms supporting working memory under normal conditions have been described and include persistent, high-frequency activity of prefrontal cortical neurons. However, little is known about the molecular and cellular basis of working memory dysfunction in the context of neuropsychiatric disorders. To elucidate synaptic and n...

  19. Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

    Shupliakov, Oleg; Bloom, Ona; Gustafsson, Jenny S;

    2002-01-01

    the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin...... fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses. Our data provide...

  20. Statistical Modelling of Synaptic Vesicles Distribution and Analysing their Physical Characteristics

    Khanmohammadi, Mahdieh

    transmission electron microscopy is used to acquire images from two experimental groups of rats: 1) rats subjected to a behavioral model of stress and 2) rats subjected to sham stress as the control group. The synaptic vesicle distribution and interactions are modeled by employing a point process approach. The......This Ph.D. thesis deals with mathematical and statistical modeling of synaptic vesicle distribution, shape, orientation and interactions. The first major part of this thesis treats the problem of determining the effect of stress on synaptic vesicle distribution and interactions. Serial section...... differences of statistical measures in section and the same measures in between sections. Three-dimensional (3D) datasets are reconstructed by using image registration techniques and estimated thicknesses. We distinguish the effect of stress by estimating the synaptic vesicle densities and modeling their...

  1. Synaptic vesicles studied by small-angle X-ray scattering

    The heterogeneous structure of synaptic vesicles isolated from rat brain is investigated considering solution small-angle X-ray scattering data in combination with data obtained by cryogenic electron microscopy, dynamic light scattering and biochemical analysis. Overall low resolution structural models of the entire functional synaptic vesicle are proposed, elucidating details on the density profile of the membrane, including contributions from the lipids and the proteins, as well as addressing the average conformation and overall lateral organization of proteins in micro-domains on the average synaptic vesicle under quasi-physiological conditions. Entropic contributions to free energy due to possible protein cluster formation and disintegration on the synaptic vesicle are investigated. Further, cell free fusion systems are characterized employing dynamic light scattering and applicability of small-angle X-ray scattering is considered for investigating membrane fusion processes.

  2. Nonmuscle Myosin II helps regulate synaptic vesicle mobility at the Drosophila neuromuscular junction

    Qiu Xinping

    2010-03-01

    Full Text Available Abstract Background Although the mechanistic details of the vesicle transport process from the cell body to the nerve terminal are well described, the mechanisms underlying vesicle traffic within nerve terminal boutons is relatively unknown. The actin cytoskeleton has been implicated but exactly how actin or actin-binding proteins participate in vesicle movement is not clear. Results In the present study we have identified Nonmuscle Myosin II as a candidate molecule important for synaptic vesicle traffic within Drosophila larval neuromuscular boutons. Nonmuscle Myosin II was found to be localized at the Drosophila larval neuromuscular junction; genetics and pharmacology combined with the time-lapse imaging technique FRAP were used to reveal a contribution of Nonmuscle Myosin II to synaptic vesicle movement. FRAP analysis showed that vesicle dynamics were highly dependent on the expression level of Nonmuscle Myosin II. Conclusion Our results provide evidence that Nonmuscle Myosin II is present presynaptically, is important for synaptic vesicle mobility and suggests a role for Nonmuscle Myosin II in shuttling vesicles at the Drosophila neuromuscular junction. This work begins to reveal the process by which synaptic vesicles traverse within the bouton.

  3. Size distribution and radial density profile of synaptic vesicles by SAXS and light scattering

    Castorph, Simon; Salditt, Tim [Institute for X-ray Physics, Goettingen (Germany); Holt, Matthew; Jahn, Reinhard [Max Plank Institute for Biophysical Chemistry, Goettingen (Germany); Sztucki, Michael [European Synchrotron Radiation Facility, Grenoble (France)

    2008-07-01

    Synaptic vesicles are small membraneous organelles within the nerve terminal, encapsulating neurotransmitters by a lipid bilayer. The transport of the neurotransmitter, the fusion at the plasma membrane, and the release of the stored neurotransmitters into the synaptic cleft are since long know as essential step in nerve conduction of the chemical synapse. A detailed structural view of these molecular mechanisms is still lacking, not withstanding the enormous progress in the field during recent years. From measurements and quantitative fitting of small angle X-ray scattering curves and dynamic light scattering the averaged structural properties of synaptic vesicles can be determined. We present SAXS measurements and fits revealing the width of the size distribution function and details of the radial scattering length profile of synaptic vesicles from rat brain. Representative values for the inner and outer radius and the size polydispersity as well as the density and width of the outer protein layer are obtained.

  4. Differential regulation of synaptic vesicle tethering and docking by UNC-18 and TOM-1

    Elena O Gracheva

    2010-10-01

    Full Text Available The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18, unc-64(syntaxin and tom-1(tomosyn. We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin

  5. Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue

    1981-01-01

    Two different monoclonal antibodies, characterized initially as binding synaptic terminal regions of rat brain, bind a 65,000-dalton protein, which is exposed on the outer surface of brain synaptic vesicles. Immunocytochemical experiments at the electron microscope level demonstrate that these antibodies bind the vesicles in many different types of nerve terminals. The antibodies have been used successfully to purify synaptic vesicles from crude brain homogenates by immunoprecipitation onto t...

  6. Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion

    Petrov, Alexey M., E-mail: fysio@rambler.ru; Zakyrjanova, Guzalija F., E-mail: guzik121192@mail.ru; Yakovleva, Anastasia A., E-mail: nastya1234qwer@mail.ru; Zefirov, Andrei L., E-mail: zefiroval@rambler.ru

    2015-01-02

    Highlights: • We examine the involvement of PKC in MCD induced synaptic vesicle exocytosis. • PKC inhibitor does not decrease the effect MCD on MEPP frequency. • PKC inhibitor prevents MCD induced FM1-43 unloading. • PKC activation may switch MCD induced exocytosis from kiss-and-run to a full mode. • Inhibition of phospholipase C does not lead to similar change in exocytosis. - Abstract: Previous studies demonstrated that depletion of membrane cholesterol by 10 mM methyl-beta-cyclodextrin (MCD) results in increased spontaneous exocytosis at both peripheral and central synapses. Here, we investigated the role of protein kinase C in the enhancement of spontaneous exocytosis at frog motor nerve terminals after cholesterol depletion using electrophysiological and optical methods. Inhibition of the protein kinase C by myristoylated peptide and chelerythrine chloride prevented MCD-induced increases in FM1-43 unloading, whereas the frequency of spontaneous postsynaptic events remained enhanced. The increase in FM1-43 unloading still could be observed if sulforhodamine 101 (the water soluble FM1-43 quencher that can pass through the fusion pore) was added to the extracellular solution. This suggests a possibility that exocytosis of synaptic vesicles under these conditions could occur through the kiss-and-run mechanism with the formation of a transient fusion pore. Inhibition of phospholipase C did not lead to similar change in MCD-induced exocytosis.

  7. Synaptic vesicle cycling is not impaired in a glutamatergic and a cholinergic synapse that exhibit deficits in acidification and filling

    Bento João Abreu; Luciana Ferreira Leite; Débora Lopes Oliveira; Ernani Amaral

    2012-01-01

    The purpose of the present work was to investigate synaptic vesicle trafficking when vesicles exhibit alterations in filling and acidification in two different synapses: a cholinergic frog neuromuscular junction and a glutamatergic ribbon-type nerve terminal in the retina. These synapses display remarkable structural and functional differences, and the mechanisms regulating synaptic vesicle cycling might also differ between them. The lipophilic styryl dye FM1-43 was used to monitor vesicle tr...

  8. Synaptic-like vesicles and candidate transduction channels in mechanosensory terminals.

    Bewick, Guy S

    2015-08-01

    This article summarises progress to date over an exciting and very enjoyable first 15 years of collaboration with Bob Banks. Our collaboration began when I contacted him with (to me) an unexpected observation that a dye used to mark recycling synaptic vesicle membrane at efferent terminals also labelled muscle spindle afferent terminals. This observation led to the re-discovery of a system of small clear vesicles present in all vertebrate primary mechanosensory nerve terminals. These synaptic-like vesicles (SLVs) have been, and continue to be, the major focus of our work. This article describes our characterisation of the properties and functional significance of these SLVs, combining our complementary skills: Bob's technical expertise and encyclopaedic knowledge of mechanosensation with my experience of synaptic vesicles and the development of the styryl pyridinium dyes, of which the most widely used is FM1-43. On the way we have found that SLVs seem to be part of a constitutive glutamate secretory system necessary to maintain the stretch-sensitivity of spindle endings. The glutamate activates a highly unusual glutamate receptor linked to phospholipase D activation, which we have termed the PLD-mGluR. It has a totally distinct pharmacology first described in the hippocampus nearly 20 years ago but, like the SLVs that were first described over 50 years ago, has since been little researched. Yet, our evidence and literature searches suggest this glutamate/SLV/PLD-mGluR system is a ubiquitous feature of mechanosensory endings and, at least for spindles, is essential for maintaining mechanosensory function. This article summarises how this system integrates with the classical model of mechanosensitive channels in spindles and other mechanosensory nerve terminals, including hair follicle afferents and baroreceptors controlling blood pressure. Finally, in this time when there is an imperative to show translational relevance, I describe how this fascinating system might

  9. UNC-41/stonin functions with AP2 to recycle synaptic vesicles in Caenorhabditis elegans.

    Gregory P Mullen

    Full Text Available The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2 have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.

  10. SUMOylation of synapsin Ia maintains synaptic vesicle availability and is reduced in an autism mutation

    Tang, Leo T. -H.; Tim J Craig; Henley, Jeremy M.

    2015-01-01

    Synapsins are key components of the presynaptic neurotransmitter release machinery. Their main role is to cluster synaptic vesicles (SVs) to each other and anchor them to the actin cytoskeleton to establish the reserve vesicle pool, and then release them in response to appropriate membrane depolarization. Here we demonstrate that SUMOylation of synapsin Ia (SynIa) at K687 is necessary for SynIa function. Replacement of endogenous SynIa with a non-SUMOylatable mutant decreases the size of the ...

  11. CAPS1 stabilizes the state of readily releasable synaptic vesicles to fusion competence at CA3–CA1 synapses in adult hippocampus

    Shinoda, Yo; Ishii, Chiaki; Fukazawa, Yugo; Sadakata, Tetsushi; Ishii, Yuki; Sano, Yoshitake; Iwasato, Takuji; Itohara, Shigeyoshi; Furuichi, Teiichi

    2016-01-01

    Calcium-dependent activator protein for secretion 1 (CAPS1) regulates exocytosis of dense-core vesicles in neuroendocrine cells and of synaptic vesicles in neurons. However, the synaptic function of CAPS1 in the mature brain is unclear because Caps1 knockout (KO) results in neonatal death. Here, using forebrain-specific Caps1 conditional KO (cKO) mice, we demonstrate, for the first time, a critical role of CAPS1 in adult synapses. The amplitude of synaptic transmission at CA3–CA1 synapses was strongly reduced, and paired-pulse facilitation was significantly increased, in acute hippocampal slices from cKO mice compared with control mice, suggesting a perturbation in presynaptic function. Morphological analysis revealed an accumulation of synaptic vesicles in the presynapse without any overall morphological change. Interestingly, however, the percentage of docked vesicles was markedly decreased in the Caps1 cKO. Taken together, our findings suggest that CAPS1 stabilizes the state of readily releasable synaptic vesicles, thereby enhancing neurotransmitter release at hippocampal synapses. PMID:27545744

  12. 38,000-DALTON MEMBRANE PROTEIN (P38) PRESENT IN SYNAPTIC VESICLES

    A protein with an apparent molecular mass of 38,000 daltons designated p38 was found in synaptic vesicles from rat brain. The subcellular distribution of p38 and some of its properties were determined with the aid of polyclonal and monoclonal antibodies. The subcellular distribut...

  13. CHARACTERIZATION OF A NOVEL RADIOTRACER TARGETING SYNAPTIC VESICLE PROTEIN 2A (SV2A)

    Warnock, Geoffrey; Aerts, Joël; Bahri, Mohamed Ali; Bretin, Florian; Buchanan, T; Klitgaard, H; Mestdagh, N; A. Valade; Mercier, J.; Seret, Alain; Luxen, André; Salmon, Eric; Plenevaux, Alain

    2012-01-01

    Synaptic vesicle protein 2A (SV2A) has been identified as the binding site of the antiepileptic levetiracetam (Keppra) [1]. SV2 proteins are critical for proper nervous system function and have been demonstrated to be involved in vesicle trafficking. Their implication in epilepsy makes them an interesting therapeutic target, and the widespread distribution of SV2A in particular may provide an opportunity to develop a PET-based measure of neuronal function in brain diseases. [18F]UCB-H i...

  14. Synaptic vesicle recycling at the calyx of Held

    Lei XUE; Yan-ai MEI

    2011-01-01

    Efficient endocytosis is crucial for maintaining synaptic transmission because of its role in retrieving constituent membrane and associated proteins. In the past three decades three modes of endocytosis have been proposed involving the central nervous system: clathrin-mediated endocytosis, kiss-and-run endocytosis and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. Here, we review the existence and initiation of all three modes of endocytosis at a giant glutamatergic synapse, the calyx of Held. The possibility of direct electrophysiology recording in this synapse allows for accurate tracking of exocytosis and endocytosis via capacitance measurements. Future aims will be focused on identifying the molecules that undergo the different mechanisms of endocytosis and the conditions under which different forms of endocytosis predominate.

  15. Identification of a human synaptotagmin-1 mutation that perturbs synaptic vesicle cycling

    Baker, Kate; Gordon, Sarah L.; Grozeva, Detelina; Van Kogelenberg, Margriet; Roberts, Nicola Y.; Pike, Michael; Blair, Edward; Hurles, Matthew E.; Chong, W Kling; Baldeweg, Torsten; Kurian, Manju A.; Boyd, Stewart G; Cousin, Michael A; Raymond, F. Lucy

    2015-01-01

    Synaptotagmin-1 (SYT1) is a calcium-binding synaptic vesicle protein that is required for both exocytosis and endocytosis. Here, we describe a human condition associated with a rare variant in SYT1. The individual harboring this variant presented with an early onset dyskinetic movement disorder, severe motor delay, and profound cognitive impairment. Structural MRI was normal, but EEG showed extensive neurophysiological disturbances that included the unusual features of low-frequency oscillato...

  16. Identification of a human synaptotagmin-1 mutation that perturbs synaptic vesicle cycling.

    Baker, Kate; Gordon, Sarah L; Grozeva, Detelina; van Kogelenberg, Margriet; Roberts, Nicola Y; Pike, Michael; Blair, Edward; Hurles, Matthew E; Chong, W Kling; Baldeweg, Torsten; Kurian, Manju A; Boyd, Stewart G; Cousin, Michael A; Raymond, F Lucy

    2015-04-01

    Synaptotagmin-1 (SYT1) is a calcium-binding synaptic vesicle protein that is required for both exocytosis and endocytosis. Here, we describe a human condition associated with a rare variant in SYT1. The individual harboring this variant presented with an early onset dyskinetic movement disorder, severe motor delay, and profound cognitive impairment. Structural MRI was normal, but EEG showed extensive neurophysiological disturbances that included the unusual features of low-frequency oscillatory bursts and enhanced paired-pulse depression of visual evoked potentials. Trio analysis of whole-exome sequence identified a de novo SYT1 missense variant (I368T). Expression of rat SYT1 containing the equivalent human variant in WT mouse primary hippocampal cultures revealed that the mutant form of SYT1 correctly localizes to nerve terminals and is expressed at levels that are approximately equal to levels of endogenous WT protein. The presence of the mutant SYT1 slowed synaptic vesicle fusion kinetics, a finding that agrees with the previously demonstrated role for I368 in calcium-dependent membrane penetration. Expression of the I368T variant also altered the kinetics of synaptic vesicle endocytosis. Together, the clinical features, electrophysiological phenotype, and in vitro neuronal phenotype associated with this dominant negative SYT1 mutation highlight presynaptic mechanisms that mediate human motor control and cognitive development. PMID:25705886

  17. Molecular Machines Determining the Fate of Endocytosed Synaptic Vesicles in Nerve Terminals

    Fassio, Anna; Fadda, Manuela; Benfenati, Fabio

    2016-01-01

    The cycle of a synaptic vesicle (SV) within the nerve terminal is a step-by-step journey with the final goal of ensuring the proper synaptic strength under changing environmental conditions. The SV cycle is a precisely regulated membrane traffic event in cells and, because of this, a plethora of membrane-bound and cytosolic proteins are devoted to assist SVs in each step of the journey. The cycling fate of endocytosed SVs determines both the availability for subsequent rounds of release and the lifetime of SVs in the terminal and is therefore crucial for synaptic function and plasticity. Molecular players that determine the destiny of SVs in nerve terminals after a round of exo-endocytosis are largely unknown. Here we review the functional role in SV fate of phosphorylation/dephosphorylation of SV proteins and of small GTPases acting on membrane trafficking at the synapse, as they are emerging as key molecules in determining the recycling route of SVs within the nerve terminal. In particular, we focus on: (i) the cyclin-dependent kinase-5 (cdk5) and calcineurin (CN) control of the recycling pool of SVs; (ii) the role of small GTPases of the Rab and ADP-ribosylation factor (Arf) families in defining the route followed by SV in their nerve terminal cycle. These regulatory proteins together with their synaptic regulators and effectors, are molecular nanomachines mediating homeostatic responses in synaptic plasticity and potential targets of drugs modulating the efficiency of synaptic transmission. PMID:27242505

  18. MOLECULAR MACHINES DETERMINING THE FATE OF ENDOCYTOSED SYNAPTIC VESICLES IN NERVE TERMINALS

    Anna Fassio

    2016-05-01

    Full Text Available The cycle of a synaptic vesicle (SV within the nerve terminal is a step-by-step journey with the final goal of ensuring the proper synaptic strength under changing environmental conditions.The SV cycle is a precisely regulated membrane traffic event in cells and, because of this, a plethora of membrane-bound and cytosolic proteins are devoted to assist SVs in each step of the journey. The cycling fate of endocytosed SVs determines both the availability for subsequent rounds of release and the lifetime of SVs in the terminal and is therefore crucial for synaptic function and plasticity. Molecular players that determine the destiny of SVs in nerve terminals after a round of exo-endocytosis are largely unknown. Here we review the functional role in SV fate of phosphorylation/dephosphorylation of SV proteins and of small GTPases acting on membrane trafficking at the synapse, as they are emerging as key molecules in determining the recycling route of SVs within the nerve terminal. In particular, we focus on (i the cyclin-dependent kinase-5 and calcineurin control of the recycling pool of SVs; (ii the role of small GTPases of the Rab and ADP-ribosylation factor (Arf families in defining the route followed by SV in their nerve terminal cycle. These regulatory proteins together with their synaptic regulators and effectors, are molecular nanomachines mediating homeostatic responses in synaptic plasticity and potential targets of drugs modulating the efficiency of synaptic transmission.

  19. Activity-Dependent Calpain Activation Plays a Critical Role in Synaptic Facilitation and Post-Tetanic Potentiation

    Khoutorsky, Arkady; Spira, Micha E.

    2009-01-01

    Synaptic facilitation and post-tetanic potentiation (PTP) are believed to necessitate active regeneration of the release machinery and supply of synaptic vesicles to a ready-releasable site. The prevailing hypothesis assumes that synapsins play pivotal roles in these processes. Using a cholinergic synapse formed between cultured "Aplysia" neurons…

  20. Evidence that the ZNT3 protein controls the total amount of elemental zinc in synaptic vesicles

    Linkous, D.H.; Flinn, J.M.; Koh, J.Y.; Lanzirotti, A.; Bertsch, P.M.; Jones, B.F.; Giblin, L.J.; Frederickson, C.J.

    2008-01-01

    The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2- to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the "stainability" but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain. ?? The Histochemical Society, Inc.

  1. Botulinum Neurotoxins Can Enter Cultured Neurons Independent of Synaptic Vesicle Recycling

    Pellett, Sabine; Tepp, William H.; Jacob M Scherf; Eric A Johnson

    2015-01-01

    Botulinum neurotoxins (BoNTs) are the causative agent of the severe and long-lasting disease botulism. At least seven different serotypes of BoNTs (denoted A-G) have been described. All BoNTs enter human or animal neuronal cells via receptor mediated endocytosis and cleave cytosolic SNARE proteins, resulting in a block of synaptic vesicle exocytosis, leading to the flaccid paralysis characteristic of botulism. Previous data have indicated that once a neuronal cell has been intoxicated by a Bo...

  2. ATM protein is located on presynaptic vesicles and its deficit leads to failures in synaptic plasticity.

    Vail, Graham; Cheng, Aifang; Han, Yu Ray; Zhao, Teng; Du, Shengwang; Loy, Michael M T; Herrup, Karl; Plummer, Mark R

    2016-07-01

    Ataxia telangiectasia is a multisystemic disorder that includes a devastating neurodegeneration phenotype. The ATM (ataxia-telangiectasia mutated) protein is well-known for its role in the DNA damage response, yet ATM is also found in association with cytoplasmic vesicular structures: endosomes and lysosomes, as well as neuronal synaptic vesicles. In keeping with this latter association, electrical stimulation of the Schaffer collateral pathway in hippocampal slices from ATM-deficient mice does not elicit normal long-term potentiation (LTP). The current study was undertaken to assess the nature of this deficit. Theta burst-induced LTP was reduced in Atm(-/-) animals, with the reduction most pronounced at burst stimuli that included 6 or greater trains. To assess whether the deficit was associated with a pre- or postsynaptic failure, we analyzed paired-pulse facilitation and found that it too was significantly reduced in Atm(-/-) mice. This indicates a deficit in presynaptic function. As further evidence that these synaptic effects of ATM deficiency were presynaptic, we used stochastic optical reconstruction microscopy. Three-dimensional reconstruction revealed that ATM is significantly more closely associated with Piccolo (a presynaptic marker) than with Homer1 (a postsynaptic marker). These results underline how, in addition to its nuclear functions, ATM plays an important functional role in the neuronal synapse where it participates in the regulation of presynaptic vesicle physiology. PMID:27075534

  3. Measurement of quantal secretion induced by ouabain and its correlation with depletion of synaptic vesicles.

    Haimann, C; Torri-Tarelli, F; Fesce, R; Ceccarelli, B

    1985-11-01

    Ouabain (0.1 and 0.05 mM) was applied to frog cutaneous pectoris nerve-muscle preparations bathed in modified Ringer's solution containing either 1.8 mM Ca2+ (and 4 mM Mg2+) or no added Ca2+ (4 mM Mg2+ and 1 mM EGTA). During the intense quantal release of acetylcholine (ACh) induced by ouabain, the parameters of the miniature endplate potentials (mepps) were deduced from the variance, skew, and power spectra of the endplate recordings by applying a recently described modification of classical fluctuation analysis. Often the high frequency of mepps is not stationary; therefore, the signal was high-pass filtered (time constant of the resistance-capacitance filter of 2 ms) to remove the errors introduced by nonstationarity. When ouabain was applied in the presence of Ca2+, mepp frequency started to rise exponentially after a lag of 1.5-2 h, reached an average peak frequency of 1,300/s in approximately 30 min, and then suddenly subsided to low level (10/s). In Ca2+-free solution, after a shorter lag (1-1.5 h), mepp frequency rose to peak rate of 700/s in approximately 20 min and then gradually subsided. In spite of the different time course of secretion in the two experimental conditions, the cumulative quantal release was not significantly different (7.4 +/- 1.3 X 10(5) in Ca2+-containing and 8.8 +/- 2.7 X 10(5) in Ca2+-free solutions). 60 min after the peak secretion, the muscles were fixed for observation in the electron microscope. Morphometric analysis on micrographs of neuromuscular junctions revealed in both cases a profound depletion of synaptic vesicles and deep infoldings of presynaptic membrane. This rapid depletion and the lack of uptake of horseradish peroxidase suggest that ouabain impairs the recycling process that tends to conserve the vesicle population during intense secretion of neurotransmitter. The good correlation observed between the reduction in the store of synaptic vesicles and the total number of quanta of ACh secreted in the absence of a

  4. DGKθ Catalytic Activity Is Required for Efficient Recycling of Presynaptic Vesicles at Excitatory Synapses

    Hana L. Goldschmidt

    2016-01-01

    Full Text Available Synaptic transmission relies on coordinated coupling of synaptic vesicle (SV exocytosis and endocytosis. While much attention has focused on characterizing proteins involved in SV recycling, the roles of membrane lipids and their metabolism remain poorly understood. Diacylglycerol, a major signaling lipid produced at synapses during synaptic transmission, is regulated by diacylglycerol kinase (DGK. Here, we report a role for DGKθ in the mammalian CNS in facilitating recycling of presynaptic vesicles at excitatory synapses. Using synaptophysin- and vGlut1-pHluorin optical reporters, we found that acute and chronic deletion of DGKθ attenuated the recovery of SVs following neuronal stimulation. Rescue of recycling kinetics required DGKθ kinase activity. Our data establish a role for DGK catalytic activity at the presynaptic nerve terminal in SV recycling. Altogether, these data suggest that DGKθ supports synaptic transmission during periods of elevated neuronal activity.

  5. Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling.

    Baumgart, Joel P; Zhou, Zhen-Yu; Hara, Masato; Cook, Daniel C; Hoppa, Michael B; Ryan, Timothy A; Hemmings, Hugh C

    2015-09-22

    Identifying presynaptic mechanisms of general anesthetics is critical to understanding their effects on synaptic transmission. We show that the volatile anesthetic isoflurane inhibits synaptic vesicle (SV) exocytosis at nerve terminals in dissociated rat hippocampal neurons through inhibition of presynaptic Ca(2+) influx without significantly altering the Ca(2+) sensitivity of SV exocytosis. A clinically relevant concentration of isoflurane (0.7 mM) inhibited changes in [Ca(2+)]i driven by single action potentials (APs) by 25 ± 3%, which in turn led to 62 ± 3% inhibition of single AP-triggered exocytosis at 4 mM extracellular Ca(2+) ([Ca(2+)]e). Lowering external Ca(2+) to match the isoflurane-induced reduction in Ca(2+) entry led to an equivalent reduction in exocytosis. These data thus indicate that anesthetic inhibition of neurotransmitter release from small SVs occurs primarily through reduced axon terminal Ca(2+) entry without significant direct effects on Ca(2+)-exocytosis coupling or on the SV fusion machinery. Isoflurane inhibition of exocytosis and Ca(2+) influx was greater in glutamatergic compared with GABAergic nerve terminals, consistent with selective inhibition of excitatory synaptic transmission. Such alteration in the balance of excitatory to inhibitory transmission could mediate reduced neuronal interactions and network-selective effects observed in the anesthetized central nervous system. PMID:26351670

  6. Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

    Marco Pirazzini

    2014-09-01

    Full Text Available Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.

  7. Molecular Profiling of Synaptic Vesicle Docking Sites Reveals Novel Proteins but Few Differences between Glutamatergic and GABAergic Synapses

    Boyken, Janina; Gronborg, Mads; Riedel, Dietmar; Urlaub, Henning; Jahn, Reinhard; Chua, John Jia En

    2013-01-01

    Neurotransmission involves calcium-triggered fusion of docked synaptic vesicles at specialized presynaptic release sites. While many of the participating proteins have been identified, the molecular composition of these sites has not been characterized comprehensively. Here, we report a procedure to

  8. Glutamatergic modulation of synaptic-like vesicle recycling in mechanosensory lanceolate nerve terminals of mammalian hair follicles

    Banks, R.W.; Cahusac, P.M.; Graca, A.; Kain, N.; Shenton, F.; Singh, P.; Nja, A.; Simon, A.; Watson, S.; Slater, C.R.; Bewick, G.S.

    2013-01-01

    Abstract Our aim in the present study was to determine whether a glutamatergic modulatory system involving synaptic-like vesicles (SLVs) is present in the lanceolate ending of the mouse and rat hair follicle and, if so, to assess its similarity to that of the rat muscle spindle annulospiral ending w

  9. Analysis of synaptic vesicle endocytosis in synaptosomes by high-content screening.

    Daniel, James A; Malladi, Chandra S; Kettle, Emma; McCluskey, Adam; Robinson, Phillip J

    2012-08-01

    Small molecules modulating synaptic vesicle endocytosis (SVE) may ultimately be useful for diseases where pathological neurotransmission is implicated. Only a small number of specific SVE modulators have been identified to date. Slow progress is due to the laborious nature of traditional approaches to study SVE, in which nerve terminals are identified and studied in cultured neurons, typically yielding data from 10-20 synapses per experiment. We provide a protocol for a quantitative, high-throughput method for studying SVE in thousands of nerve terminals. Rat forebrain synaptosomes are attached to 96-well microplates and depolarized; SVE is then quantified by uptake of the dye FM4-64, which is imaged by high-content screening. Synaptosomes that have been frozen and stored can be used in place of fresh synaptosomes, reducing the experimental time and animal numbers required. With a supply of frozen synaptosomes, the assay can be performed within a day, including data analysis. PMID:22767087

  10. Presynaptic active zone density during development and synaptic plasticity.

    Gwenaëlle L Clarke

    2012-02-01

    Full Text Available Neural circuits transmit information through synapses, and the efficiency of synaptic transmission is closely related to the density of presynaptic active zones, where synaptic vesicles are released. The goal of this review is to highlight recent insights into the molecular mechanisms that control the number of active zones per presynaptic terminal (active zone density during developmental and stimulus-dependent changes in synaptic efficacy. At the neuromuscular junctions (NMJs, the active zone density is preserved across species, remains constant during development, and is the same between synapses with different activities. However, the NMJ active zones are not always stable, as exemplified by the change in active zone density during acute experimental manipulation or as a result of aging. Therefore, a mechanism must exist to maintain its density. In the central nervous system (CNS, active zones have restricted maximal size, exist in multiple numbers in larger presynaptic terminals, and maintain a constant density during development. These findings suggest that active zone density in the CNS is also controlled. However, in contrast to the NMJ, active zone density in the CNS can also be increased, as observed in hippocampal synapses in response to synaptic plasticity. Although the numbers of known active zone proteins and protein interactions have increased, less is known about the mechanism that controls the number or spacing of active zones. The following molecules are known to control active zone density and will be discussed herein: extracellular matrix laminins and voltage-dependent calcium channels, amyloid precursor proteins, the small GTPase Rab3, an endocytosis mechanism including synaptojanin, cytoskeleton protein spectrins and β-adducin, and a presynaptic web including spectrins. The molecular mechanisms that organize the active zone density are just beginning to be elucidated.

  11. Activation of calcineurin by phosphotidylserine containing vesicles

    Politino, M.; King, M.M.

    1986-05-01

    Calcineurin (CaN) is a Ca/sup 2 +/- and calmodulin-regulated phosphatase. Recent findings suggested an association of CaN with biological membranes and prompted the present investigation into the interactions of the phosphatase with phospholipids in vitro. In the absence of calmodulin, sonicated preparations of phosphatidylserine (PS) provided a five-fold activation of the Ni- and Mn-supported activities of CaN towards (/sup 32/P) histone Hl; activation in the presence of calmodulin was much less pronounced. Half-maximal activation in the absence of calmodulin required approximately 0.1 mg/ml of PS. Activation of CaN was also observed with mixed vesicles of phosphatidylcholine (PC) containing 20% PS but not with PC alone, or with phosphatidylethanolamine (PE). Molecular sieve chromatography on Ultrogel AcA 34 provided further evidence that CaN associates with phospholipid vesicles composed of PS, or PC containing 20% PS, but not with vesicles of PC or PE. Complete association with medium sized vesicles of PS and PC/PS required Ca/sup 2 +/ ions; in the absence of the metal ion at least 60% of the enzyme failed to interact with the lipids while the remainder preferentially migrated with larger vesicles. These results suggest a role for Ca/sup 2 +/ in regulating CaN's interaction with phospholipids.

  12. In vitro study of interaction of synaptic vesicles with lipid membranes

    Ghosh, S K; Castorph, S; Salditt, T [Institute for X-ray Physics, University of Goettingen, 37077 Goettingen (Germany); Konovalov, O [European Synchrotron Radiation Facility, 38043 Grenoble Cedex (France); Jahn, R; Holt, M, E-mail: sghosh1@gwdg.d, E-mail: mholt@gwdg.d, E-mail: tsaldit@gwdg.d [Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Goettingen (Germany)

    2010-10-15

    The fusion of synaptic vesicles (SVs) with the plasma membrane in neurons is a crucial step in the release of neurotransmitters, which are responsible for carrying signals between nerve cells. While many of the molecular players involved in this fusion process have been identified, a precise molecular description of their roles in the process is still lacking. A case in point is the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}). Although PIP{sub 2} is known to be essential for vesicle fusion, its precise role in the process remains unclear. We have re-investigated the role of this lipid in membrane structure and function using the complementary experimental techniques of x-ray reflectivity, both on lipid monolayers at an air-water interface and bilayers on a solid support, and grazing incidence x-ray diffraction on lipid monolayers. These techniques provide unprecedented access to structural information at the molecular level, and detail the profound structural changes that occur in a membrane following PIP{sub 2} incorporation. Further, we also confirm and extend previous findings that the association of SVs with membranes is enhanced by PIP{sub 2} incorporation, and reveal the structural changes that underpin this phenomenon. Further, the association is further intensified by a physiologically relevant amount of Ca{sup 2+} ions in the subphase of the monolayer, as revealed by the increase in interfacial pressure seen with the lipid monolayer system. Finally, a theoretical calculation concerning the products arising from the fusion of these SVs with proteoliposomes is presented, with which we aim to illustrate the potential future uses of this system.

  13. In vitro study of interaction of synaptic vesicles with lipid membranes

    The fusion of synaptic vesicles (SVs) with the plasma membrane in neurons is a crucial step in the release of neurotransmitters, which are responsible for carrying signals between nerve cells. While many of the molecular players involved in this fusion process have been identified, a precise molecular description of their roles in the process is still lacking. A case in point is the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Although PIP2 is known to be essential for vesicle fusion, its precise role in the process remains unclear. We have re-investigated the role of this lipid in membrane structure and function using the complementary experimental techniques of x-ray reflectivity, both on lipid monolayers at an air-water interface and bilayers on a solid support, and grazing incidence x-ray diffraction on lipid monolayers. These techniques provide unprecedented access to structural information at the molecular level, and detail the profound structural changes that occur in a membrane following PIP2 incorporation. Further, we also confirm and extend previous findings that the association of SVs with membranes is enhanced by PIP2 incorporation, and reveal the structural changes that underpin this phenomenon. Further, the association is further intensified by a physiologically relevant amount of Ca2+ ions in the subphase of the monolayer, as revealed by the increase in interfacial pressure seen with the lipid monolayer system. Finally, a theoretical calculation concerning the products arising from the fusion of these SVs with proteoliposomes is presented, with which we aim to illustrate the potential future uses of this system.

  14. Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held

    R.M. Leão

    2009-01-01

    Full Text Available The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5-7 (p5-7 or older (p10-12 rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5-7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10-12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels.

  15. Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin

    Gad, H; Ringstad, N; Löw, P;

    2000-01-01

    impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were...

  16. A Missense Mutation of the Gene Encoding Synaptic Vesicle Glycoprotein 2A (SV2A) Confers Seizure Susceptibility by Disrupting Amygdalar Synaptic GABA Release.

    Tokudome, Kentaro; Okumura, Takahiro; Terada, Ryo; Shimizu, Saki; Kunisawa, Naofumi; Mashimo, Tomoji; Serikawa, Tadao; Sasa, Masashi; Ohno, Yukihiro

    2016-01-01

    Synaptic vesicle glycoprotein 2A (SV2A) is specifically expressed in the membranes of synaptic vesicles and modulates action potential-dependent neurotransmitter release. To explore the role of SV2A in the pathogenesis of epileptic disorders, we recently generated a novel rat model (Sv2a(L174Q) rat) carrying a missense mutation of the Sv2a gene and showed that the Sv2a(L174Q) rats were hypersensitive to kindling development (Tokudome et al., 2016). Here, we further conducted behavioral and neurochemical studies to clarify the pathophysiological mechanisms underlying the seizure vulnerability in Sv2a(L174Q) rats. Sv2a(L174Q) rats were highly susceptible to pentylenetetrazole (PTZ)-induced seizures, yielding a significantly higher seizure scores and seizure incidence than the control animals. Brain mapping analysis of Fos expression, a biological marker of neural excitation, revealed that the seizure threshold level of PTZ region-specifically elevated Fos expression in the amygdala in Sv2a(L174Q) rats. In vivo microdialysis study showed that the Sv2a(L174Q) mutation preferentially reduced high K(+) (depolarization)-evoked GABA release, but not glutamate release, in the amygdala. In addition, specific control of GABA release by SV2A was supported by its predominant expression in GABAergic neurons, which were co-stained with antibodies against SV2A and glutamate decarboxylase 1. The present results suggest that dysfunction of SV2A by the missense mutation elevates seizure susceptibility in rats by preferentially disrupting synaptic GABA release in the amygdala, illustrating the crucial role of amygdalar SV2A-GABAergic system in epileptogenesis. PMID:27471467

  17. Astrocytes optimize synaptic fidelity

    Nadkarni, Suhita; Jung, Peter; Levine, Herbert

    2007-03-01

    Most neuronal synapses in the central nervous system are enwrapped by an astrocytic process. This relation allows the astrocyte to listen to and feed back to the synapse and to regulate synaptic transmission. We combine a tested mathematical model for the Ca^2+ response of the synaptic astrocyte and presynaptic feedback with a detailed model for vesicle release of neurotransmitter at active zones. The predicted Ca^2+ dependence of the presynaptic synaptic vesicle release compares favorably for several types of synapses, including the Calyx of Held. We hypothesize that the feedback regulation of the astrocyte onto the presynaptic terminal optimizes the fidelity of the synapse in terms of information transmission.

  18. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

    Benoit, Roger M.; Frey, Daniel; Hilbert, Manuel; Kevenaar, Josta T.; Wieser, Mara M.; Stirnimann, Christian U.; McMillan, David; Ceska, Tom; Lebon, Florence; Jaussi, Rolf; Steinmetz, Michel O.; Schertler, Gebhard F. X.; Hoogenraad, Casper C.; Capitani, Guido; Kammerer, Richard A.

    2014-01-01

    Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

  19. Synaptic Vesicle Tethering and the CaV2.2 Distal C-terminal

    Fiona K Wong

    2014-03-01

    Full Text Available . Evidence that synaptic vesicles (SVs can be gated by a single voltage sensitive calcium channel (CaV2.2 predict a molecular linking mechanism or ‘tether’[Stanley 1993]. Recent studies have proposed that the SV binds to the distal C-terminal on the CaV2.2 calcium channel [Kaeser et al. 2011;Wong, Li, and Stanley 2013] while genetic analysis proposed a double tether mechanism via RIM: directly to the C terminus PDZ ligand domain or indirectly via a more proximal proline rich site [Kaeser et al. 2011]. Using a novel in vitro SV-PD binding assay, we reported that SVs bind to a fusion protein comprising the C-terminal distal third (C3, aa 2137-2357 [Wong, Li, and Stanley 2013]. Here we limit the binding site further to the last 58 aa, beyond the proline rich site, by the absence of SV capture by a truncated C3 fusion protein (aa 2137-2299. To test PDZ-dependent binding we generated two C terminus-mutant C3 fusion proteins and a mimetic blocking peptide (H-WC, aa 2349-2357 and validated these by elimination of MINT-1 or RIM binding. Persistence of SV capture with all three fusion proteins or with the full length C3 protein but in the presence of the blocking peptide, demonstrated that SVs can bind to the distal C-terminal via a PDZ-independent mechanism. These results were supported in situ by normal SV turnover in H-WC-loaded synaptosomes, as assayed by a novel peptide cryoloading method. Thus, SVs tether to the CaV2.2 C-terminal within a 49 aa region immediately prior to the terminus PDZ ligand domain. Long tethers that could reflect extended C termini were imaged by electron microscopy of synaptosome ghosts. To fully account for SV tethering we propose a model where SVs are initially captured, or ‘grabbed’, from the cytoplasm by a binding site on the distal region of the channel C-terminal and are then retracted to be ‘locked’ close to the channel by a second attachment mechanism in preparation for single channel domain gating.

  20. Vesicle recycling at ribbon synapses in the finely branched axon terminals of mouse retinal bipolar neurons

    LoGiudice, Lisamarie; Sterling, Peter; Matthews, Gary

    2009-01-01

    In retinal bipolar neurons, synaptic ribbons mark the presence of exocytotic active zones in the synaptic terminal. It is unknown, however, where compensatory vesicle retrieval is localized in this cell type and by what mechanism(s) excess membrane is recaptured. To determine whether endocytosis is localized or diffuse in mouse bipolar neurons, we imaged FM4-64 to track vesicles in cells whose synaptic ribbons were tagged with a fluorescent peptide. In synaptic terminals, vesicle retrieval oc...

  1. [Peptidergic modulation of the hippocampus synaptic activity].

    Skrebitskiĭ, V G; Kondratenko, R V; Povarov, I S; Dereviagin, V I

    2011-11-01

    Effects of two newly synthesized nootropic and anxiolytic dipeptides: Noopept and Selank on inhibitory synaptic transmission in hippocampal CA1 pyramidal cells were investigated using patch-clamp technique in whole-cell configuration. Bath application of Noopept (1 microM) or Selank (2 microM) significantly increased the frequency of spike-dependent spontaneous m1PSCs, whereas spike-independent mlPSCs remained unchanged. It was suggested that both peptides mediated their effect sue to activation of inhibitory interneurons terminating on CA1 pyramidal cells. Results of current clamp recording of inhibitory interneurons residing in stratum radiatum confirmed this suggestion, at least for Noonent. PMID:22390072

  2. Calcium-dependent synaptic vesicle trafficking underlies indefatigable release at the hair cell afferent fiber synapse

    Schnee, M.E.; Santos-Sacchi, J; Castellano-Muñoz, M.; Kong, J-H.; Ricci, A.J.

    2011-01-01

    Sensory hair cell ribbon synapses respond to graded stimulation in a linear, indefatigable manner, requiring that vesicle trafficking to synapses is rapid and non rate limiting. Real time monitoring of vesicle fusion identified two release components. The first was saturable with both release rate and magnitude varying linearly with Ca2+, however the magnitude was too small to account for sustained afferent firing rates. A second superlinear release component required recruitment, in a Ca2+-d...

  3. Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms

    Lai, Ying; Diao, Jiajie; Cipriano, Daniel J.; Zhang, Yunxiang; Pfuetzner, Richard A.; Padolina, Mark S; Brunger, Axel T.

    2014-01-01

    Previously we showed that fast Ca2+-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle–vesicle lipid/content mixing assay (Diao et al., 2012). When complexin-1 was included, a more pronounced Ca2+-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same...

  4. Synaptic reverberation underlying mnemonic persistent activity.

    Wang, X J

    2001-08-01

    Stimulus-specific persistent neural activity is the neural process underlying active (working) memory. Since its discovery 30 years ago, mnemonic activity has been hypothesized to be sustained by synaptic reverberation in a recurrent circuit. Recently, experimental and modeling work has begun to test the reverberation hypothesis at the cellular level. Moreover, theory has been developed to describe memory storage of an analog stimulus (such as spatial location or eye position), in terms of continuous 'bump attractors' and 'line attractors'. This review summarizes new studies, and discusses insights and predictions from biophysically based models. The stability of a working memory network is recognized as a serious problem; stability can be achieved if reverberation is largely mediated by NMDA receptors at recurrent synapses. PMID:11476885

  5. Synaptic vesicle cycling is not impaired in a glutamatergic and a cholinergic synapse that exhibit deficits in acidification and filling

    Bento João Abreu

    2012-03-01

    Full Text Available The purpose of the present work was to investigate synaptic vesicle trafficking when vesicles exhibit alterations in filling and acidification in two different synapses: a cholinergic frog neuromuscular junction and a glutamatergic ribbon-type nerve terminal in the retina. These synapses display remarkable structural and functional differences, and the mechanisms regulating synaptic vesicle cycling might also differ between them. The lipophilic styryl dye FM1-43 was used to monitor vesicle trafficking. Both preparations were exposed to pharmacological agents that collapse ΔpH (NH4Cl and methylamine or the whole ΔµH+ (bafilomycin, a necessary situation to provide the driving force for neurotransmitter accumulation into synaptic vesicles. The results showed that FM1-43 loading and unloading in neuromuscular junctions did not differ statistically between control and experimental conditions (P > 0.05. Also, FM1-43 labeling in bipolar cell terminals proved highly similar under all conditions tested. Despite remarkable differences in both experimental models, the present findings show that acidification and filling are not required for normal vesicle trafficking in either synapse.O objetivo do presente trabalho foi investigar o tráfego de vesículas sinápticas quando estas apresentam alterações no armazenamento de neurotransmissores e acidificação em duas distintas sinapses: a junção neuromuscular colinérgica de rãs versus o terminal nervoso glutamatérgico do tipo ribbon em céulas bipolares da retina. Essas sinapses exibem notáveis diferenças estruturais e funcionais e os mecanismos de regulação de ciclo das vesículas sinápticas podem ser diferentes entre eles. Para monitorar o tráfego de vesícula, foi utilizado o marcador lipofílico FM1-43. Ambas as preparações foram expostas a agentes farmacológicos que provocam o colapso de ΔpH (NH4Cl e metilamina ou de todo ΔµH+ (bafilomicina, gradientes necessários para o ac

  6. Vesicle Priming in a SNAP

    Müller, Martin; Davis, Graeme W.

    2010-01-01

    In this issue of Neuron, Burgalossi et al. (2010) investigate synaptic vesicle priming using presynaptic Ca2+ uncaging at a small, glutamatergic, central synapse. Combining this technique with mouse genetics, the authors demonstrate that vesicle priming during ongoing neural activity can be limited by the recycling of recently used SNARE complexes.

  7. Astroglial networks scale synaptic activity and plasticity

    Pannasch, Ulrike; Vargová, Lydia; Reingruber, Jürgen; Ezan, Pascal; Holcman, David; Giaume, Christian; Syková, Eva; Rouach, Nathalie

    2011-01-01

    Astrocytes dynamically interact with neurons to regulate synaptic transmission. Although the gap junction proteins connexin 30 (Cx30) and connexin 43 (Cx43) mediate the extensive network organization of astrocytes, their role in synaptic physiology is unknown. Here we show, by inactivating Cx30 and Cx43 genes, that astroglial networks tone down hippocampal synaptic transmission in CA1 pyramidal neurons. Gap junctional networking facilitates extracellular glutamate and potassium removal during...

  8. Analysis of shape and spatial interaction of synaptic vesicles using data from focused ion beam scanning electron microscopy (FIB-SEM)

    Khanmohammadi, Mahdieh; Waagepetersen, Rasmus Plenge; Sporring, Jon

    2015-01-01

    deviations from spherical shape and systematic trends in their orientation. We studied three-dimensional representations of synapses obtained by manual annotation of focused ion beam scanning electron microscopy (FIB-SEM) images of male mouse brain. The configurations of synaptic vesicles were regarded...

  9. Syntaxin 1B, but not syntaxin 1A, is necessary for the regulation of synaptic vesicle exocytosis and of the readily releasable pool at central synapses.

    Tatsuya Mishima

    Full Text Available Two syntaxin 1 (STX1 isoforms, HPC-1/STX1A and STX1B, are coexpressed in neurons and function as neuronal target membrane (t-SNAREs. However, little is known about their functional differences in synaptic transmission. STX1A null mutant mice develop normally and do not show abnormalities in fast synaptic transmission, but monoaminergic transmissions are impaired. In the present study, we found that STX1B null mutant mice died within 2 weeks of birth. To examine functional differences between STX1A and 1B, we analyzed the presynaptic properties of glutamatergic and GABAergic synapses in STX1B null mutant and STX1A/1B double null mutant mice. We found that the frequency of spontaneous quantal release was lower and the paired-pulse ratio of evoked postsynaptic currents was significantly greater in glutamatergic and GABAergic synapses of STX1B null neurons. Deletion of STX1B also accelerated synaptic vesicle turnover in glutamatergic synapses and decreased the size of the readily releasable pool in glutamatergic and GABAergic synapses. Moreover, STX1A/1B double null neurons showed reduced and asynchronous evoked synaptic vesicle release in glutamatergic and GABAergic synapses. Our results suggest that although STX1A and 1B share a basic function as neuronal t-SNAREs, STX1B but not STX1A is necessary for the regulation of spontaneous and evoked synaptic vesicle exocytosis in fast transmission.

  10. Chloroquine causes similar electroretinogram modifications, neuronal phospholipidosis and marked impairment of synaptic vesicle transport in Albino and Pigmented Rats

    Retinal toxicity of chloroquine has been known for several years, but the mechanism(s) of toxicity remain controversial; some author support the idea that the binding of chloroquine to melanin pigments in the retinal pigmented epithelium (RPE) play a major toxic role by concentrating the drug in the eye. In our study, 12 albinos Sprague-Dawley (SD) and 12 pigmented Brown Norway (BN) rats were treated orally for 3 months with chloroquine to compare functional and pathological findings. On Flash electroretinograms (ERG) performed in scotopic conditions, similar and progressive (time-dependent) delayed onset and decreased amplitudes of oscillatory potentials (from Day 71) and b-waves (on Day 92) were identified in both BN and SD rats. In both strains, identical morphological changes consisted of neuronal phospholipidosis associated with UV auto-fluorescence without evidence of retinal degeneration and gliosis; the RPE did not show any morphological lesions or autofluorescence. IHC analyses demonstrated a decrease in GABA expression in the inner nuclear layer. In addition, a marked accumulation of synaptic vesicles coupled with a marked disruption of neurofilaments in the optic nerve fibers was identified. In conclusion, ERG observations were very similar to those described in humans. Comparable ERG modifications, histopathology and immunohistochemistry findings were observed in the retina of both rat strains suggesting that melanin pigment is unlikely involved. chloroquine-induced impairment of synaptic vesicle transport, likely related to disruption of neurofilaments was identified and non-previously reported. This new mechanism of toxicity may also be responsible for the burry vision described in humans chronically treated with chloroquine

  11. Structural and Genetic Studies Demonstrate Neurologic Dysfunction in Triosephosphate Isomerase Deficiency Is Associated with Impaired Synaptic Vesicle Dynamics

    Roland, Bartholomew P.; Zeccola, Alison M.; Larsen, Samantha B.; Amrich, Christopher G.; Talsma, Aaron D.; Stuchul, Kimberly A.; Heroux, Annie; Levitan, Edwin S.; VanDemark, Andrew P.; Palladino, Michael J.

    2016-01-01

    Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface. PMID:27031109

  12. Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

    Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S.; Dolphin, Annette C.

    2014-04-01

    Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.

  13. Phosphorylation of synapsin I by cyclin-dependent kinase-5 sets the ratio between the resting and recycling pools of synaptic vesicles at hippocampal synapses.

    Verstegen, Anne M J; Tagliatti, Erica; Lignani, Gabriele; Marte, Antonella; Stolero, Tamar; Atias, Merav; Corradi, Anna; Valtorta, Flavia; Gitler, Daniel; Onofri, Franco; Fassio, Anna; Benfenati, Fabio

    2014-05-21

    Cyclin-dependent kinase-5 (Cdk5) was reported to downscale neurotransmission by sequestering synaptic vesicles (SVs) in the release-reluctant resting pool, but the molecular targets mediating this activity remain unknown. Synapsin I (SynI), a major SV phosphoprotein involved in the regulation of SV trafficking and neurotransmitter release, is one of the presynaptic substrates of Cdk5, which phosphorylates it in its C-terminal region at Ser(549) (site 6) and Ser(551) (site 7). Here we demonstrate that Cdk5 phosphorylation of SynI fine tunes the recruitment of SVs to the active recycling pool and contributes to the Cdk5-mediated homeostatic responses. Phosphorylation of SynI by Cdk5 is physiologically regulated and enhances its binding to F-actin. The effects of Cdk5 inhibition on the size and depletion kinetics of the recycling pool, as well as on SV distribution within the nerve terminal, are virtually abolished in mouse SynI knock-out (KO) neurons or in KO neurons expressing the dephosphomimetic SynI mutants at sites 6,7 or site 7 only. The observation that the single site-7 mutant phenocopies the effects of the deletion of SynI identifies this site as the central switch in mediating the synaptic effects of Cdk5 and demonstrates that SynI is necessary and sufficient for achieving the effects of the kinase on SV trafficking. The phosphorylation state of SynI by Cdk5 at site 7 is regulated during chronic modification of neuronal activity and is an essential downstream effector for the Cdk5-mediated homeostatic scaling. PMID:24849359

  14. Activity-dependent modulation of neural circuit synaptic connectivity

    Tessier, Charles R.; Kendal Broadie

    2009-01-01

    In many nervous systems, the establishment of neural circuits is known to proceed via a two-stage process; 1) early, activity-independent wiring to produce a rough map characterized by excessive synaptic connections, and 2) subsequent, use-dependent pruning to eliminate inappropriate connections and reinforce maintained synapses. In invertebrates, however, evidence of the activity-dependent phase of synaptic refinement has been elusive, and the dogma has long been that invertebrate circ...

  15. Expression of the synaptic vesicle proteins VAMPs/synaptobrevins 1 and 2 in non-neural tissues

    Ralston, E; Beushausen, S; Ploug, Thorkil

    1994-01-01

    The VAMPs/synaptobrevins (Vp/Sybs) are small integral membrane proteins. Two isoforms, Vp/Syb 1 and Vp/Syb 2, are considered to be specific to neural tissue. They are associated with synaptic vesicles and are believed to play an important role in neurotransmitter release. A third isoform......, cellubrevin, has recently been found in non-neural tissues. We now report that the distribution of Vp/Syb 1 and Vp/Syb 2 is wider than previously thought. RNA transcripts for both Vp/Syb 1 and Vp/Syb 2 were found in rat skeletal muscle and in several other rat non-neural tissues, and antibodies specific for...... Vp/Syb 2 detected a protein in the endoplasmic reticulum-Golgi area of skeletal muscle. Thus Vp/Sybs 1 and 2 are not restricted to the nervous system but appear to be co-expressed with cellubrevin in many different tissues. This redundancy of Vp/Sybs in a single cell may be required to control the...

  16. MPTP-meditated hippocampal dopamine deprivation modulates synaptic transmission and activity-dependent synaptic plasticity

    Parkinson's disease (PD)-like symptoms including learning deficits are inducible by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Therefore, it is possible that MPTP may disturb hippocampal memory processing by modulation of dopamine (DA)- and activity-dependent synaptic plasticity. We demonstrate here that intraperitoneal (i.p.) MPTP injection reduces the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN) within 7 days. Subsequently, the TH expression level in SN and hippocampus and the amount of DA and its metabolite DOPAC in striatum and hippocampus decrease. DA depletion does not alter basal synaptic transmission and changes pair-pulse facilitation (PPF) of field excitatory postsynaptic potentials (fEPSPs) only at the 30 ms inter-pulse interval. In addition, the induction of long-term potentiation (LTP) is impaired whereas the duration of long-term depression (LTD) becomes prolonged. Since both LTP and LTD depend critically on activation of NMDA and DA receptors, we also tested the effect of DA depletion on NMDA receptor-mediated synaptic transmission. Seven days after MPTP injection, the NMDA receptor-mediated fEPSPs are decreased by about 23%. Blocking the NMDA receptor-mediated fEPSP does not mimic the MPTP-LTP. Only co-application of D1/D5 and NMDA receptor antagonists during tetanization resembled the time course of fEPSP potentiation as observed 7 days after i.p. MPTP injection. Together, our data demonstrate that MPTP-induced degeneration of DA neurons and the subsequent hippocampal DA depletion alter NMDA receptor-mediated synaptic transmission and activity-dependent synaptic plasticity. - Highlights: → I.p. MPTP-injection mediates death of dopaminergic neurons. → I.p. MPTP-injection depletes DA and DOPAC in striatum and hippocampus. → I.p. MPTP-injection does not alter basal synaptic transmission. → Reduction of LTP and enhancement of LTD after i.p. MPTP-injection. → Attenuation of NMDA-receptors mediated

  17. Disruption of adaptor protein 2mu (AP-2mu) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing

    Jung, S.; Maritzen, T.; Wichmann, C.; Jing, Z.; Neef, A.; Revelo, N.H.; Al-Moyed, H.; Meese, S.; Wojcik, S.M.; Panou, I.; Bulut, H.; Schu, P.; Ficner, R.; Reisinger, E.; Rizzoli, S.O.; Neef, J.; Strenzke, N.; Haucke, V.; Moser, T.

    2015-01-01

    Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2mu (AP-2mu) is required for release site replenishment and hearing. We show that

  18. The immediately releasable pool of mouse chromaffin cell vesicles is coupled to P/Q-type calcium channels via the synaptic protein interaction site.

    Yanina D Álvarez

    Full Text Available It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca(2+ channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca(2+ current. Accordingly, in the present work we found that the Ca(2+ current flowing through P/Q-type Ca(2+ channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels. To investigate the mechanism that underlies the coupling between the immediately releasable pool and P/Q-type channels we transiently expressed in mouse chromaffin cells peptides corresponding to the synaptic protein interaction site of Cav2.2 to competitively uncouple P/Q-type channels from the secretory vesicle release complex. This treatment reduced the efficiency of Ca(2+ current to induce exocytosis to similar values as direct inhibition of P/Q-type channels via ω-agatoxin-IVA. In addition, the same treatment markedly reduced immediately releasable pool exocytosis, but did not affect the exocytosis provoked by sustained electric or high K(+ stimulation. Together, our results indicate that the synaptic protein interaction site is a crucial factor for the establishment of the functional coupling between immediately releasable pool vesicles and P/Q-type Ca(2+ channels.

  19. Spontaneous Activity Drives Local Synaptic Plasticity In Vivo

    Winnubst, Johan; Cheyne, Juliette E; Niculescu, Dragos; Lohmann, C.

    2015-01-01

    Spontaneous activity fine-tunes neuronal connections in the developing brain. To explore the underlying synaptic plasticity mechanisms, we monitored naturally occurring changes in spontaneous activity at individual synapses with whole-cell patch-clamp recordings and simultaneous calcium imaging in t

  20. Synapses, synaptic activity and intraneuronal Aβ in Alzheimer's disease

    Davide Tampellini

    2010-05-01

    Full Text Available β-amyloid peptide accumulation plays a central role in the pathogenesis of Alzheimer’s disease. Aberrant β-amyloid buildup in the brain has been shown to be present both in the extracellular space and within neurons. Synapses are important targets of β-amyloid, and alterations in synapses better correlate with cognitive impairment than amyloid plaques or neurofibrillary tangles. The link between β-amyloid and synapses became even tighter when it was discovered that β-amyloid accumulates within synapses and that synaptic activity modulates β-amyloid secretion. Currently, a central question in Alzheimer’s disease research is what role synaptic activity plays in the disease process, and how specifically β-amyloid is involved in the synaptic dysfunction that characterizes the disease.

  1. Spontaneous Activity Drives Local Synaptic Plasticity In Vivo.

    Winnubst, Johan; Cheyne, Juliette E; Niculescu, Dragos; Lohmann, Christian

    2015-07-15

    Spontaneous activity fine-tunes neuronal connections in the developing brain. To explore the underlying synaptic plasticity mechanisms, we monitored naturally occurring changes in spontaneous activity at individual synapses with whole-cell patch-clamp recordings and simultaneous calcium imaging in the mouse visual cortex in vivo. Analyzing activity changes across large populations of synapses revealed a simple and efficient local plasticity rule: synapses that exhibit low synchronicity with nearby neighbors (depressed in their transmission frequency. Asynchronous electrical stimulation of individual synapses in hippocampal slices showed that this is due to a decrease in synaptic transmission efficiency. Accordingly, experimentally increasing local synchronicity, by stimulating synapses in response to spontaneous activity at neighboring synapses, stabilized synaptic transmission. Finally, blockade of the high-affinity proBDNF receptor p75(NTR) prevented the depression of asynchronously stimulated synapses. Thus, spontaneous activity drives local synaptic plasticity at individual synapses in an "out-of-sync, lose-your-link" fashion through proBDNF/p75(NTR) signaling to refine neuronal connectivity. VIDEO ABSTRACT. PMID:26182421

  2. A memristor SPICE model accounting for synaptic activity dependence.

    Qingjiang Li

    Full Text Available In this work, we propose a new memristor SPICE model that accounts for the typical synaptic characteristics that have been previously demonstrated with practical memristive devices. We show that this model could account for both volatile and non-volatile memristance changes under distinct stimuli. We then demonstrate that our model is capable of supporting typical STDP with simple non-overlapping digital pulse pairs. Finally, we investigate the capability of our model to simulate the activity dependence dynamics of synaptic modification and present simulated results that are in excellent agreement with biological results.

  3. Reconciling Ligase Ribozyme Activity with Fatty Acid Vesicle Stability

    Fabrizio Anella

    2014-12-01

    Full Text Available The “RNA world” and the “Lipid world” theories for the origin of cellular life are often considered incompatible due to the differences in the environmental conditions at which they can emerge. One obstacle resides in the conflicting requirements for divalent metal ions, in particular Mg2+, with respect to optimal ribozyme activity, fatty acid vesicle stability and protection against RNA strand cleavage. Here, we report on the activity of a short L1 ligase ribozyme in the presence of myristoleic acid (MA vesicles at varying concentrations of Mg2+. The ligation rate is significantly lower at low-Mg2+ conditions. However, the loss of activity is overcompensated by the increased stability of RNA leading to a larger amount of intact ligated substrate after long reaction periods. Combining RNA ligation assays with fatty acid vesicles we found that MA vesicles made of 5 mM amphiphile are stable and do not impair ligase ribozyme activity in the presence of approximately 2 mM Mg2+. These results provide a scenario in which catalytic RNA and primordial membrane assembly can coexist in the same environment.

  4. The 4p16.3 Parkinson Disease Risk Locus Is Associated with GAK Expression and Genes Involved with the Synaptic Vesicle Membrane

    Nagle, Michael W.; Latourelle, Jeanne C.; Labadorf, Adam; Dumitriu, Alexandra; Hadzi, Tiffany C.; Beach, Thomas G.; Myers, Richard H.

    2016-01-01

    Genome-wide association studies (GWAS) have identified the GAK/DGKQ/IDUA region on 4p16.3 among the top three risk loci for Parkinson’s disease (PD), but the specific gene and risk mechanism are unclear. Here, we report transcripts containing the 3’ clathrin-binding domain of GAK identified by RNA deep-sequencing in post-mortem human brain tissue as having increased expression in PD. Furthermore, carriers of 4p16.3 PD GWAS risk SNPs show decreased expression of one of these transcripts, GAK25 (Gencode Transcript 009), which correlates with the expression of genes functioning in the synaptic vesicle membrane. Together, these findings provide strong evidence for GAK clathrin-binding- and J-domain transcripts’ influence on PD pathogenicity, and for a role for GAK in regulating synaptic function in PD. PMID:27508417

  5. Intense synaptic activity enhances temporal resolution in spinal motoneurons

    Berg, Rune W; Ditlevsen, Susanne; Hounsgaard, Jørn Dybkjær

    2008-01-01

    In neurons, spike timing is determined by integration of synaptic potentials in delicate concert with intrinsic properties. Although the integration time is functionally crucial, it remains elusive during network activity. While mechanisms of rapid processing are well documented in sensory systems...

  6. Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.

    Lagow, Robert D; Hong Bao; Cohen, Evan N; Daniels, Richard W.; Aleksej Zuzek; Williams, Wade H; Gregory T Macleod; R. Bryan Sutton; Bing Zhang

    2007-01-01

    Author Summary Most living cells constantly renew their membrane compositions and frequently communicate with neighboring cells by delivering cargo molecules from small vesicles. A key step in cargo delivery requires the fusion of the vesicle membrane with the target membrane mediated by SNARE proteins. In most cellular compartments, fusion occurs constitutively, requiring little participation of other molecules. In other cellular compartments, such as synapses in the nervous system, vesicle ...

  7. Multiple Modes of Endophilin-mediated Conversion of Lipid Vesicles into Coated Tubes: IMPLICATIONS FOR SYNAPTIC ENDOCYTOSIS*

    Mizuno, Naoko; Jao, Christine C; Langen, Ralf; Steven, Alasdair C

    2010-01-01

    Endophilin A1 is a BAR (Bin/amphiphysin/Rvs) protein abundant in neural synapses that senses and induces membrane curvature, contributing to neck formation in presynaptic endocytic vesicles. To investigate its role in membrane remodeling, we used cryoelectron microscopy to characterize structural changes induced in lipid vesicles by exposure to endophilin. The vesicles convert rapidly to coated tubules whose morphology reflects the local concentration of endophilin. Their diameters and curvat...

  8. Irregular activity arises as a natural consequence of synaptic inhibition

    Irregular neuronal activity is observed in a variety of brain regions and states. This work illustrates a novel mechanism by which irregular activity naturally emerges in two-cell neuronal networks featuring coupling by synaptic inhibition. We introduce a one-dimensional map that captures the irregular activity occurring in our simulations of conductance-based differential equations and mathematically analyze the instability of fixed points corresponding to synchronous and antiphase spiking for this map. We find that the irregular solutions that arise exhibit expansion, contraction, and folding in phase space, as expected in chaotic dynamics. Our analysis shows that these features are produced from the interplay of synaptic inhibition with sodium, potassium, and leak currents in a conductance-based framework and provides precise conditions on parameters that ensure that irregular activity will occur. In particular, the temporal details of spiking dynamics must be present for a model to exhibit this irregularity mechanism and must be considered analytically to capture these effects

  9. Irregular activity arises as a natural consequence of synaptic inhibition

    Terman, D., E-mail: terman@math.ohio-state.edu [Department of Mathematics, The Ohio State University, Columbus, Ohio 43210 (United States); Rubin, J. E., E-mail: jonrubin@pitt.edu [Department of Mathematics, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 (United States); Diekman, C. O., E-mail: diekman@njit.edu [Department of Mathematical Sciences, New Jersey Institute of Technology, Newark, New Jersey 07102 (United States)

    2013-12-15

    Irregular neuronal activity is observed in a variety of brain regions and states. This work illustrates a novel mechanism by which irregular activity naturally emerges in two-cell neuronal networks featuring coupling by synaptic inhibition. We introduce a one-dimensional map that captures the irregular activity occurring in our simulations of conductance-based differential equations and mathematically analyze the instability of fixed points corresponding to synchronous and antiphase spiking for this map. We find that the irregular solutions that arise exhibit expansion, contraction, and folding in phase space, as expected in chaotic dynamics. Our analysis shows that these features are produced from the interplay of synaptic inhibition with sodium, potassium, and leak currents in a conductance-based framework and provides precise conditions on parameters that ensure that irregular activity will occur. In particular, the temporal details of spiking dynamics must be present for a model to exhibit this irregularity mechanism and must be considered analytically to capture these effects.

  10. Irregular activity arises as a natural consequence of synaptic inhibition

    Terman, D.; Rubin, J. E.; Diekman, C. O.

    2013-12-01

    Irregular neuronal activity is observed in a variety of brain regions and states. This work illustrates a novel mechanism by which irregular activity naturally emerges in two-cell neuronal networks featuring coupling by synaptic inhibition. We introduce a one-dimensional map that captures the irregular activity occurring in our simulations of conductance-based differential equations and mathematically analyze the instability of fixed points corresponding to synchronous and antiphase spiking for this map. We find that the irregular solutions that arise exhibit expansion, contraction, and folding in phase space, as expected in chaotic dynamics. Our analysis shows that these features are produced from the interplay of synaptic inhibition with sodium, potassium, and leak currents in a conductance-based framework and provides precise conditions on parameters that ensure that irregular activity will occur. In particular, the temporal details of spiking dynamics must be present for a model to exhibit this irregularity mechanism and must be considered analytically to capture these effects.

  11. Activity-dependent modulation of neural circuit synaptic connectivity

    Charles R Tessier

    2009-07-01

    Full Text Available In many nervous systems, the establishment of neural circuits is known to proceed via a two-stage process; 1 early, activity-independent wiring to produce a rough map characterized by excessive synaptic connections, and 2 subsequent, use-dependent pruning to eliminate inappropriate connections and reinforce maintained synapses. In invertebrates, however, evidence of the activity-dependent phase of synaptic refinement has been elusive, and the dogma has long been that invertebrate circuits are “hard-wired” in a purely activity-independent manner. This conclusion has been challenged recently through the use of new transgenic tools employed in the powerful Drosophila system, which have allowed unprecedented temporal control and single neuron imaging resolution. These recent studies reveal that activity-dependent mechanisms are indeed required to refine circuit maps in Drosophila during precise, restricted windows of late-phase development. Such mechanisms of circuit refinement may be key to understanding a number of human neurological diseases, including developmental disorders such as Fragile X syndrome (FXS and autism, which are hypothesized to result from defects in synaptic connectivity and activity-dependent circuit function. This review focuses on our current understanding of activity-dependent synaptic connectivity in Drosophila, primarily through analyzing the role of the fragile X mental retardation protein (FMRP in the Drosophila FXS disease model. The particular emphasis of this review is on the expanding array of new genetically-encoded tools that are allowing cellular events and molecular players to be dissected with ever greater precision and detail.

  12. A Memristor SPICE Model Accounting for Synaptic Activity Dependence

    Qingjiang Li; Alexander Serb; Themistoklis Prodromakis; Hui Xu

    2015-01-01

    In this work, we propose a new memristor SPICE model that accounts for the typical synaptic characteristics that have been previously demonstrated with practical memristive devices. We show that this model could account for both volatile and non-volatile memristance changes under distinct stimuli. We then demonstrate that our model is capable of supporting typical STDP with simple non-overlapping digital pulse pairs. Finally, we investigate the capability of our model to simulate the activity...

  13. Super-resolution microscopy of the synaptic active zone

    Markus Sauer; Kittel, Robert J.

    2015-01-01

    Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialised proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium (Ca2+) channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modelling approaches has prov...

  14. Realistic modelling of receptor activation in hippocampal excitatory synapses: analysis of multivesicular release, release location, temperature and synaptic cross-talk.

    Boucher, Jérôme; Kröger, Helmut; Sík, Attila

    2010-07-01

    Chemically mediated synaptic transmission results from fusion of synaptic vesicles with the presynaptic plasma membrane, subsequent release of the vesicular content into the cleft and binding to postsynaptic receptors. Previous modelling studies of excitatory neurotransmitter glutamate were based on simplified geometries failing to account for the biologically realistic synaptic environment, in particular, the presence of astrocytes, the geometry of extracellular space, and the neurotransmitter uptake mechanism. Using 3-dimensional reconstructions of hippocampal glutamatergic synapses including the surrounding astrocytic processes we have developed a biologically realistic model to analyse receptor activation in different conditions. We used the finite element method to simulate glutamate release, analyse glutamate diffusion following single and multiple vesicle release and binding at the postsynaptic site to AMPA and NMDA receptors. We demonstrate that: (1) the transmitter diffusion is highly temperature-sensitive; (2) release conditions and geometry more specifically affect AMPARs than NMDARs; (3) the sensitivities of AMPARs and NMDARs to simultaneous vesicular release are different; (4) in the case of multivesicle neurotransmitter release with variable delays, the binding of glutamate to AMPARs is additive up to 1 ms after the release, then becomes independent, but to NMDARs the binding is additive up to 33 ms; (5) the number of AMPARs varies more than the number of NMDRs in response to the input firing patterns; (6) the presence of astrocytes effectively blocks synaptic cross-talk; and (7) synaptic cross-talk, mediated by NMDARs but not AMPARs, is only possible after quasi-simultaneous multivesicular release at physiological temperature (35 degrees C) without intervening astrocytes, but not at 25 degrees C. Our simulations demonstrate the importance of temperature and ultrastructural synaptic environment in synaptic transmission and synaptic cross-talk. PMID

  15. Super-resolution microscopy of the synaptic active zone

    Markus Sauer

    2015-01-01

    Full Text Available Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ a variety of specialised proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium (Ca2+ channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modelling approaches has provided predictions of channel properties, numbers and even positions on the nanometre scale. However, elucidating the nanoscopic organisation of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how super-resolution microscopy can be used to obtain information on the organisation of AZ proteins.

  16. Reconciling Ligase Ribozyme Activity with Fatty Acid Vesicle Stability

    Fabrizio Anella; Christophe Danelon

    2014-01-01

    The “RNA world” and the “Lipid world” theories for the origin of cellular life are often considered incompatible due to the differences in the environmental conditions at which they can emerge. One obstacle resides in the conflicting requirements for divalent metal ions, in particular Mg2+, with respect to optimal ribozyme activity, fatty acid vesicle stability and protection against RNA strand cleavage. Here, we report on the activity of a short L1 ligase ribozyme in the presence of myristol...

  17. Liprin-α2 promotes the presynaptic recruitment and turnover of RIM1/CASK to facilitate synaptic transmission

    S.A. Spangler (Samantha); S.K. Schmitz (Sabine); J.T. Kevenaar (Josta); E. de Graaff (Esther); M. De Wit (Meike); J.A.A. Demmers (Jeroen); P.W. Toonen (Pim ); C.C. Hoogenraad (Casper)

    2013-01-01

    textabstractThe presynaptic active zone mediates synaptic vesicle exocytosis, and modulation of its molecular composition is important for many types of synaptic plasticity. Here, we identify synaptic scaffold protein liprin-α2 as a key organizer in this process. We show that liprin-α2 levels were r

  18. SYN2 is an autism predisposing gene: loss-of-function mutations alter synaptic vesicle cycling and axon outgrowth.

    Corradi, Anna; Fadda, Manuela; Piton, Amélie; Patry, Lysanne; Marte, Antonella; Rossi, Pia; Cadieux-Dion, Maxime; Gauthier, Julie; Lapointe, Line; Mottron, Laurent; Valtorta, Flavia; Rouleau, Guy A; Fassio, Anna; Benfenati, Fabio; Cossette, Patrick

    2014-01-01

    An increasing number of genes predisposing to autism spectrum disorders (ASDs) has been identified, many of which are implicated in synaptic function. This 'synaptic autism pathway' notably includes disruption of SYN1 that is associated with epilepsy, autism and abnormal behavior in both human and mice models. Synapsins constitute a multigene family of neuron-specific phosphoproteins (SYN1-3) present in the majority of synapses where they are implicated in the regulation of neurotransmitter release and synaptogenesis. Synapsins I and II, the major Syn isoforms in the adult brain, display partially overlapping functions and defects in both isoforms are associated with epilepsy and autistic-like behavior in mice. In this study, we show that nonsense (A94fs199X) and missense (Y236S and G464R) mutations in SYN2 are associated with ASD in humans. The phenotype is apparent in males. Female carriers of SYN2 mutations are unaffected, suggesting that SYN2 is another example of autosomal sex-limited expression in ASD. When expressed in SYN2  knockout neurons, wild-type human Syn II fully rescues the SYN2 knockout phenotype, whereas the nonsense mutant is not expressed and the missense mutants are virtually unable to modify the SYN2 knockout phenotype. These results identify for the first time SYN2  as a novel predisposing gene for ASD and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies ASD. PMID:23956174

  19. Circuito eléctrico equivalente de una vesícula sináptica Electric Circuit Equivalent to a Synaptic Vesicle

    Cortés Xaira

    2003-06-01

    Full Text Available En el presente trabajo se desarrolla un modelo eléctrico de uno de los elementosprimordiales en la sinapsis nerviosa: la vesícula sináptica. Dicha vesícula se consideracomo un organelo esferoidal, despojada de neurotransmisores y se asume, además, quesu lumen, su membrana y el citoplasma neuronal se comportan como medios lineales,homogéneos e isotrópicos caracterizados por conductividades y permitividades especí-ficas. El método utilizado será la aplicación teórica de un campo eléctrico (que varía enel tiempo a bajas frecuencias sobre esta vesícula, lo que induce a través de su membra-na una diferencia de potencial cuya caracterización se obtiene a partir de las ecuacionesde Maxwell sometidas a condiciones de contorno adecuadas, en la denominada aproxi-mación cuasi-estacionaria. A su vez, mediante aplicación de la Transformada de Laplacea las expresiones resultantes se obtiene la FUNCIÓN DE TRANSFERENCIA, que condu-ce a sintetizar un circuito RLC equivalente de la vesícula en estudio. El modelo predicevalores de capacitancia para vesículas esféricas individuales que, al ser contrastados conlos que presenta la literatura existente derivada de procesos experimentales previos,alienta la perseverancia en este enfoque teórico germinal.In the present work an electrical model of the synaptic vesicle is developed. The vesicleis considered as a spheroidal organelle without neurotransmitters in its inner space. Inaddition, its lumen, its membrane and the neuronal cytoplasm behave like linear,homogenous and isotropic media characterized by specific conductivities and permi-tivities. The theoretical approach considers the application of an electric field (varying intime at low frequencies on this vesicle. A transmembrane potential difference is inducedand its characterization is obtained from Maxwell's equations subject to appropriateboundary conditions, in the so-called quasi-stationary approach. By applying theLaplace Transform to

  20. Widespread alterations in the synaptic proteome of the adolescent cerebral cortex following prenatal immune activation in rats.

    Györffy, Balázs A; Gulyássy, Péter; Gellén, Barbara; Völgyi, Katalin; Madarasi, Dóra; Kis, Viktor; Ozohanics, Olivér; Papp, Ildikó; Kovács, Péter; Lubec, Gert; Dobolyi, Árpád; Kardos, József; Drahos, László; Juhász, Gábor; Kékesi, Katalin A

    2016-08-01

    An increasing number of studies have revealed associations between pre- and perinatal immune activation and the development of schizophrenia and autism spectrum disorders (ASDs). Accordingly, neuroimmune crosstalk has a considerably large impact on brain development during early ontogenesis. While a plethora of heterogeneous abnormalities have already been described in established maternal immune activation (MIA) rodent and primate animal models, which highly correlate to those found in human diseases, the underlying molecular background remains obscure. In the current study, we describe the long-term effects of MIA on the neocortical pre- and postsynaptic proteome of adolescent rat offspring in detail. Molecular differences were revealed in sub-synaptic fractions, which were first thoroughly characterized using independent methods. The widespread proteomic examination of cortical samples from offspring exposed to maternal lipopolysaccharide administration at embryonic day 13.5 was conducted via combinations of different gel-based proteomic techniques and tandem mass spectrometry. Our experimentally validated proteomic data revealed more pre- than postsynaptic protein level changes in the offspring. The results propose the relevance of altered synaptic vesicle recycling, cytoskeletal structure and energy metabolism in the presynaptic region in addition to alterations in vesicle trafficking, the cytoskeleton and signal transduction in the postsynaptic compartment in MIA offspring. Differing levels of the prominent signaling regulator molecule calcium/calmodulin-dependent protein kinase II in the postsynapse was validated and identified specifically in the prefrontal cortex. Finally, several potential common molecular regulators of these altered proteins, which are already known to be implicated in schizophrenia and ASD, were identified and assessed. In summary, unexpectedly widespread changes in the synaptic molecular machinery in MIA rats were demonstrated which

  1. SNAP-29-mediated Modulation of Synaptic Transmission in Cultured Hippocampal Neurons*

    Pan, Ping-Yue; Cai, Qian; Lin, Lin; Lu, Pei-Hua; Duan, Shumin; Sheng, Zu-Hang

    2005-01-01

    Identifying the molecules that regulate both the recycling of synaptic vesicles and the SNARE components required for fusion is critical for elucidating the molecular mechanisms underlying synaptic plasticity. SNAP-29 was initially isolated as a syntaxin-binding and ubiquitously expressed protein. Previous studies have suggested that SNAP-29 inhibits SNARE complex disassembly, thereby reducing synaptic transmission in cultured superior cervical ganglion neurons in an activity-dependent manner...

  2. Serotonin increases synaptic activity in olfactory bulb glomeruli.

    Brill, Julia; Shao, Zuoyi; Puche, Adam C; Wachowiak, Matt; Shipley, Michael T

    2016-03-01

    Serotoninergic fibers densely innervate olfactory bulb glomeruli, the first sites of synaptic integration in the olfactory system. Acting through 5HT2A receptors, serotonin (5HT) directly excites external tufted cells (ETCs), key excitatory glomerular neurons, and depolarizes some mitral cells (MCs), the olfactory bulb's main output neurons. We further investigated 5HT action on MCs and determined its effects on the two major classes of glomerular interneurons: GABAergic/dopaminergic short axon cells (SACs) and GABAergic periglomerular cells (PGCs). In SACs, 5HT evoked a depolarizing current mediated by 5HT2C receptors but did not significantly impact spike rate. 5HT had no measurable direct effect in PGCs. Serotonin increased spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) in PGCs and SACs. Increased sEPSCs were mediated by 5HT2A receptors, suggesting that they are primarily due to enhanced excitatory drive from ETCs. Increased sIPSCs resulted from elevated excitatory drive onto GABAergic interneurons and augmented GABA release from SACs. Serotonin-mediated GABA release from SACs was action potential independent and significantly increased miniature IPSC frequency in glomerular neurons. When focally applied to a glomerulus, 5HT increased MC spontaneous firing greater than twofold but did not increase olfactory nerve-evoked responses. Taken together, 5HT modulates glomerular network activity in several ways: 1) it increases ETC-mediated feed-forward excitation onto MCs, SACs, and PGCs; 2) it increases inhibition of glomerular interneurons; 3) it directly triggers action potential-independent GABA release from SACs; and 4) these network actions increase spontaneous MC firing without enhancing responses to suprathreshold sensory input. This may enhance MC sensitivity while maintaining dynamic range. PMID:26655822

  3. SOFT MALLEABLE VESICLES TAILORED FOR ENHANCED DELIVERY OF ACTIVE AGENTS THROUGH THE SKIN: AN UPDATE

    Sandeep Kumar Parihar*, Mithun Bhowmick, Rajeev Kumar and Balkrishna Dubey

    2013-01-01

    Full Text Available Ethosomes are noninvasive delivery carriers that enable drugs to reach the deep skin layers and/or the systemic circulation. These are soft, malleable vesicles tailored for enhanced delivery of active agents. They are composed mainly of phospholipids, high concentration of ethanol and water. The high concentration of ethanol makes the ethosomes unique, as ethanol is known for its disturbance of skin lipid bilayer organization; therefore, when integrated into a vesicle membrane, it gives that vesicle the ability to penetrate the stratum corneum. Also, because of their high ethanol concentration, the lipid membrane is packed less tightly than conventional vesicles but has equivalent stability, allowing a more malleable structure and improves drug distribution ability in stratum corneum lipids. The Ethosomes were found to be suitable for various applications within the pharmaceutical, biotechnology, veterinary, cosmetic, and nutraceutical markets. These “soft vesicles” represents novel vesicular carrier for enhanced delivery to/through skin.

  4. Synaptic network activity induces neuronal differentiation of adult hippocampal precursor cells through BDNF signaling

    HarishBabu

    2009-09-01

    Full Text Available Adult hippocampal neurogenesis is regulated by activity. But how do neural precursor cells in the hippocampus respond to surrounding network activity and translate increased neural activity into a developmental program? Here we show that long-term potential (LTP-like synaptic activity within a cellular network of mature hippocampal neurons promotes neuronal differentiation of newly generated cells. In co-cultures of precursor cells with primary hippocampal neurons, LTP-like synaptic plasticity induced by addition of glycine in Mg2+-free media for 5 min, produced synchronous network activity and subsequently increased synaptic strength between neurons. Furthermore, this synchronous network activity led to a significant increase in neuronal differentiation from the co-cultured neural precursor cells. When applied directly to precursor cells, glycine and Mg2+-free solution did not induce neuronal differentiation. Synaptic plasticity-induced neuronal differentiation of precursor cells was observed in the presence of GABAergic neurotransmission blockers but was dependent on NMDA-mediated Ca2+ influx. Most importantly, neuronal differentiation required the release of brain-derived neurotrophic factor (BDNF from the underlying substrate hippocampal neurons as well as TrkB receptor phosphorylation in precursor cells. This suggests that activity-dependent stem cell differentiation within the hippocampal network is mediated via synaptically evoked BDNF signaling.

  5. Synaptic plasticity and phosphorylation

    Lee, Hey-Kyoung

    2006-01-01

    A number of neuronal functions, including synaptic plasticity, depend on proper regulation of synaptic proteins, many of which can be rapidly regulated by phosphorylation. Neuronal activity controls the function of these synaptic proteins by exquisitely regulating the balance of various protein kinase and protein phosphatase activity. Recent understanding of synaptic plasticity mechanisms underscores important roles that these synaptic phosphoproteins play in regulating both pre- and post-syn...

  6. Synaptic refinement during development and its effect on slow-wave activity: a computational study.

    Hoel, Erik P; Albantakis, Larissa; Cirelli, Chiara; Tononi, Giulio

    2016-04-01

    Recent evidence suggests that synaptic refinement, the reorganization of synapses and connections without significant change in their number or strength, is important for the development of the visual system of juvenile rodents. Other evidence in rodents and humans shows that there is a marked drop in sleep slow-wave activity (SWA) during adolescence. Slow waves reflect synchronous transitions of neuronal populations between active and inactive states, and the amount of SWA is influenced by the connection strength and organization of cortical neurons. In this study, we investigated whether synaptic refinement could account for the observed developmental drop in SWA. To this end, we employed a large-scale neural model of primary visual cortex and sections of the thalamus, capable of producing realistic slow waves. In this model, we reorganized intralaminar connections according to experimental data on synaptic refinement: during prerefinement, local connections between neurons were homogenous, whereas in postrefinement, neurons connected preferentially to neurons with similar receptive fields and preferred orientations. Synaptic refinement led to a drop in SWA and to changes in slow-wave morphology, consistent with experimental data. To test whether learning can induce synaptic refinement, intralaminar connections were equipped with spike timing-dependent plasticity. Oriented stimuli were presented during a learning period, followed by homeostatic synaptic renormalization. This led to activity-dependent refinement accompanied again by a decline in SWA. Together, these modeling results show that synaptic refinement can account for developmental changes in SWA. Thus sleep SWA may be used to track noninvasively the reorganization of cortical connections during development. PMID:26843602

  7. Glucose is necessary to maintain neurotransmitter homeostasis during synaptic activity in cultured glutamatergic neurons

    Bak, Lasse K; Schousboe, Arne; Sonnewald, Ursula;

    2006-01-01

    Glucose is the primary energy substrate for the adult mammalian brain. However, lactate produced within the brain might be able to serve this purpose in neurons. In the present study, the relative significance of glucose and lactate as substrates to maintain neurotransmitter homeostasis was...... was unaffected by the choice of substrate. In conclusion, the present study shows that glucose is a necessary substrate to maintain neurotransmitter homeostasis during synaptic activity and that synaptic activity does not induce an upregulation of lactate metabolism in glutamatergic neurons....

  8. Activity-dependent synaptic plasticity modulates the critical phase of brain development.

    Chaudhury, Sraboni; Sharma, Vikram; Kumar, Vivek; Nag, Tapas C; Wadhwa, Shashi

    2016-04-01

    Plasticity or neuronal plasticity is a unique and adaptive feature of nervous system which allows neurons to reorganize their interactions in response to an intrinsic or extrinsic stimulation and shapes the formation and maintenance of a functional neuronal circuit. Synaptic plasticity is the most important form of neural plasticity and plays critical role during the development allowing the formation of precise neural connectivity via the process of pruning. In the sensory systems-auditory and visual, this process is heavily dependent on the external cues perceived during the development. Environmental enrichment paradigms in an activity-dependent manner result in early maturation of the synapses and more efficient trans-synaptic signaling or communication flow. This has been extensively observed in the avian auditory system. On the other hand, stimuli results in negative effect can cause alterations in the synaptic connectivity and strength resulting in various developmental brain disorders including autism, fragile X syndrome and rett syndrome. In this review we discuss the role of different forms of activity (spontaneous or environmental) during the development of the nervous system in modifying synaptic plasticity necessary for shaping the adult brain. Also, we try to explore various factors (molecular, genetic and epigenetic) involved in altering the synaptic plasticity in positive and negative way. PMID:26515724

  9. Effects of active conductance distribution over dendrites on the synaptic integration in an identified nonspiking interneuron.

    Akira Takashima

    Full Text Available The synaptic integration in individual central neuron is critically affected by how active conductances are distributed over dendrites. It has been well known that the dendrites of central neurons are richly endowed with voltage- and ligand-regulated ion conductances. Nonspiking interneurons (NSIs, almost exclusively characteristic to arthropod central nervous systems, do not generate action potentials and hence lack voltage-regulated sodium channels, yet having a variety of voltage-regulated potassium conductances on their dendritic membrane including the one similar to the delayed-rectifier type potassium conductance. It remains unknown, however, how the active conductances are distributed over dendrites and how the synaptic integration is affected by those conductances in NSIs and other invertebrate neurons where the cell body is not included in the signal pathway from input synapses to output sites. In the present study, we quantitatively investigated the functional significance of active conductance distribution pattern in the spatio-temporal spread of synaptic potentials over dendrites of an identified NSI in the crayfish central nervous system by computer simulation. We systematically changed the distribution pattern of active conductances in the neuron's multicompartment model and examined how the synaptic potential waveform was affected by each distribution pattern. It was revealed that specific patterns of nonuniform distribution of potassium conductances were consistent, while other patterns were not, with the waveform of compound synaptic potentials recorded physiologically in the major input-output pathway of the cell, suggesting that the possibility of nonuniform distribution of potassium conductances over the dendrite cannot be excluded as well as the possibility of uniform distribution. Local synaptic circuits involving input and output synapses on the same branch or on the same side were found to be potentially affected under

  10. Synapse geometry and receptor dynamics modulate synaptic strength.

    Dominik Freche

    Full Text Available Synaptic transmission relies on several processes, such as the location of a released vesicle, the number and type of receptors, trafficking between the postsynaptic density (PSD and extrasynaptic compartment, as well as the synapse organization. To study the impact of these parameters on excitatory synaptic transmission, we present a computational model for the fast AMPA-receptor mediated synaptic current. We show that in addition to the vesicular release probability, due to variations in their release locations and the AMPAR distribution, the postsynaptic current amplitude has a large variance, making a synapse an intrinsic unreliable device. We use our model to examine our experimental data recorded from CA1 mice hippocampal slices to study the differences between mEPSC and evoked EPSC variance. The synaptic current but not the coefficient of variation is maximal when the active zone where vesicles are released is apposed to the PSD. Moreover, we find that for certain type of synapses, receptor trafficking can affect the magnitude of synaptic depression. Finally, we demonstrate that perisynaptic microdomains located outside the PSD impacts synaptic transmission by regulating the number of desensitized receptors and their trafficking to the PSD. We conclude that geometrical modifications, reorganization of the PSD or perisynaptic microdomains modulate synaptic strength, as the mechanisms underlying long-term plasticity.

  11. Prenatal immune activation causes hippocampal synaptic deficits in the absence of overt microglia anomalies.

    Giovanoli, Sandra; Weber-Stadlbauer, Ulrike; Schedlowski, Manfred; Meyer, Urs; Engler, Harald

    2016-07-01

    Prenatal exposure to infectious or inflammatory insults can increase the risk of developing neuropsychiatric disorder in later life, including schizophrenia, bipolar disorder, and autism. These brain disorders are also characterized by pre- and postsynaptic deficits. Using a well-established mouse model of maternal exposure to the viral mimetic polyriboinosinic-polyribocytidilic acid [poly(I:C)], we examined whether prenatal immune activation might cause synaptic deficits in the hippocampal formation of pubescent and adult offspring. Based on the widely appreciated role of microglia in synaptic pruning, we further explored possible associations between synaptic deficits and microglia anomalies in offspring of poly(I:C)-exposed and control mothers. We found that prenatal immune activation induced an adult onset of presynaptic hippocampal deficits (as evaluated by synaptophysin and bassoon density). The early-life insult further caused postsynaptic hippocampal deficits in pubescence (as evaluated by PSD95 and SynGAP density), some of which persisted into adulthood. In contrast, prenatal immune activation did not change microglia (or astrocyte) density, nor did it alter their activation phenotypes. The prenatal manipulation did also not cause signs of persistent systemic inflammation. Despite the absence of overt glial anomalies or systemic inflammation, adult offspring exposed to prenatal immune activation displayed increased hippocampal IL-1β levels. Taken together, our findings demonstrate that age-dependent synaptic deficits and abnormal pro-inflammatory cytokine expression can occur during postnatal brain maturation in the absence of microglial anomalies or systemic inflammation. PMID:26408796

  12. CaMKII Activity in the Ventral Tegmental Area Gates Cocaine-Induced Synaptic Plasticity in the Nucleus Accumbens

    Liu, Xiaojie; Liu, Yong; Zhong, Peng; Wilkinson, Brianna; Qi, Jinshun; Olsen, Christopher M; Bayer, K. Ulrich; Liu, Qing-song

    2013-01-01

    Addictive drugs such as cocaine induce synaptic plasticity in discrete regions of the reward circuit. The aim of the present study is to investigate whether cocaine-evoked synaptic plasticity in the ventral tegmental area (VTA) and nucleus accumbens (NAc) is causally linked. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a central regulator of long-term synaptic plasticity, learning, and drug addiction. We examined whether blocking CaMKII activity in the VTA affected cocaine conditio...

  13. Cholinergic-mediated IP3-receptor activation induces long-lasting synaptic enhancement in CA1 pyramidal neurons

    Fernández de Sevilla, D.; Núñez Molina, Ángel; Borde, M.; Malinow, R.; Buño, Washinton

    2008-01-01

    Cholinergic-glutamatergic interactions influence forms of synaptic plasticity that are thought to mediate memory and learning. We tested in vitro the induction of long-lasting synaptic enhancement at Schaffer collaterals by acetylcholine (ACh) at the apical dendrite of CA1 pyramidal neurons and in vivo by stimulation of cholinergic afferents. In vitro ACh induced a Ca2+ wave and synaptic enhancement mediated by insertion of AMPA receptors in spines. Activation of muscarinic ACh receptors (mAC...

  14. A trans-synaptic nanocolumn aligns neurotransmitter release to receptors.

    Tang, Ai-Hui; Chen, Haiwen; Li, Tuo P; Metzbower, Sarah R; MacGillavry, Harold D; Blanpied, Thomas A

    2016-08-11

    Synaptic transmission is maintained by a delicate, sub-synaptic molecular architecture, and even mild alterations in synapse structure drive functional changes during experience-dependent plasticity and pathological disorders. Key to this architecture is how the distribution of presynaptic vesicle fusion sites corresponds to the position of receptors in the postsynaptic density. However, while it has long been recognized that this spatial relationship modulates synaptic strength, it has not been precisely described, owing in part to the limited resolution of light microscopy. Using localization microscopy, here we show that key proteins mediating vesicle priming and fusion are mutually co-enriched within nanometre-scale subregions of the presynaptic active zone. Through development of a new method to map vesicle fusion positions within single synapses in cultured rat hippocampal neurons, we find that action-potential-evoked fusion is guided by this protein gradient and occurs preferentially in confined areas with higher local density of Rab3-interacting molecule (RIM) within the active zones. These presynaptic RIM nanoclusters closely align with concentrated postsynaptic receptors and scaffolding proteins, suggesting the existence of a trans-synaptic molecular 'nanocolumn'. Thus, we propose that the nanoarchitecture of the active zone directs action-potential-evoked vesicle fusion to occur preferentially at sites directly opposing postsynaptic receptor-scaffold ensembles. Remarkably, NMDA receptor activation triggered distinct phases of plasticity in which postsynaptic reorganization was followed by trans-synaptic nanoscale realignment. This architecture suggests a simple organizational principle of central nervous system synapses to maintain and modulate synaptic efficiency. PMID:27462810

  15. Long lasting protein synthesis- and activity-dependent spine shrinkage and elimination after synaptic depression.

    Yazmín Ramiro-Cortés

    Full Text Available Neuronal circuits modify their response to synaptic inputs in an experience-dependent fashion. Increases in synaptic weights are accompanied by structural modifications, and activity dependent, long lasting growth of dendritic spines requires new protein synthesis. When multiple spines are potentiated within a dendritic domain, they show dynamic structural plasticity changes, indicating that spines can undergo bidirectional physical modifications. However, it is unclear whether protein synthesis dependent synaptic depression leads to long lasting structural changes. Here, we investigate the structural correlates of protein synthesis dependent long-term depression (LTD mediated by metabotropic glutamate receptors (mGluRs through two-photon imaging of dendritic spines on hippocampal pyramidal neurons. We find that induction of mGluR-LTD leads to robust and long lasting spine shrinkage and elimination that lasts for up to 24 hours. These effects depend on signaling through group I mGluRs, require protein synthesis, and activity. These data reveal a mechanism for long lasting remodeling of synaptic inputs, and offer potential insights into mental retardation.

  16. Activity-dependent synaptic plasticity of a chalcogenide electronic synapse for neuromorphic systems.

    Li, Yi; Zhong, Yingpeng; Zhang, Jinjian; Xu, Lei; Wang, Qing; Sun, Huajun; Tong, Hao; Cheng, Xiaoming; Miao, Xiangshui

    2014-01-01

    Nanoscale inorganic electronic synapses or synaptic devices, which are capable of emulating the functions of biological synapses of brain neuronal systems, are regarded as the basic building blocks for beyond-Von Neumann computing architecture, combining information storage and processing. Here, we demonstrate a Ag/AgInSbTe/Ag structure for chalcogenide memristor-based electronic synapses. The memristive characteristics with reproducible gradual resistance tuning are utilised to mimic the activity-dependent synaptic plasticity that serves as the basis of memory and learning. Bidirectional long-term Hebbian plasticity modulation is implemented by the coactivity of pre- and postsynaptic spikes, and the sign and degree are affected by assorted factors including the temporal difference, spike rate and voltage. Moreover, synaptic saturation is observed to be an adjustment of Hebbian rules to stabilise the growth of synaptic weights. Our results may contribute to the development of highly functional plastic electronic synapses and the further construction of next-generation parallel neuromorphic computing architecture. PMID:24809396

  17. ACTIVE CALCIUM TRANSPORT IN PLASMA MEMBRANE VESICLES FROM DEVELOPING COTYLEDONS OF COMMON BEAN

    黄建中; 陈子元

    1995-01-01

    Plasma membrane vesicles were prepared from the developing cotyledons of common bean (Phaseolus vulgaris L cv Diyundou)by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes.The putative plasma membrane fraction was minimalyy contaminated by membranes other than plasma membrane and hence was of high purity,It exhibited a Ca2+-dependent ATPase activity,which was inhibited by 1umol/L EB and promoted by calcium ionophore A23187.Such an activity was responsible for the observed ATP dependent 45Ca2+ uptake into inside-out plasma membrane vesicles.This process was stimulated by 0.5μmol/L CaM and 20μmol/L IAA but inhibited by 2μmol/L ABA and abolished by A23187,Possible role of cytoplasmic Ca2+ in mediating phytohormones activity is discussed.

  18. Active calcium transport in plasma membrane vesicles from developing cotyledons of common bean

    Plasma membrane vesicles were prepared from the developing cotyledons of common bean (Phaseolus vulgaris L cv Diyundou) by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes. The putative plasma membrane fraction was minimally contaminated by membranes other than plasma membrane and hence was of high purity. It exhibited a Ca2+-dependent ATPase activity, which was inhibited by 1 μmol/L EB and promoted by calcium ionophore A23187. Such an activity was responsible for the observed ATP-dependent 45Ca2+ uptake into inside-out plasma membrane vesicles. This process was stimulated by 0.6 μmol/L CaM and 20 μmol/L IAA but inhibited by 2 μmol/L ABA and abolished by A23187. Possible role of cytoplasmic Ca2+ in mediating phytohormones activity is discussed

  19. Enhanced Synaptic Transmission at the Squid Giant Synapse by Artificial Seawater Based on Physically Modified Saline

    Kerry D Walton

    2014-02-01

    Full Text Available Superfusion of the squid giant synapse with artificial seawater (ASW based on isotonic saline containing oxygen nanobubbles (RNS60 ASW generates an enhancement of synaptic transmission. This was determined by examining the postsynaptic response to single and repetitive presynaptic spike activation, spontaneous transmitter release, and presynaptic voltage clamp studies. In the presence of RNS60 ASW single presynaptic stimulation elicited larger postsynaptic potentials (PSP and more robust recovery from high frequency stimulation than in control ASW. Analysis of postsynaptic noise revealed an increase in spontaneous transmitter release with modified noise kinetics in RNS60 ASW. Presynaptic voltage clamp demonstrated an increased EPSP, without an increase in presynaptic ICa⁺⁺ amplitude during RNS60 ASW superfusion. Synaptic release enhancement reached stable maxima within 5 to 10 minutes of RNS60 ASW superfusion and was maintained for the entire recording time, up to one hour. Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles and large endosome-like vesicles near junctional sites. Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW. After ATP block the number of vesicles at the active zone and clathrin-coated vesicles was reduced, with an increase in large vesicles. The possibility that RNS60 ASW acts by increasing mitochondrial ATP synthesis was tested by direct determination of ATP levels in both presynaptic and postsynaptic structures. This was implemented using luciferin/luciferase photon emission, which demonstrated a marked increase in ATP synthesis following RNS60 administration. It is concluded that RNS60 positively modulates synaptic transmission by up-regulating ATP synthesis, thus leading to synaptic

  20. Calcium-sensing receptor activation depresses synaptic transmission

    Phillips, Cecilia G.; Harnett, Mark T.; Chen, Wenyan; Smith, Stephen M.

    2008-01-01

    At excitatory synapses, decreases in cleft [Ca] arising from activity-dependent transmembrane Ca flux reduce the probability of subsequent transmitter release. Intense neural activity, induced by physiological and pathological stimuli, disturb the external microenvironment reducing extracellular [Ca] ([Ca]o) and thus may impair neurotransmission. Increases in [Ca]o activate the extracellular calcium sensing receptor (CaSR) which in turn inhibits non-selective cation channels (NSCC) at the maj...

  1. Similar oxysterols may lead to opposite effects on synaptic transmission: Olesoxime versus 5α-cholestan-3-one at the frog neuromuscular junction.

    Kasimov, M R; Zakyrjanova, G F; Giniatullin, A R; Zefirov, A L; Petrov, A M

    2016-07-01

    Cholesterol oxidation products frequently have a high biological activity. In the present study, we have used microelectrode recording of end plate currents and FM-based optical detection of synaptic vesicle exo-endocytosis to investigate the effects of two structurally similar oxysterols, olesoxime (cholest-4-en-3-one, oxime) and 5ɑ-cholestan-3-one (5ɑCh3), on neurotransmission at the frog neuromuscular junction. Olesoxime is an exogenous, potentially neuroprotective, substance and 5ɑCh3 is an intermediate product in cholesterol metabolism, which is elevated in the case of cerebrotendinous xanthomatosis. We found that olesoxime slightly increased evoked neurotransmitter release in response to a single stimulus and significantly reduced synaptic depression during high frequency activity. The last effect was due to an increase in both the number of synaptic vesicles involved in exo-endocytosis and the rate of synaptic vesicle recycling. In contrast, 5ɑCh3 reduced evoked neurotransmitter release during the low- and high frequency synaptic activities. The depressant action of 5ɑCh3 was associated with a reduction in the number of synaptic vesicles participating in exo- and endocytosis during high frequency stimulation, without a change in rate of the synaptic vesicle recycling. Of note, olesoxime increased the staining of synaptic membranes with the B-subunit of cholera toxin and the formation of fluorescent ganglioside GM1 clusters, and decreased the fluorescence of 22-NBD-cholesterol, while 5ɑCh3 had the opposite effects, suggesting that the two oxysterols have different effects on lipid raft stability. Taken together, these data show that these two structurally similar oxysterols induce marked different changes in neuromuscular transmission which are related with the alteration in synaptic vesicle cycle. PMID:27102612

  2. Mutation in AP-3 delta in the mocha mouse links endosomal transport to storage deficiency in platelets, melanosomes, and synaptic vesicles.

    Kantheti, P; Qiao, X; Diaz, M E; Peden, A A; Meyer, G E; Carskadon, S L; Kapfhamer, D; Sufalko, D; Robinson, M S; Noebels, J L; Burmeister, M

    1998-07-01

    The mouse mutant mocha, a model for the Hermansky-Pudlak storage pool deficiency syndrome, is characterized by defective platelets, coat and eye color dilution, lysosomal abnormalities, inner ear degeneration, and neurological deficits. Here, we show that mocha is a null allele of the delta subunit of the adaptor-like protein complex AP-3, which is associated with coated vesicles budding from the trans-Golgi network, and that AP-3 is missing in mocha tissues. In mocha brain, the ZnT-3 transporter is reduced, resulting in a lack of zinc-associated Timm historeactivity in hippocampal mossy fibers. Our results demonstrate that the AP-3 complex is responsible for cargo selection to lysosome-related organelles such as melanosomes and platelet dense granules as well as to neurotransmitter vesicles. PMID:9697856

  3. Synaptic development in the injured spinal cord cavity following co-transplantation of fetal spinal cord cells and autologous activated Schwann cells

    Wendong Ruan; Yuan Xue; Ninghua Li; Xiaotao Zhao; Huajian Zhao; Peng Li

    2010-01-01

    Transplantation of activated transgenic Schwann cells or a fetal spinal cord cell suspension has been widely used to treat spinal cord injury. However, little is known regarding the effects of co-transplantation. In the present study, autologous Schwann cells in combination with a fetal spinal cord cell suspension were transplanted into adult Wistar rats with spinal cord injury, and newly generated axonal connections were observed ultrastructurally. Transmission electron microscopic observations showed that the neuroblast first presented cytoplasmic processes, followed by pre- and postsynaptic membranes with low electron density forming a dense projection. The number and types of synaptic vesicles were increased. Synaptic connections developed from single cell body-dendritic synapses into multiple cell body-dendritic anddendrite-dendritic synapses. In addition, the cell organs of the transplanted neuroblast, oligodendroblast and astroblast matured gradually. The blood-brain barrier appeared subsequently. Moreover, neurofilament, histamine, calcitonin-gene-related peptides, and glial fibrillary acidic protein positive fibers were observed in the transplant region. These findings demonstrate that fetal spinal cord cells in the presence of autologous activated Schwann cells can develop into mature synapses in the cavity of injured spinal cords, suggesting the possibility of information exchange through the reconstructed synapse between fetal spinal cord cells and the host.

  4. SOFT MALLEABLE VESICLES TAILORED FOR ENHANCED DELIVERY OF ACTIVE AGENTS THROUGH THE SKIN: AN UPDATE

    Sandeep Kumar Parihar*, Mithun Bhowmick, Rajeev Kumar and Balkrishna Dubey

    2013-01-01

    Ethosomes are noninvasive delivery carriers that enable drugs to reach the deep skin layers and/or the systemic circulation. These are soft, malleable vesicles tailored for enhanced delivery of active agents. They are composed mainly of phospholipids, high concentration of ethanol and water. The high concentration of ethanol makes the ethosomes unique, as ethanol is known for its disturbance of skin lipid bilayer organization; therefore, when integrated into ...

  5. NMDA-receptor activation but not ion flux is required for amyloid-beta induced synaptic depression.

    Albert Tamburri

    Full Text Available Alzheimer disease is characterized by a gradual decrease of synaptic function and, ultimately, by neuronal loss. There is considerable evidence supporting the involvement of oligomeric amyloid-beta (Aβ in the etiology of Alzheimer's disease. Historically, AD research has mainly focused on the long-term changes caused by Aβ rather than analyzing its immediate effects. Here we show that acute perfusion of hippocampal slice cultures with oligomeric Aβ depresses synaptic transmission within 20 minutes. This depression is dependent on synaptic stimulation and the activation of NMDA-receptors, but not on NMDA-receptor mediated ion flux. It, therefore, appears that Aβ dependent synaptic depression is mediated through a use-dependent metabotropic-like mechanism of the NMDA-receptor, but does not involve NMDA-receptor mediated synaptic transmission, i.e. it is independent of calcium flux through the NMDA-receptor.

  6. Importance of being Nernst: Synaptic activity andfunctional relevance in stem cell-derived neurons

    2015-01-01

    Functional synaptogenesis and network emergence aresignature endpoints of neurogenesis. These behaviorsprovide higher-order confirmation that biochemicaland cellular processes necessary for neurotransmitterrelease, post-synaptic detection and network propagation of neuronal activity have been properly expressed andcoordinated among cells. The development of synapticneurotransmission can therefore be considered a definingproperty of neurons. Although dissociated primaryneuron cultures readily form functioning synapsesand network behaviors in vitro , continuously culturedneurogenic cell lines have historically failed to meet thesecriteria. Therefore, in vitro -derived neuron models thatdevelop synaptic transmission are critically needed for awide array of studies, including molecular neuroscience,developmental neurogenesis, disease research andneurotoxicology. Over the last decade, neurons derivedfrom various stem cell lines have shown varying ability todevelop into functionally mature neurons. In this review,we will discuss the neurogenic potential of various stemcells populations, addressing strengths and weaknessesof each, with particular attention to the emergenceof functional behaviors. We will propose methods tofunctionally characterize new stem cell-derived neuron(SCN) platforms to improve their reliability as physiologicalrelevant models. Finally, we will review howsynaptically active SCNs can be applied to accelerateresearch in a variety of areas. Ultimately, emphasizingthe critical importance of synaptic activity and networkresponses as a marker of neuronal maturation is anticipatedto result in in vitro findings that better translateto efficacious clinical treatments.

  7. The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection.

    Dieudé, Mélanie; Bell, Christina; Turgeon, Julie; Beillevaire, Deborah; Pomerleau, Luc; Yang, Bing; Hamelin, Katia; Qi, Shijie; Pallet, Nicolas; Béland, Chanel; Dhahri, Wahiba; Cailhier, Jean-François; Rousseau, Matthieu; Duchez, Anne-Claire; Lévesque, Tania; Lau, Arthur; Rondeau, Christiane; Gingras, Diane; Muruve, Danie; Rivard, Alain; Cardinal, Héloise; Perreault, Claude; Desjardins, Michel; Boilard, Éric; Thibault, Pierre; Hébert, Marie-Josée

    2015-12-16

    Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. PMID:26676607

  8. Synaptic and functional linkages between spinal premotor interneurons and hand-muscle activity during precision grip

    Tomohiko Takei

    2013-04-01

    Full Text Available Grasping is a highly complex movement that requires the coordination of a number of hand joints and muscles. Previous studies showed that spinal premotor interneurons (PreM-INs in the primate cervical spinal cord have divergent synaptic effects on hand motoneurons and that they might contribute to hand-muscle synergies. However, the extent to which these PreM-IN synaptic connections functionally contribute to modulating hand-muscle activity is not clear. In this paper, we explored the contribution of spinal PreM-INs to hand-muscle activation by quantifying the synaptic linkage (SL and functional linkage (FL of the PreM-INs with hand-muscle activities. The activity of 23 PreM-INs was recorded from the cervical spinal cord (C6–T1, with EMG signals measured simultaneously from hand and arm muscles in two macaque monkeys performing a precision grip task. Spike-triggered averages (STAs of rectified EMGs were compiled for 456 neuron–muscle pairs; 63 pairs showed significant post-spike effects (i.e., SL. Conversely, 231 of 456 pairs showed significant cross-correlations between the IN firing rate and rectified EMG (i.e., FL. Importantly, a greater proportion of the neuron–muscle pairs with SL showed FL (43/63 pairs, 68% compared with the pairs without SL (203/393, 52%, and the presence of SL was significantly associated with that of FL. However, a significant number of pairs had SL without FL (SL∩!FL, n = 20 or FL without SL (!SL∩FL, n = 203, and the proportions of these incongruities exceeded the number expected by chance. These results suggested that spinal PreM-INs function to significantly modulate hand-muscle activity during precision grip, but the contribution of other neural structures is also needed to recruit an adequate combination of hand-muscle motoneurons.

  9. Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting.

    Higginbotham, James N; Zhang, Qin; Jeppesen, Dennis K; Scott, Andrew M; Manning, H Charles; Ochieng, Josiah; Franklin, Jeffrey L; Coffey, Robert J

    2016-01-01

    Exosomes are small, 40-130 nm secreted extracellular vesicles that recently have become the subject of intense focus as agents of intercellular communication, disease biomarkers and potential vehicles for drug delivery. It is currently unknown whether a cell produces different populations of exosomes with distinct cargo and separable functions. To address this question, high-resolution methods are needed. Using a commercial flow cytometer and directly labelled fluorescent antibodies, we show the feasibility of using fluorescence-activated vesicle sorting (FAVS) to analyse and sort individual exosomes isolated by sequential ultracentrifugation from the conditioned medium of DiFi cells, a human colorectal cancer cell line. EGFR and the exosomal marker, CD9, were detected on individual DiFi exosomes by FAVS; moreover, both markers were identified by high-resolution stochastic optical reconstruction microscopy on individual, approximately 100 nm vesicles from flow-sorted EGFR/CD9 double-positive exosomes. We present evidence that the activation state of EGFR can be assessed in DiFi-derived exosomes using a monoclonal antibody (mAb) that recognizes "conformationally active" EGFR (mAb 806). Using human antigen-specific antibodies, FAVS was able to detect human EGFR and CD9 on exosomes isolated from the plasma of athymic nude mice bearing DiFi tumour xenografts. Multicolour FAVS was used to simultaneously identify CD9, EGFR and an EGFR ligand, amphiregulin (AREG), on human plasma-derived exosomes from 3 normal individuals. These studies demonstrate the feasibility of FAVS to both analyse and sort individual exosomes based on specific cell-surface markers. We propose that FAVS may be a useful tool to monitor EGFR and AREG in circulating exosomes from individuals with colorectal cancer and possibly other solid tumours. PMID:27345057

  10. Astrocytic vesicles and gliotransmitters: Slowness of vesicular release and synaptobrevin2-laden vesicle nanoarchitecture.

    Zorec, R; Verkhratsky, A; Rodríguez, J J; Parpura, V

    2016-05-26

    Neurotransmitters released at synapses activate neighboring astrocytes, which in turn, modulate neuronal activity by the release of diverse neuroactive substances that include classical neurotransmitters such as glutamate, GABA or ATP. Neuroactive substances are released from astrocytes through several distinct molecular mechanisms, for example, by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release regulated by a stimulus-mediated increase in cytosolic calcium involves soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE)-dependent merger of the vesicle membrane with the plasmalemma. Up to 25 molecules of synaptobrevin 2 (Sb2), a SNARE complex protein, reside at a single astroglial vesicle; an individual neuronal, i.e. synaptic, vesicle contains ∼70 Sb2 molecules. It is proposed that this paucity of Sb2 molecules in astrocytic vesicles may determine the slow secretion. In the present essay we shall overview multiple aspects of vesicular architecture and types of vesicles based on their cargo and dynamics in astroglial cells. PMID:25727638

  11. Hemoglobin-Vesicles as Oxygen Carriers : Influence on Phagocytic Activity and Histopathological Changes in Reticuloendothelial System

    Sakai, Hiromi; Horinouchi, Hirohisa; Tomiyama, Kenichi; IKEDA, EIJI; Takeoka, Shinji; Kobayashi, Koichi; Tsuchida, Eishun

    2001-01-01

    Hemoglobin-vesicles (HbV) have been developed for use as artificial oxygen carriers (particle diameter, 250 nm) in which a purified Hb solution is encapsulated with a phospholipid bilayer membrane. The influence of HbV on the reticuloendothelial system was studied by carbon clearance measurements and histopathological examination. The HbV suspension ([Hb] = 10 g/dl) was intravenously infused in male Wistar rats at dose rates of 10 and 20 ml/kg, and the phagocytic activity was measured by moni...

  12. Destruction of giant cluster-like vesicles by an ultrasonically activated device

    Yahagi, Ryosuke; Yoshida, Kenji; Zhang, Yiting; Ebata, Masahiko; Toyota, Taro; Yamaguchi, Tadashi; Hayashi, Hideki

    2016-07-01

    In this paper, we propose a technically simple method of destroying a tissue marker composed of giant cluster-like vesicles (GCVs) to facilitate laparoscopic surgeries; the method releases various biological tracers contained in GCVs. An ultrasonically activated device (USAD) emitting 55.5 kHz ultrasound was employed for this purpose. Optical microscopy and fluorospectrophotometry revealed the destruction of GCVs after ultrasound irradiation when the blade tip was set 1.0 mm or closer to, but not directly in contact with, a GCV-containing cell. This means that USAD could be safely used for destroying this GCV tissue marker in clinical settings.

  13. Vesicle Photonics

    Vasdekis, Andreas E.; Scott, E. A.; Roke, Sylvie; Hubbell, J. A.; Psaltis, D.

    2013-04-03

    Thin membranes, under appropriate boundary conditions, can self-assemble into vesicles, nanoscale bubbles that encapsulate and hence protect or transport molecular payloads. In this paper, we review the types and applications of light fields interacting with vesicles. By encapsulating light-emitting molecules (e.g. dyes, fluorescent proteins, or quantum dots), vesicles can act as particles and imaging agents. Vesicle imaging can take place also under second harmonic generation from vesicle membrane, as well as employing mass spectrometry. Light fields can also be employed to transport vesicles using optical tweezers (photon momentum) or directly pertrurbe the stability of vesicles and hence trigger the delivery of the encapsulated payload (photon energy).

  14. Aurora A drives early signalling and vesicle dynamics during T-cell activation

    Blas-Rus, Noelia; Bustos-Morán, Eugenio; Pérez de Castro, Ignacio; de Cárcer, Guillermo; Borroto, Aldo; Camafeita, Emilio; Jorge, Inmaculada; Vázquez, Jesús; Alarcón, Balbino; Malumbres, Marcos; Martín-Cófreces, Noa B.; Sánchez-Madrid, Francisco

    2016-01-01

    Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal. PMID:27091106

  15. Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation

    Komeili, Arash; Vali, Hojatollah; Beveridge, Terrance J.; Newman, Dianne K.

    2004-01-01

    Bacterial magnetosomes are intracellular compartments that house highly ordered magnetite crystals. By using Magnetospirillum sp. AMB-1 as a model system, we show that magnetosome vesicles exist in the absence of magnetite, biomineralization of magnetite proceeds simultaneously in multiple vesicles, and biomineralization proceeds from the same location in each vesicle. The magnetosome-associated protein, MamA, is required for the formation of functional magnetosome vesicles and displays a dyn...

  16. Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting

    Higginbotham, James N.; Zhang, Qin; Jeppesen, Dennis K.; Scott, Andrew M.; Manning, H. Charles; Ochieng, Josiah; Franklin, Jeffrey L.; Coffey, Robert J.

    2016-01-01

    Exosomes are small, 40–130 nm secreted extracellular vesicles that recently have become the subject of intense focus as agents of intercellular communication, disease biomarkers and potential vehicles for drug delivery. It is currently unknown whether a cell produces different populations of exosomes with distinct cargo and separable functions. To address this question, high-resolution methods are needed. Using a commercial flow cytometer and directly labelled fluorescent antibodies, we show the feasibility of using fluorescence-activated vesicle sorting (FAVS) to analyse and sort individual exosomes isolated by sequential ultracentrifugation from the conditioned medium of DiFi cells, a human colorectal cancer cell line. EGFR and the exosomal marker, CD9, were detected on individual DiFi exosomes by FAVS; moreover, both markers were identified by high-resolution stochastic optical reconstruction microscopy on individual, approximately 100 nm vesicles from flow-sorted EGFR/CD9 double-positive exosomes. We present evidence that the activation state of EGFR can be assessed in DiFi-derived exosomes using a monoclonal antibody (mAb) that recognizes “conformationally active” EGFR (mAb 806). Using human antigen-specific antibodies, FAVS was able to detect human EGFR and CD9 on exosomes isolated from the plasma of athymic nude mice bearing DiFi tumour xenografts. Multicolour FAVS was used to simultaneously identify CD9, EGFR and an EGFR ligand, amphiregulin (AREG), on human plasma-derived exosomes from 3 normal individuals. These studies demonstrate the feasibility of FAVS to both analyse and sort individual exosomes based on specific cell-surface markers. We propose that FAVS may be a useful tool to monitor EGFR and AREG in circulating exosomes from individuals with colorectal cancer and possibly other solid tumours. PMID:27345057

  17. Investigation of nano lipid vesicles of methotrexate for anti-rheumatoid activity

    Prabhu P

    2012-01-01

    Full Text Available Prabhakara Prabhu1, Rakshith Shetty1, Marina Koland1, K Vijayanarayana3, KK Vijayalakshmi2, M Harish Nairy1, GS Nisha11Department of Pharmaceutics, Nitte University, NGSM Institute of Pharmaceutical Sciences, Paneer, Deralakatte, Mangalore, Karnataka, India; 2Department of Applied Zoology, Mangalore University, Konaje, Mangalore, Karnataka, India; 3Department of Pharmacy Practice, Manipal University, Manipal College of Pharmaceutical Sciences, Manipal, Karnataka, IndiaBackground: The purpose of this study was to formulate and evaluate nano lipid vesicles of methotrexate (MTX for its anti-rheumatoid activity.Methods: In this study the principle of both active as well as passive targeting using MTX-loaded stealth liposomes as per the magic gun approach was followed. Stealth liposomes of MTX were prepared by thin-film hydration method using a PEGylated phospholipid-like DSPE-MPEG 2000. Similarly, conventional liposomes were prepared using phospholipids like DPPC and DSPC. Conventional liposomes were coated with a hydrophilic biocompatible polymer like chitosan. They were investigated for their physical properties and in vitro release profile. Further, in vivo screening of the formulations for their anti-rheumatoid efficacy was carried out in rats. Rheumatoid arthritis was induced in male Wistar-Lewis rats using complete Freund’s adjuvant (1 mg/mL Mycobacterium tuberculosis, heat killed in mineral oil.Results: It was found that chitosan coating of the conventional liposomes increased the physical stability of the liposomal suspension as well as its entrapment efficiency. The size of the unsonicated lipid vesicles was found to be in the range of 8–10 µm, and the sonicated lipid vesicles in the range of 210–260 nm, with good polydispersity index. Further, chitosan-coated conventional liposomes and the PEGylated liposomes released the drug for a prolonged period of time, compared to the uncoated conventional liposomes. It was found that there

  18. Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-01-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

  19. Docking of secretory vesicles is syntaxin dependent.

    Heidi de Wit

    Full Text Available Secretory vesicles dock at the plasma membrane before they undergo fusion. Molecular docking mechanisms are poorly defined but believed to be independent of SNARE proteins. Here, we challenged this hypothesis by acute deletion of the target SNARE, syntaxin, in vertebrate neurons and neuroendocrine cells. Deletion resulted in fusion arrest in both systems. No docking defects were observed in synapses, in line with previous observations. However, a drastic reduction in morphologically docked secretory vesicles was observed in chromaffin cells. Syntaxin-deficient chromaffin cells showed a small reduction in total and plasma membrane staining for the docking factor Munc18-1, which appears insufficient to explain the drastic reduction in docking. The sub-membrane cortical actin network was unaffected by syntaxin deletion. These observations expose a docking role for syntaxin in the neuroendocrine system. Additional layers of regulation may have evolved to make syntaxin redundant for docking in highly specialized systems like synaptic active zones.

  20. Effect of vanadate on glucose transporter (GLUT4) intrinsic activity in skeletal muscle plasma membrane giant vesicles

    Kristiansen, S; Youn, J; Richter, Erik

    1996-01-01

    vanadate (NaVO3) on glucose transporter (GLUT4) intrinsic activity (V(max) = intrinsic activity x [GLUT4 protein]) was studied in muscle plasma membrane giant vesicles. Giant vesicles (average diameter 7.6 microns) were produced by collagenase treatment of rat skeletal muscle. The vesicles were incubated......) 55% and 60%, respectively, compared with control. The plasma membrane GLUT4 protein content was not changed in response to vanadate. It is concluded that vanadate decreased glucose transport per GLUT4 (intrinsic activity). This finding suggests that regulation of glucose transport in skeletal muscle......Maximally effective concentrations of vanadate (a phosphotyrosine phosphatase inhibitor) increase glucose transport in muscle less than maximal insulin stimulation. This might be due to vanadate-induced decreased intrinsic activity of GLUT4 accompanying GLUT4 translocation. Thus, the effect of...

  1. Melatonin receptor activation increases glutamatergic synaptic transmission in the rat medial lateral habenula.

    Evely, Katherine M; Hudson, Randall L; Dubocovich, Margarita L; Haj-Dahmane, Samir

    2016-05-01

    Melatonin (MLT) is secreted from the pineal gland and mediates its physiological effects through activation of two G protein-coupled receptors, MT1 and MT2 . These receptors are expressed in several brain areas, including the habenular complex, a pair of nuclei that relay information from forebrain to midbrain and modulate a plethora of behaviors, including sleep, mood, and pain. However, so far, the precise mechanisms by which MLT control the function of habenula neurons remain unknown. Using whole cell recordings from male rat brain slices, we examined the effects of MLT on the excitability of medial lateral habenula (MLHb) neurons. We found that MLT had no significant effects on the intrinsic excitability of MLHb neurons, but profoundly increased the amplitude of glutamate-mediated evoked excitatory post-synaptic currents (EPSC). The increase in strength of glutamate synapses onto MLHb neurons was mediated by an increase in glutamate release. The MLT-induced increase in glutamatergic synaptic transmission was blocked by the competitive MT1 /MT2 receptor antagonist luzindole (LUZ). These results unravel a potential cellular mechanism by which MLT receptor activation enhances the excitability of MLHb neurons. The MLT-mediated control of glutamatergic inputs to the MLHb may play a key role in the modulation of various behaviors controlled by the habenular complex. PMID:26799638

  2. Reduced synaptic activity in neuronal networks derived from embryonic stem cells of murine Rett syndrome model.

    Barth, Lydia; Sütterlin, Rosmarie; Nenniger, Markus; Vogt, Kaspar E

    2014-01-01

    Neurodevelopmental diseases such as the Rett syndrome (RTT) have received renewed attention, since the mechanisms involved may underlie a broad range of neuropsychiatric disorders such as schizophrenia and autism. In vertebrates early stages in the functional development of neurons and neuronal networks are difficult to study. Embryonic stem cell-derived neurons provide an easily accessible tool to investigate neuronal differentiation and early network formation. We used in vitro cultures of neurons derived from murine embryonic stem cells missing the methyl-CpG-binding protein 2 (MECP2) gene (MeCP2-/y) and from wild type cells of the corresponding background. Cultures were assessed using whole-cell patch-clamp electrophysiology and immunofluorescence. We studied the functional maturation of developing neurons and the activity of the synaptic connections they formed. Neurons exhibited minor differences in the developmental patterns for their intrinsic parameters, such as resting membrane potential and excitability; with the MeCP2-/y cells showing a slightly accelerated development, with shorter action potential half-widths at early stages. There was no difference in the early phase of synapse development, but as the cultures matured, significant deficits became apparent, particularly for inhibitory synaptic activity. MeCP2-/y embryonic stem cell-derived neuronal cultures show clear developmental deficits that match phenotypes observed in slice preparations and thus provide a compelling tool to further investigate the mechanisms behind RTT pathophysiology. PMID:24723848

  3. Reduced synaptic activity in neuronal networks derived from embryonic stem cells of murine Rett syndrome model

    Kaspar Emanuel Vogt

    2014-03-01

    Full Text Available Neurodevelopmental diseases such as the Rett syndrome have received renewed attention, since the mechanisms involved may underlie a broad range of neuropsychiatric disorders such as schizophrenia and autism. In vertebrates early stages in the functional development of neurons and neuronal networks are difficult to study. Embryonic stem cell-derived neurons provide an easily accessible tool to investigate neuronal differentiation and early network formation. We used in vitro cultures of neurons derived from murine embryonic stem cells missing the methyl-CpG-binding protein 2 (MECP2 gene (MeCP2-/y and from wild type cells of the corresponding background. Cultures were assessed using whole-cell patch-clamp electrophysiology and immunofluorescence. We studied the functional maturation of developing neurons and the activity of the synaptic connections they formed. Neurons exhibited minor differences in the developmental patterns for their intrinsic parameters, such as resting membrane potential and excitability; with the MeCP2-/y cells showing a slightly accelerated development, with shorter action potential half-widths at early stages. There was no difference in the early phase of synapse development, but as the cultures matured, significant deficits became apparent, particularly for inhibitory synaptic activity. MeCP2-/y embryonic stem cell-derived neuronal cultures show clear developmental deficits that match phenotypes observed in slice preparations and thus provide a compelling tool to further investigate the mechanisms behind Rett syndrome pathophysiology.

  4. Self healing of open circuit faults: With active re-configurability and mimicry of synaptic plasticity

    Yaswant, Vaddi; Kumar, Amit; Sambandan, Sanjiv

    2016-07-01

    We discuss the self-repair of open faults in circuits using electrically conductive particles dispersed in an insulating fluid. The repair is triggered by the electric field developed across the open circuit in a current carrying interconnect and results in the formation of a bridge of particles across the gap. We illustrate and model the dynamics of the resistance of the self-healed route, Rb, in low field conditions. Furthermore, active control of Rb and active re-wiring are also demonstrated. Considering Rb to be akin to weights between nodes, the formation and re-wiring of routes and the control of Rb mimic synaptic plasticity in biological systems and open interesting possibilities for computing.

  5. Spontaneous Synaptic Activation of Muscarinic Receptors by Striatal Cholinergic Neuron Firing.

    Mamaligas, Aphroditi A; Ford, Christopher P

    2016-08-01

    Cholinergic interneurons (CHIs) play a major role in motor and learning functions of the striatum. As acetylcholine does not directly evoke postsynaptic events at most striatal synapses, it remains unclear how postsynaptic cholinergic receptors encode the firing patterns of CHIs in the striatum. To examine the dynamics of acetylcholine release, we used optogenetics and paired recordings from CHIs and medium spiny neurons (MSNs) virally overexpressing G-protein-activated inwardly rectifying potassium (GIRK) channels. Due to the efficient coupling between endogenous muscarinic receptors and GIRK channels, we found that firing of individual CHIs resulted in monosynaptic spontaneous inhibitory post-synaptic currents (IPSCs) in MSNs. Paired CHI-MSN recordings revealed that the high probability of acetylcholine release at these synapses allowed muscarinic receptors to faithfully encode physiological activity patterns from individual CHIs without failure. These results indicate that muscarinic receptors in striatal output neurons reliably decode CHI firing. PMID:27373830

  6. Synaptic Ribbons Require Ribeye for Electron Density, Proper Synaptic Localization, and Recruitment of Calcium Channels.

    Lv, Caixia; Stewart, William J; Akanyeti, Otar; Frederick, Courtney; Zhu, Jie; Santos-Sacchi, Joseph; Sheets, Lavinia; Liao, James C; Zenisek, David

    2016-06-21

    Synaptic ribbons are structures made largely of the protein Ribeye that hold synaptic vesicles near release sites in non-spiking cells in some sensory systems. Here, we introduce frameshift mutations in the two zebrafish genes encoding for Ribeye and thus remove Ribeye protein from neuromast hair cells. Despite Ribeye depletion, vesicles collect around ribbon-like structures that lack electron density, which we term "ghost ribbons." Ghost ribbons are smaller in size but possess a similar number of smaller vesicles and are poorly localized to synapses and calcium channels. These hair cells exhibit enhanced exocytosis, as measured by capacitance, and recordings from afferent neurons post-synaptic to hair cells show no significant difference in spike rates. Our results suggest that Ribeye makes up most of the synaptic ribbon density in neuromast hair cells and is necessary for proper localization of calcium channels and synaptic ribbons. PMID:27292637

  7. In Vivo Activation of Azipropofol Prolongs Anesthesia and Reveals Synaptic Targets*

    Weiser, Brian P.; Kelz, Max B.; Eckenhoff, Roderic G.

    2013-01-01

    General anesthetic photolabels have been instrumental in discovering and confirming protein binding partners and binding sites of these promiscuous ligands. We report the in vivo photoactivation of meta-azipropofol, a potent analog of propofol, in Xenopus laevis tadpoles. Covalent adduction of meta-azipropofol in vivo prolongs the primary pharmacologic effect of general anesthetics in a behavioral phenotype we termed “optoanesthesia.” Coupling this behavior with a tritiated probe, we performed unbiased, time-resolved gel proteomics to identify neuronal targets of meta-azipropofol in vivo. We have identified synaptic binding partners, such as synaptosomal-associated protein 25, as well as voltage-dependent anion channels as potential facilitators of the general anesthetic state. Pairing behavioral phenotypes elicited by the activation of efficacious photolabels in vivo with time-resolved proteomics provides a novel approach to investigate molecular mechanisms of general anesthetics. PMID:23184948

  8. In vivo activation of azipropofol prolongs anesthesia and reveals synaptic targets.

    Weiser, Brian P; Kelz, Max B; Eckenhoff, Roderic G

    2013-01-11

    General anesthetic photolabels have been instrumental in discovering and confirming protein binding partners and binding sites of these promiscuous ligands. We report the in vivo photoactivation of meta-azipropofol, a potent analog of propofol, in Xenopus laevis tadpoles. Covalent adduction of meta-azipropofol in vivo prolongs the primary pharmacologic effect of general anesthetics in a behavioral phenotype we termed "optoanesthesia." Coupling this behavior with a tritiated probe, we performed unbiased, time-resolved gel proteomics to identify neuronal targets of meta-azipropofol in vivo. We have identified synaptic binding partners, such as synaptosomal-associated protein 25, as well as voltage-dependent anion channels as potential facilitators of the general anesthetic state. Pairing behavioral phenotypes elicited by the activation of efficacious photolabels in vivo with time-resolved proteomics provides a novel approach to investigate molecular mechanisms of general anesthetics. PMID:23184948

  9. The central role of heat shock factor 1 in synaptic fidelity and memory consolidation.

    Hooper, Philip L; Durham, Heather D; Török, Zsolt; Hooper, Paul L; Crul, Tim; Vígh, László

    2016-09-01

    Networks of neuronal synapses are the fundamental basis for making and retaining memory. Reduced synapse number and quality correlates with loss of memory in dementia. Heat shock factor 1 (HSF1), the major transcription factor regulating expression of heat shock genes, plays a central role in proteostasis, in establishing and sustaining synaptic fidelity and function, and in memory consolidation. Support for this thesis is based on these observations: (1) heat shock induces improvements in synapse integrity and memory consolidation; (2) synaptic depolarization activates HSF1; (3) activation of HSF1 alone (independent of the canonical heat shock response) augments formation of essential synaptic elements-neuroligands, vesicle transport, synaptic scaffolding proteins, lipid rafts, synaptic spines, and axodendritic synapses; (4) HSF1 coalesces and activates memory receptors in the post-synaptic dendritic spine; (5) huntingtin or α-synuclein accumulation lowers HSF1 while HSF1 lowers huntingtin and α-synuclein aggregation-a potential vicious cycle; and (6) HSF1 agonists (including physical activity) can improve cognitive function in dementia models. Thus, via direct gene expression of synaptic elements, production of HSPs that assure high protein fidelity, and activation of other neuroprotective signaling pathways, HSF1 agonists could provide breakthrough therapy for dementia-associated disease. PMID:27283588

  10. Persistent ERK Activation Maintains Learning-Induced Long-Lasting Modulation of Synaptic Connectivity

    Cohen-Matsliah, Sivan Ida; Seroussi, Yaron; Rosenblum, Kobi; Barkai, Edi

    2008-01-01

    Pyramidal neurons in the piriform cortex from olfactory-discrimination (OD) trained rats undergo synaptic modifications that last for days after learning. A particularly intriguing modification is reduced paired-pulse facilitation (PPF) in the synapses interconnecting these cells; a phenomenon thought to reflect enhanced synaptic release. The…

  11. Valine but not leucine or isoleucine supports neurotransmitter glutamate synthesis during synaptic activity in cultured cerebellar neurons

    Bak, Lasse Kristoffer; Johansen, Maja L.; Schousboe, Arne;

    2012-01-01

    group nitrogen donors for synthesis of vesicular neurotransmitter glutamate was investigated in cultured mouse cerebellar (primarily glutamatergic) neurons. The cultures were superfused in the presence of (15) N-labeled BCAAs, and synaptic activity was induced by pulses of N-methyl-D-aspartate (300 µ...

  12. Prenatal alcohol exposure alters synaptic activity of adult hippocampal dentate granule cells under conditions of enriched environment.

    Kajimoto, Kenta; Valenzuela, C Fernando; Allan, Andrea M; Ge, Shaoyu; Gu, Yan; Cunningham, Lee Anna

    2016-08-01

    Prenatal alcohol exposure (PAE) results in fetal alcohol spectrum disorder (FASD), which is characterized by a wide range of cognitive and behavioral deficits that may be linked to impaired hippocampal function and adult neurogenesis. Preclinical studies in mouse models of FASD indicate that PAE markedly attenuates enrichment-mediated increases in the number of adult-generated hippocampal dentate granule cells (aDGCs), but whether synaptic activity is also affected has not been studied. Here, we utilized retroviral birth-dating coupled with whole cell patch electrophysiological recordings to assess the effects of PAE on enrichment-mediated changes in excitatory and inhibitory synaptic activity as a function of DGC age. We found that exposure to an enriched environment (EE) had no effect on baseline synaptic activity of 4- or 8-week-old aDGCs from control mice, but significantly enhanced the excitatory/inhibitory ratio of synaptic activity in 8-week-old aDGCs from PAE mice. In contrast, exposure to EE significantly enhanced the excitatory/inhibitory ratio of synaptic activity in older pre-existing DGCs situated in the outer dentate granule cell layer (i.e., those generated during embryonic development; dDGCs) in control mice, an effect that was blunted in PAE mice. These findings indicate distinct electrophysiological responses of hippocampal DGCs to behavioral challenge based on cellular ontogenetic age, and suggest that PAE disrupts EE-mediated changes in overall hippocampal network activity. These findings may have implications for future therapeutic targeting of hippocampal dentate circuitry in clinical FASD. © 2016 Wiley Periodicals, Inc. PMID:27009742

  13. Studying the cytolytic activity of gas plasma with self-signalling phospholipid vesicles dispersed within a gelatin matrix

    A synthetic biological sensor was developed to monitor the interaction of plasma with soft, hydrated biological material. It comprises phospholipid vesicles in a hydrated proteinaceous environment comprising 5% (w/v) gelatin. The vesicles contained a self-quenched dye, which was activated by vesicle destruction giving a clear fluorescent switch on. The interaction of bacterial toxins with the sensor was measured in a proof of principle experiment, then the effect of atmospheric plasma jets with the sensor, was studied in order to assess the cytolytic effect of plasma jets in biological systems. When the plasma contacted the gelatin surface perpendicular to the surface, the treatment resulted in the formation of a star-shaped pattern of microchannels that radiated out from the centre of the treatment area within the gelatin matrix, and locally damaged vesicles within the microchannels at a depth of 150 µm below the gelatin surface. Plasma jets applied in parallel to the surface of the matrix resulted in the formation of a single microchannel with damage to the vesicles only evident at the walls of the channel, and a much reduced penetration depth within the gelatin. Our data show that the effects of plasma can be deep in the gelatin material and that the angle of treatment significantly influenced the nature and level of damage to the gelatin and vesicles. Potentially this gelatin model can be used to unravel the roles of different plasma species and the direct effect of whole plasma contact, from those of primary and secondary species—i.e. primary, those emanating directly from the plasma and secondary, those species created in the ‘target’ tissue. This type of insight could be useful in the future development of safe and effective plasma medical technologies. (paper)

  14. EDITORIAL: Synaptic electronics Synaptic electronics

    Demming, Anna; Gimzewski, James K.; Vuillaume, Dominique

    2013-09-01

    Conventional computers excel in logic and accurate scientific calculations but make hard work of open ended problems that human brains handle easily. Even von Neumann—the mathematician and polymath who first developed the programming architecture that forms the basis of today's computers—was already looking to the brain for future developments before his death in 1957 [1]. Neuromorphic computing uses approaches that better mimic the working of the human brain. Recent developments in nanotechnology are now providing structures with very accommodating properties for neuromorphic approaches. This special issue, with guest editors James K Gimzewski and Dominique Vuillaume, is devoted to research at the serendipitous interface between the two disciplines. 'Synaptic electronics', looks at artificial devices with connections that demonstrate behaviour similar to synapses in the nervous system allowing a new and more powerful approach to computing. Synapses and connecting neurons respond differently to incident signals depending on the history of signals previously experienced, ultimately leading to short term and long term memory behaviour. The basic characteristics of a synapse can be replicated with around ten simple transistors. However with the human brain having around 1011 neurons and 1015 synapses, artificial neurons and synapses from basic transistors are unlikely to accommodate the scalability required. The discovery of nanoscale elements that function as 'memristors' has provided a key tool for the implementation of synaptic connections [2]. Leon Chua first developed the concept of the 'The memristor—the missing circuit element' in 1971 [3]. In this special issue he presents a tutorial describing how memristor research has fed into our understanding of synaptic behaviour and how they can be applied in information processing [4]. He also describes, 'The new principle of local activity, which uncovers a minuscule life-enabling "Goldilocks zone", dubbed the

  15. Coarse-grained modeling of vesicle responses to active rotational nanoparticles

    Zhang, Liuyang; Wang, Xianqiao

    2015-08-01

    In recent years, magnetically-driven-rotating superparamagnetic nanoparticles have been emerging as a valuable component in designing targeted drug delivery carriers and cellular killers via membranes' physical rupture. The lack of an in-depth understanding of how to control the interaction of rotational nanoparticles (RNPs) with vesicles has hindered progress in the development of their relevant biomedical applications. Here we perform dissipative particle dynamics simulations to analyze the rotation frequencies, size, and coating patterns of the RNPs as they interact with the vesicle so as to provide novel designs of drug delivery applications. Results have revealed that the RNPs are capable of triggering local disturbance around the vesicle and therefore promoting the vesicle translocation toward the RNPs. By investigating the translocation time and driving forces required for RNPs to enter inside the vesicle at various rotation frequencies as well as the interaction energy between coated RNPs and the vesicle, we have tuned the coating pattern of the ligands on the surface of RNPs to open a specified channel in the vesicle for promoting drug delivery. Our findings can provide useful guidelines for the molecular design of patterned RNPs for controllable bio/inorganic interfaces and help establish qualitative rules for the organization and optimization of ligands on the surface of the desired drug delivery carriers.

  16. Phospholipase A2 activation enhances inhibitory synaptic transmission in rat substantia gelatinosa neurons.

    Liu, Tao; Fujita, Tsugumi; Nakatsuka, Terumasa; Kumamoto, Eiichi

    2008-03-01

    Phospholipase A(2) (PLA(2)) activation enhances glutamatergic excitatory synaptic transmission in substantia gelatinosa (SG) neurons, which play a pivotal role in regulating nociceptive transmission in the spinal cord. By using melittin as a tool to activate PLA(2), we examined the effect of PLA(2) activation on spontaneous inhibitory postsynaptic currents (sIPSCs) recorded at 0 mV in SG neurons of adult rat spinal cord slices by use of the whole cell patch-clamp technique. Melittin enhanced the frequency and amplitude of GABAergic and glycinergic sIPSCs. The enhancement of GABAergic but not glycinergic transmission was largely depressed by Na(+) channel blocker tetrodotoxin or glutamate-receptor antagonists (6-cyano-7-nitroquinoxaline-2,3-dione and/or dl-2-amino-5-phosphonovaleric acid) and also in a Ca(2+)-free Krebs solution. The effects of melittin on glycinergic sIPSC frequency and amplitude were dose-dependent with an effective concentration of approximately 0.7 microM for half-maximal effect and were depressed by PLA(2) inhibitor 4-bromophenacyl bromide or aristolochic acid. The melittin-induced enhancement of glycinergic transmission was depressed by lipoxygenase inhibitor nordihydroguaiaretic acid but not cyclooxygenase inhibitor indomethacin. These results indicate that the activation of PLA(2) in the SG enhances GABAergic and glycinergic inhibitory transmission in SG neurons. The former action is mediated by glutamate-receptor activation and neuronal activity increase, possibly the facilitatory effect of PLA(2) activation on excitatory transmission, whereas the latter action is due to PLA(2) and subsequent lipoxygenase activation and is independent of extracellular Ca(2+). It is suggested that PLA(2) activation in the SG could enhance not only excitatory but also inhibitory transmission, resulting in the modulation of nociception. PMID:18216222

  17. Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain

    Kurbatskaya, Ksenia; Phillips, Emma Claire; Croft, Cara Louise; Dentoni, Giacomo; Hughes, Martina; Wade, Matthew Austen James; Al-Sarraj, Safa; Troakes, Claire; O'Neill, Michael; Gomez Perez-Nievas, Beatriz; Hanger, Diane Pamela; Noble, Wendy Jane

    2016-01-01

    Alterations in calcium homeostasis are widely reported to contribute to synaptic degeneration and neuronal loss in Alzheimer’s disease. Elevated cytosolic calcium concentrations lead to activation of the calcium-sensitive cysteine protease, calpain, which has a number of substrates known to be abnormally regulated in disease. Analysis of human brain has shown that calpain activity is elevated in AD compared to controls, and that calpain-mediated proteolysis regulates the activity of important...

  18. EDITORIAL: Synaptic electronics Synaptic electronics

    Demming, Anna; Gimzewski, James K.; Vuillaume, Dominique

    2013-09-01

    Conventional computers excel in logic and accurate scientific calculations but make hard work of open ended problems that human brains handle easily. Even von Neumann—the mathematician and polymath who first developed the programming architecture that forms the basis of today's computers—was already looking to the brain for future developments before his death in 1957 [1]. Neuromorphic computing uses approaches that better mimic the working of the human brain. Recent developments in nanotechnology are now providing structures with very accommodating properties for neuromorphic approaches. This special issue, with guest editors James K Gimzewski and Dominique Vuillaume, is devoted to research at the serendipitous interface between the two disciplines. 'Synaptic electronics', looks at artificial devices with connections that demonstrate behaviour similar to synapses in the nervous system allowing a new and more powerful approach to computing. Synapses and connecting neurons respond differently to incident signals depending on the history of signals previously experienced, ultimately leading to short term and long term memory behaviour. The basic characteristics of a synapse can be replicated with around ten simple transistors. However with the human brain having around 1011 neurons and 1015 synapses, artificial neurons and synapses from basic transistors are unlikely to accommodate the scalability required. The discovery of nanoscale elements that function as 'memristors' has provided a key tool for the implementation of synaptic connections [2]. Leon Chua first developed the concept of the 'The memristor—the missing circuit element' in 1971 [3]. In this special issue he presents a tutorial describing how memristor research has fed into our understanding of synaptic behaviour and how they can be applied in information processing [4]. He also describes, 'The new principle of local activity, which uncovers a minuscule life-enabling "Goldilocks zone", dubbed the

  19. Progressive brain damage, synaptic reorganization and NMDA activation in a model of epileptogenic cortical dysplasia.

    Francesca Colciaghi

    Full Text Available Whether severe epilepsy could be a progressive disorder remains as yet unresolved. We previously demonstrated in a rat model of acquired focal cortical dysplasia, the methylazoxymethanol/pilocarpine - MAM/pilocarpine - rats, that the occurrence of status epilepticus (SE and subsequent seizures fostered a pathologic process capable of modifying the morphology of cortical pyramidal neurons and NMDA receptor expression/localization. We have here extended our analysis by evaluating neocortical and hippocampal changes in MAM/pilocarpine rats at different epilepsy stages, from few days after onset up to six months of chronic epilepsy. Our findings indicate that the process triggered by SE and subsequent seizures in the malformed brain i is steadily progressive, deeply altering neocortical and hippocampal morphology, with atrophy of neocortex and CA regions and progressive increase of granule cell layer dispersion; ii changes dramatically the fine morphology of neurons in neocortex and hippocampus, by increasing cell size and decreasing both dendrite arborization and spine density; iii induces reorganization of glutamatergic and GABAergic networks in both neocortex and hippocampus, favoring excitatory vs inhibitory input; iv activates NMDA regulatory subunits. Taken together, our data indicate that, at least in experimental models of brain malformations, severe seizure activity, i.e., SE plus recurrent seizures, may lead to a widespread, steadily progressive architectural, neuronal and synaptic reorganization in the brain. They also suggest the mechanistic relevance of glutamate/NMDA hyper-activation in the seizure-related brain pathologic plasticity.

  20. Effects of Cortical Spreading Depression on Synaptic Activity, Blood Flow and Oxygen Consumption in Rat Cerebral Cortex

    Hansen, Henning Piilgaard

    2010-01-01

    As the title of this thesis indicates I have during my PhD studied the effects of cortical spreading depression (CSD) on synaptic activity, blood flow and oxygen consumption in rat cerebral cortex. This was performed in vivo using an open cranial window approach in anesthetized rats. I applied...... two different sets of interneurons. Our data imply that for a given cortical area the amplitude of vascular signals will depend critically on the type of input and hence on the type of neurons activated. In the second study I investigated the effect of cortical spreading depression (CSD) on the evoked...... Laser-Doppler Flowmetry for measurements of cerebral blood flow, glass microelectrodes for recording of synaptic activity – local field potentials – and ongoing cortical electrical activity and a Clark type electrode for measurements of tissue partial pressure of oxygen (tpO2). Offline calculations of...

  1. Active uptake of tetracycline by membrane vesicles from susceptible Escherichia coli.

    McMurry, L M; Cullinane, J C; Petrucci, R E; Levy, S. B.

    1981-01-01

    A major portion of tetracycline accumulation by susceptible bacterial cells is energy dependent. Inner membrane vesicles prepared from susceptible Escherichia coli cells concentrated tetracycline 2.5 to 5 times above the external concentration when the electron transport substrate D-lactate or reduced phenazine methosulfate was added. This stimulation was reversed by cyanide, 2,4-dinitrophenol, and carbonyl cyanide m-chlorophenyl hydrazone. These vesicles data showed that proton motive force ...

  2. Activation of 5-hyrdoxytryptamine 7 receptors within the rat nucleus tractus solitarii modulates synaptic properties.

    Matott, Michael P; Kline, David D

    2016-03-15

    Serotonin (5-HT) is a potent neuromodulator with multiple receptor types within the cardiorespiratory system, including the nucleus tractus solitarii (nTS) - the central termination site of visceral afferent fibers. The 5-HT7 receptor facilitates cardiorespiratory reflexes through its action in the brainstem and likely in the nTS. However, the mechanism and site of action for these effects is not clear. In this study, we examined the expression and function of 5-HT7 receptors in the nTS of Sprague-Dawley rats. 5-HT7 receptor mRNA and protein were identified across the rostrocaudal extent of the nTS. To determine 5-HT7 receptor function, we examined nTS synaptic properties following 5-HT7 receptor activation in monosynaptic nTS neurons in the in vitro brainstem slice preparation. Application of 5-HT7 receptor agonists altered tractus solitarii evoked and spontaneous excitatory postsynaptic currents which were attenuated with a selective 5-HT7 receptor antagonist. 5-HT7 receptor-mediated changes in excitatory postsynaptic currents were also altered by block of 5-HT1A and GABAA receptors. Interestingly, 5-HT7 receptor activation also reduced the amplitude but not frequency of GABAA-mediated inhibitory currents. Together these results indicate a complex role for 5-HT7 receptors in the nTS that mediate its diverse effects on cardiorespiratory parameters. PMID:26779891

  3. Dopaminergic modulation of synaptic transmission and neuronal activity patterns in the zebrafish homolog of olfactory cortex

    Schärer, Yan-Ping Zhang; Shum, Jennifer; Moressis, Anastasios; Friedrich, Rainer W.

    2012-01-01

    Dopamine (DA) is an important modulator of synaptic transmission and plasticity that is causally involved in fundamental brain functions and dysfunctions. We examined the dopaminergic modulation of synaptic transmission and sensory responses in telencephalic area Dp of zebrafish, the homolog of olfactory cortex. By combining anatomical tracing and immunohistochemistry, we detected no DA neurons in Dp itself but long-range dopaminergic input from multiple other brain areas. Whole-cell recordin...

  4. Dopaminergic modulation of synaptic transmission and neuronal activity patterns in the zebrafish homolog of olfactory cortex

    Friedrich, Rainer W.

    2012-01-01

    Dopamine (DA) is an important modulator of synaptic transmission and plasticity that is causally involved in fundamental brain functions and dysfunctions. We examined the dopaminergic modulation of synaptic transmission and sensory responses in telencephalic area Dp of zebrafish, the homologue of olfactory cortex. By combining anatomical tracing and immunohistochemistry, we detected no DA neurons in Dp itself but long-range dopaminergic input from multiple other brain areas. Whole-cell record...

  5. A quantitative method to assess extrasynaptic NMDA receptor function in the protective effect of synaptic activity against neurotoxicity

    Bading Hilmar

    2008-01-01

    Full Text Available Abstract Background Extrasynaptic NMDA receptors couple to a CREB shut-off pathway and cause cell death, whereas synaptic NMDA receptors and nuclear calcium signaling promote CREB-mediated transcription and neuronal survival. The distribution of NMDA receptors (synaptic versus extrasynaptic may be an important parameter that determines the susceptibility of neurons to toxic insults. Changes in receptor surface expression towards more extrasynaptic NMDA receptors may lead to neurodegeneration, whereas a reduction of extrasynaptic NMDA receptors may render neurons more resistant to death. A quantitative assessment of extrasynaptic NMDA receptors in individual neurons is needed in order to investigate the role of NMDA receptor distribution in neuronal survival and death. Results Here we refined and verified a protocol previously used to isolate the effects of extrasynaptic NMDA receptors using the NMDA receptor open channel blocker, MK-801. Using this method we investigated the possibility that the known neuroprotective shield built up in hippocampal neurons after a period of action potential bursting and stimulation of synaptic NMDA receptors is due to signal-induced trafficking of extrasynaptic NMDA receptors or a reduction in extrasynaptic NMDA receptor function. We found that extrasynaptic NMDA receptor-mediated calcium responses and whole cell currents recorded under voltage clamp were surprisingly invariable and did not change even after prolonged (16 to 24 hours periods of bursting and synaptic NMDA receptor activation. Averaging a large number of calcium imaging traces yielded a small (6% reduction of extrasynaptic NMDA receptor-mediated responses in hippocampal neurons that were pretreated with prolonged bursting. Conclusion The slight reduction in extrasynaptic NMDA receptor function following action potential bursting and synaptic NMDA receptor stimulation could contribute to but is unlikely to fully account for activity

  6. Preparation of wheat root plasma membrane vesicles and effect of water stress on 45Ca2+ transport activity

    The wheat roots plasma membrane (PM) vesicles were obtained by sucrose gradient centrifugation. The experiment results shows that the wheat roots of Zhengyin No.1 PM H+-ATPase latent activity was 24%, and PM inside-out vesicle (IOV) accounts for 76%. With -1.0 MPa stress of 24h, PM Ca2+-ATPase activity of both orientation wheat roots were increased. Under normal water condition and PEG stress, 62% and 53% of the enzyme activity was inhibited respectively by EGTA, radioactive calcium-45 transport amount was 22.09 nmol/mg pro and 4.17 nmol/mg pro. respectively with PM-IOV.PEG stress results in a decrease of 45Ca2+ transport amount of wheat roots PM-IOV by 81%

  7. Syncrip/hnRNP Q influences synaptic transmission and regulates BMP signaling at the Drosophila neuromuscular synapse

    James M. Halstead

    2014-08-01

    Full Text Available Synaptic plasticity involves the modulation of synaptic connections in response to neuronal activity via multiple pathways. One mechanism modulates synaptic transmission by retrograde signals from the post-synapse that influence the probability of vesicle release in the pre-synapse. Despite its importance, very few factors required for the expression of retrograde signals, and proper synaptic transmission, have been identified. Here, we identify the conserved RNA binding protein Syncrip as a new factor that modulates the efficiency of vesicle release from the motoneuron and is required for correct synapse structure. We show that syncrip is required genetically and its protein product is detected only in the muscle and not in the motoneuron itself. This unexpected non-autonomy is at least partly explained by the fact that Syncrip modulates retrograde BMP signals from the muscle back to the motoneuron. We show that Syncrip influences the levels of the Bone Morphogenic Protein ligand Glass Bottom Boat from the post-synapse and regulates the pre-synapse. Our results highlight the RNA-binding protein Syncrip as a novel regulator of synaptic output. Given its known role in regulating translation, we propose that Syncrip is important for maintaining a balance between the strength of presynaptic vesicle release and postsynaptic translation.

  8. Astrocytes: Orchestrating synaptic plasticity?

    De Pittà, M; Brunel, N; Volterra, A

    2016-05-26

    Synaptic plasticity is the capacity of a preexisting connection between two neurons to change in strength as a function of neural activity. Because synaptic plasticity is the major candidate mechanism for learning and memory, the elucidation of its constituting mechanisms is of crucial importance in many aspects of normal and pathological brain function. In particular, a prominent aspect that remains debated is how the plasticity mechanisms, that encompass a broad spectrum of temporal and spatial scales, come to play together in a concerted fashion. Here we review and discuss evidence that pinpoints to a possible non-neuronal, glial candidate for such orchestration: the regulation of synaptic plasticity by astrocytes. PMID:25862587

  9. Plasmonic Vesicles of Amphiphilic Nanocrystals: Optically Active Multifunctional Platform for Cancer Diagnosis and Therapy.

    Song, Jibin; Huang, Peng; Duan, Hongwei; Chen, Xiaoyuan

    2015-09-15

    Vesicular structures with compartmentalized, water-filled cavities, such as liposomes of natural and synthetic amphiphiles, have tremendous potential applications in nanomedicine. When block copolymers self-assemble, the result is polymersomes with tailored structural properties and built-in releasing mechanisms, controlled by stimuli-responsive polymer building blocks. More recently, chemists are becoming interested in multifunctional hybrid vesicles containing inorganic nanocrystals with unique optical, electronic, and magnetic properties. In this Account, we review our recent progress in assembling amphiphilic plasmonic nanostructures to create a new class of multifunctional hybrid vesicles and applying them towards cancer diagnosis and therapy. Localized surface plasmon resonance (LSPR) gives plasmonic nanomaterials a unique set of optical properties that are potentially useful for both biosensing and nanomedicine. For instance, the strong light scattering at their LSPR wavelength opens up the applications of plasmonic nanostructures in single particle plasmonic imaging. Their superior photothermal conversion properties, on the other hand, make them excellent transducers for photothermal ablation and contrast agents for photoacoustic imaging. Of particular note for ultrasensitive detection is that the confined electromagnetic field resulting from excitation of LSPR can give rise to highly efficient surface enhanced Raman scattering (SERS) for molecules in close proximity. We have explored several ways to combine well-defined plasmonic nanocrystals with amphiphilic polymer brushes of diverse chemical functionalities. In multiple systems, we have shown that the polymer grafts impart amphiphilicity-driven self-assembly to the hybrid nanoparticles. This has allowed us to synthesize well-defined vesicles in which we have embedded plasmonic nanocrystals in the shell of collapsed hydrophobic polymers. The hydrophilic brushes extend into external and interior aqueous

  10. Lattice-gas model for active vesicle transport by molecular motors with opposite polarities

    Muhuri, Sudipto; Pagonabarraga, Ignacio

    2010-08-01

    We introduce a multispecies lattice-gas model for motor protein driven collective cargo transport on cellular filaments. We use this model to describe and analyze the collective motion of interacting vesicle cargos being carried by oppositely directed molecular motors, moving on a single biofilament. Building on a totally asymmetric exclusion process to characterize the motion of the interacting cargos, we allow for mass exchange with the environment, input, and output at filament boundaries and focus on the role of interconversion rates and how they affect the directionality of the net cargo transport. We quantify the effect of the various different competing processes in terms of nonequilibrium phase diagrams. The interplay of interconversion rates, which allow for flux reversal and evaporation-deposition processes, introduces qualitatively unique features in the phase diagrams. We observe regimes of three-phase coexistence, the possibility of phase re-entrance, and a significant flexibility in how the different phase boundaries shift in response to changes in control parameters. The moving steady-state solutions of this model allows for different possibilities for the spatial distribution of cargo vesicles, ranging from homogeneous distribution of vesicles to polarized distributions, characterized by inhomogeneities or shocks. Current reversals due to internal regulation emerge naturally within the framework of this model. We believe that this minimal model will clarify the understanding of many features of collective vesicle transport, apart from serving as the basis for building more exact quantitative models for vesicle transport relevant to various in vivo situations.

  11. Age-related deficits in synaptic plasticity rescued by activating PKA or PKC in sensory neurons of Aplysia californica

    Andrew T Kempsell

    2015-09-01

    Full Text Available Brain aging is associated with declines in synaptic function that contribute to memory loss, including reduced postsynaptic response to neurotransmitters and decreased neuronal excitability. To understand how aging affects memory in a simple neural circuit, we studied neuronal proxies of memory for sensitization in mature versus advanced age Aplysia. Glutamate- (L-Glu- evoked excitatory currents were facilitated by the neuromodulator serotonin (5-HT in sensory neurons (SN isolated from mature but not aged animals. Activation of PKA and PKC signaling rescued facilitation of L-Glu currents in aged SN. Similarly, PKA and PKC activators restored increased excitability in aged tail SN. These results suggest that altered synaptic plasticity during aging involves defects in second messenger systems

  12. Deletion of the secretory vesicle proteins IA-2 and IA-2β disrupts circadian rhythms of cardiovascular and physical activity

    Kim, Soo Mi; Power, Andrea; Brown, Timothy M.; Constance, Cara M.; Coon, Steven L.; Nishimura, Takuya; Hirai, Hiroki; Cai, Tao; Eisner, Christoph; David R Weaver; Piggins, Hugh D.; Klein, David C.; Schnermann, Jürgen; Notkins, Abner L.

    2009-01-01

    Targeted deletion of IA-2 and IA-2β, major autoantigens in type 1 diabetes and transmembrane secretory vesicle proteins, results in impaired secretion of hormones and neurotransmitters. In the present study, we evaluated the effect of these deletions on daily rhythms in blood pressure, heart rate, core body temperature, and spontaneous physical and neuronal activity. We found that deletion of both IA-2 and IA-2β profoundly disrupts the usual diurnal variation of each of these parameters, wher...

  13. Gender differences in spatial learning, synaptic activity, and long-term potentiation in the hippocampus in rats: molecular mechanisms.

    Monfort, Pilar; Gomez-Gimenez, Belen; Llansola, Marta; Felipo, Vicente

    2015-08-19

    In tests of spatial ability, males outperform females both in rats and in humans. The mechanism underlying this gender differential learning ability and memory in spatial tasks remains unknown. Long-term potentiation (LTP) in the hippocampus is considered the basis for spatial learning and memory. The aims of this work were (a) to assess spatial learning and memory in male and female rats in the radial and Morris mazes; (b) to assess whether basal synaptic activity and LTP in the hippocampus are different in male and female rats; and (c) to identify the molecular mechanisms responsible for the gender differences in LTP. We analyzed in young male and female rats (a) performance in spatial tasks in the radial and Morris water mazes; (b) basal synaptic activity in hippocampal slices; and (c) LTP and some mechanisms modulating its magnitude. The results reported allow us to conclude that female rats show larger AMPA receptor-mediate synaptic responses under basal conditions, likely due to enhanced phosphorylation of GluR2 in Ser880 and increased amounts of GluR2-containing AMPA receptors in postsynaptic densities. In contrast, the magnitude of tetanus-induced LTP was lower in females than in males. This is due to reduced activation of soluble guanylate cyclase and the formation of cGMP, leading to lower activation of cGMP-dependent protein kinase and phosphorylation of GluR1 in Ser845, which results in lower insertion of AMPA receptors in the synaptic membrane and a lower magnitude of LTP. These mechanisms may contribute to the reduced performance of females in the radial and Morris water mazes. PMID:26098845

  14. CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors.

    Loebrich, Sven; Benoit, Marc Robert; Konopka, Jaclyn Aleksandra; Cottrell, Jeffrey Richard; Gibson, Joanne; Nedivi, Elly

    2016-02-01

    Internalization of glutamate receptors at the postsynaptic membrane via clathrin-mediated endocytosis (CME) is a key mechanism for regulating synaptic strength. A role for the F-actin cytoskeleton in CME is well established, and recently, PKA-dependent association of candidate plasticity gene 2 (CPG2) with the spine-cytoskeleton has been shown to mediate synaptic glutamate receptor internalization. Yet, how the endocytic machinery is physically coupled to the actin cytoskeleton to facilitate glutamate receptor internalization has not been demonstrated. Moreover, there has been no distinction of endocytic-machinery components that are specific to activity-dependent versus constitutive glutamate receptor internalization. Here, we show that CPG2, through a direct physical interaction, recruits endophilin B2 (EndoB2) to F-actin, thus anchoring the endocytic machinery to the spine cytoskeleton and facilitating glutamate receptor internalization. Regulation of CPG2 binding to the actin cytoskeleton by protein kinase A directly impacts recruitment of EndoB2 and clathrin. Specific disruption of EndoB2 or the CPG2-EndoB2 interaction impairs activity-dependent, but not constitutive, internalization of both NMDA- and AMPA-type glutamate receptors. These results demonstrate that, through direct interactions with F-actin and EndoB2, CPG2 physically bridges the spine cytoskeleton and the endocytic machinery, and this tripartite association is critical specifically for activity-dependent CME of synaptic glutamate receptors. PMID:26776730

  15. Propofol, but not etomidate, increases corticosterone levels and induces long-term alteration in hippocampal synaptic activity in neonatal rats.

    Xu, Changqing; Seubert, Christoph N; Gravenstein, Nikolaus; Martynyuk, Anatoly E

    2016-04-01

    Animal studies provide strong evidence that general anesthetics (GAs), administered during the early postnatal period, induce long-term cognitive and neurological abnormalities. Because the brain growth spurt in rodents is delayed compared to that in humans, a fundamental question is whether the postnatal human brain is similarly vulnerable. Sevoflurane and propofol, GAs that share positive modulation of the gamma-aminobutyric acid type A receptor (GABAAR) function cause marked increase in corticosterone levels and induce long-term developmental alterations in synaptic activity in rodents. If synaptogenesis is affected, investigation of mechanisms of the synaptic effects of GAs is of high interest because synaptogenesis in humans continues for several years after birth. Here, we compared long-term synaptic effects of etomidate with those of propofol. Etomidate and propofol both positively modulate GABAAR activity, but in contrast to propofol, etomidate inhibits the adrenal synthesis of corticosterone. Postnatal day (P) 4, 5, or 6 rats received five injections of etomidate, propofol, or vehicle control during 5h of maternal separation. Endocrine effects of the anesthetics were evaluated by measuring serum levels of corticosterone immediately after anesthesia or maternal separation. The frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) in hippocampal CA1 pyramidal neurons were measured at P24-40 and P≥80. Only propofol caused a significant increase in serum corticosterone levels (F(4.26)=17.739, P<0.001). In contrast to increased frequency of mIPSCs in the propofol group (F(4.23)=8.731, p<0.001), mIPSC activity in the etomidate group was not different from that in the vehicle groups. The results of this study together with previously published data suggest that anesthetic-caused increase in corticosterone levels is required for GABAergic GAs to induce synaptic effects in the form of a long-term increase in the frequency of hippocampal m

  16. Paradoxical effects of VEGF on synaptic activity partially involved in notch1 signaling in the mouse hippocampus.

    Yang, Jiajia; Yang, Chunxiao; Liu, Chunhua; Zhang, Tao; Yang, Zhuo

    2016-05-01

    It is well known that the neuronal effects of vascular endothelial growth factor (VEGF) include modulating learning and memory, plasticity of mature neurons, and synaptic transmission in addition to neurogenesis. However, there is conflicting evidence particularly of its role in the regulation of excitatory synaptic activity. In this study, application of the patch-clamp technique revealed that lower doses (10 and 50 ng/mL) of VEGF enhanced excitatory neurotransmission in hippocampal slices of mice through both presynaptic and postsynaptic mechanisms. However, the effects were reversed by higher doses of VEGF (>100 ng/mL), which inhibited excitatory neurotransmission via a presynaptic mechanism. These competing, concentration-dependent effects of VEGF suggested that different pathways were involved. The involvement of the Notch1 receptor was tested in the modulation of VEGF on synaptic activity by using heterozygous Notch1(+/-) mice. Notch1 knockdown did not influence the inhibitory effect of high VEGF doses (200 ng/mL) but reduced the enhancement effects of low concentration of VEGF (50 ng/mL) at the postsynaptic level, which might be due to the decreased level of VEGF receptor. The results indicate that the Notch1 receptor plays a role in VEGF-induced modulation of synaptic activity, which provides new insights into a complex VEGF/Notch signaling cross-talk. These findings set the groundwork for understanding new mechanisms of Notch signaling and the neurotrophic effects of VEGF, which is beneficial to develop new therapeutic targets to the VEGF/Notch axis and improve current treatments for neural diseases. PMID:26482652

  17. Coated vesicles contain a phosphatidylinositol kinase

    When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. The authors have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of the CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism

  18. Active auxin uptake by zucchini membrane vesicles: quantitation using ESR volume and delta pH determinations

    Lomax, T.L.; Mehlhorn, R.J.; Briggs, W.R.

    1985-10-01

    Closed and pH-tight membrane vesicles prepared from hypocotyls of 5-day-old dark-grown seedlings of Cucurbita pepo accumulate the plant growth hormone indole-3-acetic acid along an imposed proton gradient (pH low outside, high inside). The use of electron paramagnetic spin probes permitted quantitation both of apparent vesicle volume and magnitude of the pH gradient. Under the experimental conditions used, hormone accumulation was at minimum 20-fold, a value 4 times larger than what one would predict if accumulation reflected only diffusional equilibrium at the measured pH gradient. It is concluded that hormone uptake is an active process, with each protonated molecule of hormone accompanied by an additional proton. Experiments with ionophores confirm that it is the pH gradient itself which drives the uptake.

  19. Phosphatidylinositol turnover (PI) during synaptic activation results from the release of a stimulatory and in inhibitory agonist

    PI has been implicated in the process of synaptic transmission and is increased by agonists. It has been suggested that PI is involved in cellular Ca++ mobilization and the process represents a series of hydrolytic reactions with inositol as the final product. Hence, the rate of release of 3H-inositol (3H-Ins) from prelabelled inositol phospholipids can be used as an index of PI. In the 3H-inositol prelabelled frog sympathetic ganglia, they studied the effect of synaptic activity on PI. PI did not change during orthodromic stimulation (20 Hz, 5 min). However, upon cessation of the stimulation, PI increased rapidly and remained elevated for at least 30 min. This increase in PI was reduced by suffusing the ganglia with either acetylcholine or adenosine. In the presence of atropine (5 μM), orthodromic stimulation increased PI. They hypothesized that synaptic activation releases a long-lasting stimulatory agonist and a short-lived inhibitory (Ach/adenosine) agonist(s) affecting PI. To test this idea, 2 sympathetic ganglia were used. One was prelabelled with 3H-inositol and the other was not. The two ganglia were placed together in a 5 μl drop of Ringers solution containing atropine. Orthodromic stimuli were applied to the non-labelled ganglion and elicited release of 3H-Ins from the non-stimulated ganglion

  20. Dopaminergic modulation of synaptic transmission and neuronal activity patterns in the zebrafish homolog of olfactory cortex

    Rainer W. Friedrich

    2012-10-01

    Full Text Available Dopamine (DA is an important modulator of synaptic transmission and plasticity that is causally involved in fundamental brain functions and dysfunctions. We examined the dopaminergic modulation of synaptic transmission and sensory responses in telencephalic area Dp of zebrafish, the homologue of olfactory cortex. By combining anatomical tracing and immunohistochemistry, we detected no DA neurons in Dp itself but long-range dopaminergic input from multiple other brain areas. Whole-cell recordings revealed no obvious effects of DA on membrane potential or input resistance in the majority of Dp neurons. Electrical stimulation of the olfactory tracts produced a complex sequence of synaptic currents in Dp neurons. DA selectively decreased inhibitory currents with little or no effect on excitatory components. Multiphoton calcium imaging showed that population responses of Dp neurons to olfactory tract stimulation or odor application were enhanced by DA, consistent with its effect on inhibitory synaptic transmission. These effects of DA were blocked by an antagonist of D2-like receptors. DA therefore disinhibits and reorganizes sensory responses in Dp. This modulation may affect sensory perception and could be involved in the experience-dependent modification of odor representations.

  1. Modulation of synaptic depression of the calyx of Held synapse by GABAB receptors and spontaneous activity

    Wang, T.; Rusu, S. I.; Hrušková, Bohdana; Tureček, Rostislav; Borst, J. G.

    2013-01-01

    Roč. 591, č. 19 (2013), s. 4877-4894. ISSN 0022-3751 R&D Projects: GA ČR(CZ) GAP303/11/0131 Institutional support: RVO:68378041 Keywords : GABAB * synaptic transmission * auditory Subject RIV: FH - Neurology Impact factor: 4.544, year: 2013

  2. Kidins220/ARMS is a novel modulator of short-term synaptic plasticity in hippocampal GABAergic neurons.

    Joachim Scholz-Starke

    Full Text Available Kidins220 (Kinase D interacting substrate of 220 kDa/ARMS (Ankyrin Repeat-rich Membrane Spanning is a scaffold protein highly expressed in the nervous system. Previous work on neurons with altered Kidins220/ARMS expression suggested that this protein plays multiple roles in synaptic function. In this study, we analyzed the effects of Kidins220/ARMS ablation on basal synaptic transmission and on a variety of short-term plasticity paradigms in both excitatory and inhibitory synapses using a recently described Kidins220 full knockout mouse. Hippocampal neuronal cultures prepared from embryonic Kidins220(-/- (KO and wild type (WT littermates were used for whole-cell patch-clamp recordings of spontaneous and evoked synaptic activity. Whereas glutamatergic AMPA receptor-mediated responses were not significantly affected in KO neurons, specific differences were detected in evoked GABAergic transmission. The recovery from synaptic depression of inhibitory post-synaptic currents in WT cells showed biphasic kinetics, both in response to paired-pulse and long-lasting train stimulation, while in KO cells the respective slow components were strongly reduced. We demonstrate that the slow recovery from synaptic depression in WT cells is caused by a transient reduction of the vesicle release probability, which is absent in KO neurons. These results suggest that Kidins220/ARMS is not essential for basal synaptic transmission and various forms of short-term plasticity, but instead plays a novel role in the mechanisms regulating the recovery of synaptic strength in GABAergic synapses.

  3. Visual input controls the functional activity of goldfish Mauthner neuron through the reciprocal synaptic mechanism.

    Moshkov, Dmitry A; Shtanchaev, Rashid S; Mikheeva, Irina B; Bezgina, Elena N; Kokanova, Nadezhda A; Mikhailova, Gulnara Z; Tiras, Nadezhda R; Pavlik, Lyubov' L

    2013-03-01

    Goldfish are known to exhibit motor asymmetry due to functional asymmetry of their Mauthner neurons that induce the turns to the right or left during free swimming. It has been previously found that if the less active neuron is subjected to prolonged aimed visual stimulation via its ventral dendrite, the motor asymmetry of goldfish is inverted, testifying that this neuron becomes functionally dominant, while the size of the ventral dendrite under these conditions is reduced 2-3 times compared to its counterpart in mirror neuron. Earlier it has been also revealed that training optokinetic stimulation induces adaptation, a substantial resistance of both fish motor asymmetry and morphofunctional state of Mauthner neurons against prolonged optokinetic stimulation. The aim of this work was to study the cellular mechanisms of the effect of an unusual visual afferent input on goldfish motor asymmetry and Mauthner neuron function in norm and under adaptation. It was shown that serotonin applied onto Mauthner neurons greatly reduces their activity whereas its antagonist ondansetron increases it. Against the background of visual stimulation, serotonin strengthens functional asymmetry between neurons whereas ondansetron smoothes it. Taken together these data suggest the involvement of serotonergic excitatory synaptic transmission in the regulation of Mauthner neurons by vision. Ultrastructural study of the ventral dendrites after prolonged optokinetic stimulation has revealed depletions of numeral axo-axonal synapses with specific morphology, identified by means of immunogold label as serotonergic ones. These latter in turn are situated mainly on shaft boutons, which according to specific ultrastructural features are assigned to axo-dendritic inhibitory synapses. Thus, the excitatory serotonergic synapses seem to affect Mauthner neuron indirectly through inhibitory synapses. Further, it was morphometrically established that adaptation is accompanied by the significant

  4. Curcumin Inhibits Glutamate Release from Rat Prefrontal Nerve Endings by Affecting Vesicle Mobilization

    Shu Kuei Huang

    2012-07-01

    Full Text Available Curcumin, one of the major constituents of Curcuma longa, has been shown to inhibit depolarization-evoked glutamate release from rat prefrontocortical nerve terminals by reducing voltage-dependent Ca2+ entry. This study showed that curcumin inhibited ionomycin-induced glutamate release and KCl-evoked FM1-43 release, suggesting that some steps after Ca2+ entry are regulated by curcumin. Furthermore, disrupting the cytoskeleton organization using cytochalasin D abolished the inhibitory action of curcumin on ionomycin-induced glutamate release. Mitogen-activated protein kinase kinase (MEK inhibition also prevented the inhibitory effect of curcumin on ionomycin-induced glutamate release. Western blot analyses showed that curcumin decreased the ionomycin-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2 and synaptic vesicle-associated protein synapsin I, the main presynaptic target of ERK. These results show that curcumin-mediated inhibition of glutamate release involves modulating downstream events by controlling synaptic vesicle recruitment and exocytosis, possibly through a decrease of MAPK/ERK activation and synapsin I phosphorylation, thereby decreasing synaptic vesicle availability for exocytosis.

  5. Nanostructure, solvation dynamics, and nanotemplating of plasmonically active SERS substrate in reverse vesicles

    Saha, Ranajay; Rakshit, Surajit [S.N. Bose National Centre for Basic Sciences, Department of Chemical, Biological and Macromolecular Sciences (India); Majumdar, Dipanwita; Singha, Achintya [Bose Institute, Department of Physics (India); Mitra, Rajib Kumar; Pal, Samir Kumar, E-mail: skpal@bose.res.in [S.N. Bose National Centre for Basic Sciences, Department of Chemical, Biological and Macromolecular Sciences (India)

    2013-04-15

    Reverse vesicles (RVs) are the organic counterparts to vesicles and are spherical containers in oils consisting of an oily core surrounded by reverse bilayers with water layers present in between. We present here a facile route for forming stable RV from nontoxic surfactants and oil components. The RV formation is characterized by dynamic light scattering and further confirmed by transmission electron microscopic (TEM) techniques. The water channels present in between the bilayers are found to be a potential template for inorganic nanoparticles' (NPs) synthesis. Both the UV-Vis absorption spectroscopy and the TEM study reveal successful formation of highly clustered silver NPs within the water layers of the RVs. X-ray powder diffraction analyzes the crystalline nature of the NPs. FTIR spectroscopy shows the signature of different kinds of water molecules in between the RV bilayers. The dynamical description of the templating water, dictating the controlled formation of the NPs in the RV, is well revealed in the picosecond-resolved solvation dynamics study of a hydrophilic fluorescence probe 2 Prime -(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl) -benzimidazo-2-yl-benzimidazole] (H258). The rotational anisotropy study successfully describes geometrical restriction of the probe molecule in the RV. Notably, this study provides the first proof-of-concept data for the ability of the RV to be a template of synthesizing metal NPs. The as-prepared NP clusters are evaluated to be potential surface-enhanced Raman scattering substrate in solution using crystal violet as a model analyte. The present study offers a new RV, which is a prospective nontoxic nanotemplate and is believed to contribute potentially in the emerging NP-vesicle hybrid assembly-based plasmonic applications.Nanotemplating of metal clusters for the efficient SERS detection in liquid phase is reported in a new nontoxic reverse vesicle.

  6. Nanostructure, solvation dynamics, and nanotemplating of plasmonically active SERS substrate in reverse vesicles

    Reverse vesicles (RVs) are the organic counterparts to vesicles and are spherical containers in oils consisting of an oily core surrounded by reverse bilayers with water layers present in between. We present here a facile route for forming stable RV from nontoxic surfactants and oil components. The RV formation is characterized by dynamic light scattering and further confirmed by transmission electron microscopic (TEM) techniques. The water channels present in between the bilayers are found to be a potential template for inorganic nanoparticles’ (NPs) synthesis. Both the UV–Vis absorption spectroscopy and the TEM study reveal successful formation of highly clustered silver NPs within the water layers of the RVs. X-ray powder diffraction analyzes the crystalline nature of the NPs. FTIR spectroscopy shows the signature of different kinds of water molecules in between the RV bilayers. The dynamical description of the templating water, dictating the controlled formation of the NPs in the RV, is well revealed in the picosecond-resolved solvation dynamics study of a hydrophilic fluorescence probe 2′-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl) -benzimidazo-2-yl-benzimidazole] (H258). The rotational anisotropy study successfully describes geometrical restriction of the probe molecule in the RV. Notably, this study provides the first proof-of-concept data for the ability of the RV to be a template of synthesizing metal NPs. The as-prepared NP clusters are evaluated to be potential surface-enhanced Raman scattering substrate in solution using crystal violet as a model analyte. The present study offers a new RV, which is a prospective nontoxic nanotemplate and is believed to contribute potentially in the emerging NP-vesicle hybrid assembly-based plasmonic applications.Nanotemplating of metal clusters for the efficient SERS detection in liquid phase is reported in a new nontoxic reverse vesicle.

  7. Proteolytic activity within Lysosomes and turnover of pinocytic vesicles: a kinetic analysis

    Degradation of exogenous [125I] ribonuclease by renal lysosomes follows first-order kinetics in ribonuclease concentration. To demonstrate this, it was necessary to apply corrections for the presence of labeled but digestively inactive particles, either pinocytic vesicles or lysosomes damaged during preparation. Such kinetics were not observed under conditions favoring lysosmal breakdown, i.e., in isotonic KCl, or in the absence of EDTA. The kinetic analysis allows determination of half-times for lysosomal protein digestion. This facilitates comparison of different lysosome preparations, or of in vitro degradation rates with results of in vivo metabolism studies. Degradation of [125I] ribonuclease showed a half-time of about 11 and one-half minutes in isotonic sucrose or saline media. This is less than the half-time for decrease of kidney radioactivity in vivo after uptake of [125I] ribonuclease. The proportion of exogenous labeled protein contained within secondary lysosomes was determined as a function of time after injection of ribonuclease to monitor transfer of the protein from pinocytic vesicles to lysosomes. Ribonuclease molecules remained in pinocytic vesicles for approximately three minutes after uptake, before passage into the lysosomes

  8. A2A adenosine receptor antagonism enhances synaptic and motor effects of cocaine via CB1 cannabinoid receptor activation.

    Alessandro Tozzi

    Full Text Available BACKGROUND: Cocaine increases the level of endogenous dopamine (DA in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. PRINCIPAL FINDINGS: Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. CONCLUSIONS: The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine.

  9. Blockade of N-methyl-d-aspartate receptor activation suppresses learning-induced synaptic elimination

    Bock, Jörg; Braun, Katharina

    1999-01-01

    Auditory filial imprinting in the domestic chicken is accompanied by a dramatic loss of spine synapses in two higher associative forebrain areas, the mediorostral neostriatum/hyperstriatum ventrale (MNH) and the dorsocaudal neostriatum (Ndc). The cellular mechanisms that underlie this learning-induced synaptic reorganization are unclear. We found that local pharmacological blockade of N-methyl-d-aspartate (NMDA) receptors in the MNH, a manipulation that has been shown previously to impair aud...

  10. Activity-Dependent Synaptic Plasticity of a Chalcogenide Electronic Synapse for Neuromorphic Systems

    Yi Li; Yingpeng Zhong; Jinjian Zhang; Lei Xu; Qing Wang; Huajun Sun; Hao Tong; Xiaoming Cheng; Xiangshui Miao

    2014-01-01

    Nanoscale inorganic electronic synapses or synaptic devices, which are capable of emulating the functions of biological synapses of brain neuronal systems, are regarded as the basic building blocks for beyond-Von Neumann computing architecture, combining information storage and processing. Here, we demonstrate a Ag/AgInSbTe/Ag structure for chalcogenide memristor-based electronic synapses. The memristive characteristics with reproducible gradual resistance tuning are utilised to mimic the act...

  11. The Interplay between Synaptic Activity and Neuroligin Function in the CNS

    2015-01-01

    Neuroligins (NLs) are postsynaptic transmembrane cell-adhesion proteins that play a key role in the regulation of excitatory and inhibitory synapses. Previous in vitro and in vivo studies have suggested that NLs contribute to synapse formation and synaptic transmission. Consistent with their localization, NL1 and NL3 selectively affect excitatory synapses, whereas NL2 specifically affects inhibitory synapses. Deletions or mutations in NL genes have been found in patients with autism spectrum ...

  12. The impact of synapsins on synaptic plasticity and cognitive behaviors

    Lin ZHANG; Zhong-Xin ZHAO

    2006-01-01

    Synapsins are a family of phosphoproteins specifically associated with the cytoplasmic surface of the synaptic vesicle membrane, appearing to regulate neurotransmitter release, the formation and maintenance of synaptic contacts.They could induce the change of the synaptic plasticity to regulate various adaptation reactions, and change the cognitive behaviors. So we presume that if some cognitive behavior are damaged, synapsins would be changed as well. This gives us a new recognition of better diagnosis and therapy of cognitive disorder desease.

  13. The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons1

    Willets, Jonathon M.; Nelson, Carl P.; Nahorski, Stefan R; Challiss, R.A. John

    2007-01-01

    To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC50 concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M1 mACh r...

  14. Activity-induced synaptic delivery of the GluN2A-containing NMDA receptor is dependent on endoplasmic reticulum chaperone Bip and involved in fear memory.

    Zhang, Xiao-min; Yan, Xun-yi; Zhang, Bin; Yang, Qian; Ye, Mao; Cao, Wei; Qiang, Wen-bin; Zhu, Li-jun; Du, Yong-lan; Xu, Xing-xing; Wang, Jia-sheng; Xu, Fei; Lu, Wei; Qiu, Shuang; Yang, Wei; Luo, Jian-hong

    2015-07-01

    The N-methyl-D-aspartate receptor (NMDAR) in adult forebrain is a heterotetramer mainly composed of two GluN1 subunits and two GluN2A and/or GluN2B subunits. The synaptic expression and relative numbers of GluN2A- and GluN2B-containing NMDARs play critical roles in controlling Ca(2+)-dependent signaling and synaptic plasticity. Previous studies have suggested that the synaptic trafficking of NMDAR subtypes is differentially regulated, but the precise molecular mechanism is not yet clear. In this study, we demonstrated that Bip, an endoplasmic reticulum (ER) chaperone, selectively interacted with GluN2A and mediated the neuronal activity-induced assembly and synaptic incorporation of the GluN2A-containing NMDAR from dendritic ER. Furthermore, the GluN2A-specific synaptic trafficking was effectively disrupted by peptides interrupting the interaction between Bip and GluN2A. Interestingly, fear conditioning in mice was disrupted by intraperitoneal injection of the interfering peptide before training. In summary, we have uncovered a novel mechanism for the activity-dependent supply of synaptic GluN2A-containing NMDARs, and demonstrated its relevance to memory formation. PMID:26088419

  15. Altered active zones, vesicle pools, nerve terminal conductivity, and morphology during experimental MuSK myasthenia gravis.

    Vishwendra Patel

    Full Text Available Recent studies demonstrate reduced motor-nerve function during autoimmune muscle-specific tyrosine kinase (MuSK myasthenia gravis (MG. To further understand the basis of motor-nerve dysfunction during MuSK-MG, we immunized female C57/B6 mice with purified rat MuSK ectodomain. Nerve-muscle preparations were dissected and neuromuscular junctions (NMJs studied electrophysiologically, morphologically, and biochemically. While all mice produced antibodies to MuSK, only 40% developed respiratory muscle weakness. In vitro study of respiratory nerve-muscle preparations isolated from these affected mice revealed that 78% of NMJs produced endplate currents (EPCs with significantly reduced quantal content, although potentiation and depression at 50 Hz remained qualitatively normal. EPC and mEPC amplitude variability indicated significantly reduced number of vesicle-release sites (active zones and reduced probability of vesicle release. The readily releasable vesicle pool size and the frequency of large amplitude mEPCs also declined. The remaining NMJs had intermittent (4% or complete (18% failure of neurotransmitter release in response to 50 Hz nerve stimulation, presumably due to blocked action potential entry into the nerve terminal, which may arise from nerve terminal swelling and thinning. Since MuSK-MG-affected muscles do not express the AChR γ subunit, the observed prolongation of EPC decay time was not due to inactivity-induced expression of embryonic acetylcholine receptor, but rather to reduced catalytic activity of acetylcholinesterase. Muscle protein levels of MuSK did not change. These findings provide novel insight into the pathophysiology of autoimmune MuSK-MG.

  16. Selective optical control of synaptic transmission in the subcortical visual pathway by activation of viral vector-expressed halorhodopsin.

    Katsuyuki Kaneda

    Full Text Available The superficial layer of the superior colliculus (sSC receives visual inputs via two different pathways: from the retina and the primary visual cortex. However, the functional significance of each input for the operation of the sSC circuit remains to be identified. As a first step toward understanding the functional role of each of these inputs, we developed an optogenetic method to specifically suppress the synaptic transmission in the retino-tectal pathway. We introduced enhanced halorhodopsin (eNpHR, a yellow light-sensitive, membrane-targeting chloride pump, into mouse retinal ganglion cells (RGCs by intravitreously injecting an adeno-associated virus serotype-2 vector carrying the CMV-eNpHR-EYFP construct. Several weeks after the injection, whole-cell recordings made from sSC neurons in slice preparations revealed that yellow laser illumination of the eNpHR-expressing retino-tectal axons, putatively synapsing onto the recorded cells, effectively inhibited EPSCs evoked by electrical stimulation of the optic nerve layer. We also showed that sSC spike activities elicited by visual stimulation were significantly reduced by laser illumination of the sSC in anesthetized mice. These results indicate that photo-activation of eNpHR expressed in RGC axons enables selective blockade of retino-tectal synaptic transmission. The method established here can most likely be applied to a variety of brain regions for studying the function of individual inputs to these regions.

  17. Nonequivalent release sites govern synaptic depression.

    Wen, Hua; McGinley, Matthew J; Mandel, Gail; Brehm, Paul

    2016-01-19

    Synaptic depression is prominent among synapses, but the underlying mechanisms remain uncertain. Here, we use paired patch clamp recording to study neuromuscular transmission between the caudal primary motor neuron and target skeletal muscle in zebrafish. This synapse has an unusually low number of release sites, all with high probabilities of release in response to low-frequency stimulation. During high-frequency stimulation, the synapse undergoes short-term depression and reaches steady-state levels of transmission that sustain the swimming behavior. To determine the release parameters underlying this steady state, we applied variance analysis. Our analysis revealed two functionally distinct subclasses of release sites differing by over 60-fold in rates of vesicle reloading. A slow reloading class requires seconds to recover and contributes to depression onset but not the steady-state transmission. By contrast, a fast reloading class recovers within tens of milliseconds and is solely responsible for steady-state transmission. Thus, in contrast to most current models that assign levels of steady-state depression to vesicle availability, our findings instead assign this function to nonuniform release site kinetics. The duality of active-site properties accounts for the highly nonlinear dependence of steady-state depression levels on frequency. PMID:26715759

  18. The influence of transition and heavy metal ions on ATP-ases activity in rat synaptic plasma membranes

    VESNA VASIC

    2004-07-01

    Full Text Available The influence of transition metal (Cu2+, Zn2+, Fe2+ and Co2+ and heavy metal ions (Hg2+, Pb2+ and Cd2+ on the activities of Na+/K+-ATPase and Mg2+-ATPase isolated from rat synaptic plasma membranes (SPM was investigated. The aim of the study was to elucidate the inhibition of both ATPase activities by exposure to the considered metal ions as a function of their affinity to bind to the –SH containing ligand L-cysteine, as a model system. The half-maximum inhibitory activities (IC50 of the enzymes were determined as parameters of rectangular hyperbolas and correlated with the stability constant (Ks of the respective metal-ion-L-cysteine complex. The linear Dixon plots indicate equilibrium binding of the investigated ions to both enzymes.

  19. SENSITIVE EFFECTS OF POTASSIUM AND CALCIUM CHANNEL BLOCKING AND ATP-SENSITIVE POTASSIUM CHANNEL ACTIVATORS ON SEMINAL VESICLE SMOOTH MUSCLE CONTRACTIONS

    H SADRAEI

    2000-12-01

    Full Text Available Background. Seminal vesicle smooth muscle contraction is mediated through sympathetic and parasympathetic neurons activity. Although seminal vesicle plays an important role in male fertility, but little attention is given to mechanism involved in contraction of this organ.
    Methods. In this study effects of drugs which activate ATP - sensitive K channels and blockers of K and Ca channels were examined on contraction of guinea - pig isolated seminal vesicle due to electrical filled stimulation (EFS, noradrenaline, carbachol and KCI.
    Results. The K channel blocker tetraethyl ammonium potentate the EFS responses at all frequencies, while, the ATP - sensitive K channel inhibitor glibenclamide and the K channel opener levcromakalim, diazoxide, minoxidil and Ca channel blocker nifedipine all had relaxant effect on guinea - pig seminal vesicle.
    Discussion. This study indicate that activities of K and Ca channels is important in regulation of seminal vesicle contraction due to nerve stimulation, noradrenaline or carbachol.

  20. Mechanisms, pools, and sites of spontaneous vesicle release at synapses of rod and cone photoreceptors.

    Cork, Karlene M; Van Hook, Matthew J; Thoreson, Wallace B

    2016-08-01

    Photoreceptors have depolarized resting potentials that stimulate calcium-dependent release continuously from a large vesicle pool but neurons can also release vesicles without stimulation. We characterized the Ca(2+) dependence, vesicle pools, and release sites involved in spontaneous release at photoreceptor ribbon synapses. In whole-cell recordings from light-adapted horizontal cells (HCs) of tiger salamander retina, we detected miniature excitatory post-synaptic currents (mEPSCs) when no stimulation was applied to promote exocytosis. Blocking Ca(2+) influx by lowering extracellular Ca(2+) , by application of Cd(2+) and other agents reduced the frequency of mEPSCs but did not eliminate them, indicating that mEPSCs can occur independently of Ca(2+) . We also measured release presynaptically from rods and cones by examining quantal glutamate transporter anion currents. Presynaptic quantal event frequency was reduced by Cd(2+) or by increased intracellular Ca(2+) buffering in rods, but not in cones, that were voltage clamped at -70 mV. By inhibiting the vesicle cycle with bafilomycin, we found the frequency of mEPSCs declined more rapidly than the amplitude of evoked excitatory post-synaptic currents (EPSCs) suggesting a possible separation between vesicle pools in evoked and spontaneous exocytosis. We mapped sites of Ca(2+) -independent release using total internal reflectance fluorescence (TIRF) microscopy to visualize fusion of individual vesicles loaded with dextran-conjugated pHrodo. Spontaneous release in rods occurred more frequently at non-ribbon sites than evoked release events. The function of Ca(2+) -independent spontaneous release at continuously active photoreceptor synapses remains unclear, but the low frequency of spontaneous quanta limits their impact on noise. PMID:27255664

  1. Time-dependent reversal of synaptic plasticity induced by physiological concentrations of oligomeric Aβ42: an early index of Alzheimer’s disease

    Koppensteiner, Peter; Trinchese, Fabrizio; Fà, Mauro; Puzzo, Daniela; Gulisano, Walter; Yan, Shijun; Poussin, Arthur; Liu, Shumin; Orozco, Ian; Dale, Elena; Teich, Andrew F.; Palmeri, Agostino; Ninan, Ipe; Boehm, Stefan; Arancio, Ottavio

    2016-01-01

    The oligomeric amyloid-β (Aβ) peptide is thought to contribute to the subtle amnesic changes in Alzheimer’s disease (AD) by causing synaptic dysfunction. Here, we examined the time course of synaptic changes in mouse hippocampal neurons following exposure to Aβ42 at picomolar concentrations, mimicking its physiological levels in the brain. We found opposite effects of the peptide with short exposures in the range of minutes enhancing synaptic plasticity, and longer exposures lasting several hours reducing it. The plasticity reduction was concomitant with an increase in the basal frequency of spontaneous neurotransmitter release, a higher basal number of functional presynaptic release sites, and a redistribution of synaptic proteins including the vesicle-associated proteins synapsin I, synaptophysin, and the post-synaptic glutamate receptor I. These synaptic alterations were mediated by cytoskeletal changes involving actin polymerization and p38 mitogen-activated protein kinase. These in vitro findings were confirmed in vivo with short hippocampal infusions of picomolar Aβ enhancing contextual memory and prolonged infusions impairing it. Our findings provide a model for initiation of synaptic dysfunction whereby exposure to physiologic levels of Aβ for a prolonged period of time causes microstructural changes at the synapse which result in increased transmitter release, failure of synaptic plasticity, and memory loss. PMID:27581852

  2. A Model of Synaptic Reconsolidation

    Kastner, David B.; Schwalger, Tilo; Ziegler, Lorric; Gerstner, Wulfram

    2016-01-01

    Reconsolidation of memories has mostly been studied at the behavioral and molecular level. Here, we put forward a simple extension of existing computational models of synaptic consolidation to capture hippocampal slice experiments that have been interpreted as reconsolidation at the synaptic level. The model implements reconsolidation through stabilization of consolidated synapses by stabilizing entities combined with an activity-dependent reservoir of stabilizing entities that are immune to protein synthesis inhibition (PSI). We derive a reduced version of our model to explore the conditions under which synaptic reconsolidation does or does not occur, often referred to as the boundary conditions of reconsolidation. We find that our computational model of synaptic reconsolidation displays complex boundary conditions. Our results suggest that a limited resource of hypothetical stabilizing molecules or complexes, which may be implemented by protein phosphorylation or different receptor subtypes, can underlie the phenomenon of synaptic reconsolidation. PMID:27242410

  3. Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus? [v1; ref status: indexed, http://f1000r.es/TYJStu

    Miriam Matamales

    2012-12-01

    Full Text Available Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance.

  4. Stress-induced enhancement of mouse amygdalar synaptic plasticity depends on glucocorticoid and ß-adrenergic activity.

    Ratna Angela Sarabdjitsingh

    Full Text Available BACKGROUND: Glucocorticoid hormones, in interaction with noradrenaline, enable the consolidation of emotionally arousing and stressful experiences in rodents and humans. Such interaction is thought to occur at least partly in the basolateral nucleus of the amygdala (BLA which is crucially involved in emotional memory formation. Extensive evidence points to long-term synaptic potentiation (LTP as a mechanism contributing to memory formation. Here we determined in adolescent C57/Bl6 mice the effects of stress on LTP in the LA-BLA pathway and the specific roles of corticosteroid and β-adrenergic receptor activation in this process. PRINCIPAL FINDINGS: Exposure to 20 min of restraint stress (compared to control treatment prior to slice preparation enhanced subsequent LTP induction in vitro, without affecting baseline fEPSP responses. The role of glucocorticoid receptors, mineralocorticoid receptors and β2-adrenoceptors in the effects of stress was studied by treating mice with the antagonists mifepristone, spironolactone or propranolol respectively (or the corresponding vehicles prior to stress or control treatment. In undisturbed controls, mifepristone and propranolol administration in vivo did not influence LTP induced in vitro. By contrast, spironolactone caused a gradually attenuating form of LTP, both in unstressed and stressed mice. Mifepristone treatment prior to stress strongly reduced the ability to induce LTP in vitro. Propranolol normalized the stress-induced enhancement of LTP to control levels during the first 10 min after high frequency stimulation, after which synaptic responses further declined. CONCLUSIONS: Acute stress changes BLA electrical properties such that subsequent LTP induction is facilitated. Both β-adrenergic and glucocorticoid receptors are involved in the development of these changes. Mineralocorticoid receptors are important for the maintenance of LTP in the BLA, irrespective of stress-induced changes in the

  5. Calmodulin as a major calcium buffer shaping vesicular release and short-term synaptic plasticity: facilitation through buffer dislocation

    Yulia Timofeeva

    2015-07-01

    Full Text Available Action potential-dependent release of synaptic vesicles and short-term synaptic plasticity are dynamically regulated by the endogenous Ca2+ buffers that shape [Ca2+] profiles within a presynaptic bouton. Calmodulin is one of the most abundant presynaptic proteins and it binds Ca2+ faster than any other characterized endogenous neuronal Ca2+ buffer. Direct effects of calmodulin on fast presynaptic Ca2+ dynamics and vesicular release however have not been studied in detail. Using experimentally constrained three-dimensional diffusion modeling of Ca2+ influx–exocytosis coupling at small excitatory synapses we show that, at physiologically relevant concentrations, Ca2+ buffering by calmodulin plays a dominant role in inhibiting vesicular release and in modulating short-term synaptic plasticity. We also propose a novel and potentially powerful mechanism for short-term facilitation based on Ca2+-dependent dynamic dislocation of calmodulin molecules from the plasma membrane within the active zone.

  6. Effects of La3+ on ATPase Activities of Plasma Membrane Vesicles Isolated from Casuarina Equisetifolia Seedlings under Acid Rain Stress

    李裕红; 严重玲; 刘景春; 陈英华; 胡俊; 薛博

    2003-01-01

    The effects of La3+ on the growth and the ATPases activities of plasma membrane(PM) vesicles isolated from Casuarina equisetifolia seedlings under artificial acid rain(pH 4.5) stress were studied. The results show that the height, length of roots, fresh weight and PM H+-ATPase activites of Casuarina equisetifolia seedlings increase by the treatments of soaking seeds in LaCl3 solutions with lower concentrations, and those can reach their peak values by treating with 200 mg·L-1 La3+. However, in comparison with the CK, those are inhibited by the higher La3+ concentrations; PM Ca2+-ATPase activity is inhibited with the treatments of La3+. The results also reveal that the H+-ATPase activity and the growth of cell enlarge have a remarkable positive correlation, and La3+ activating H+-ATPase can facilitate plant growth. La3+ also can alleviate cytosolic acidification of plant under acid rain stress and indirectly maintain the stability of intracellular environment. In order to resistant to acid rain and accelerate the growth of Casuarina equisetifolia, the suitable range of La3+ concentrations to soak seeds for 8 h is 50~200 mg*L-1.

  7. Astrocytes Potentiate Synaptic Transmission

    Nadkarni, Suhita

    2005-03-01

    A recent experimental study shows that astrocytes, a subtype of glia, are able to influence the spontaneous activity in the brain via calcium dependent glutamate release. We model the coupling mechanism between an astrocyte and a neuron based on experimental data. This coupling is dynamic and bi-directional, such that the modulations in intracellular calcium concentrations in astrocytes affect neuronal excitability and vice versa via a glutamatergic pathway. We demonstrate through simple neural-glial circuits that increases in the intracellular calcium concentration in astrocytes nearby can enhance spontaneous activity in a neuron, a significant mechanism said to be involved in plasticity and learning. The pattern of this marked increase in spontaneous firing rate in our model quantitatively follows that observed in the experiment. Further, depending on the type of synaptic connections diverging from the neuron, it can either inhibit or excite the ensuing dynamics and potentiate synaptic transmission, thus reinstating the integral role played by astrocytes in normal neuronal dynamics.

  8. Cytotoxic and Inflammatory Responses Induced by Outer Membrane Vesicle-Associated Biologically Active Proteases from Vibrio cholerae.

    Mondal, Ayan; Tapader, Rima; Chatterjee, Nabendu Sekhar; Ghosh, Amit; Sinha, Ritam; Koley, Hemanta; Saha, Dhira Rani; Chakrabarti, Manoj K; Wai, Sun Nyunt; Pal, Amit

    2016-05-01

    Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses. PMID:26930702

  9. Proteasome Inhibition Triggers Activity-Dependent Increase in the Size of the Recycling Vesicle Pool in Cultured Hippocampal Neurons

    Willeumier, Kristen; Pulst, Stefan M.; Schweizer, Felix E.

    2006-01-01

    The ubiquitin proteasome system, generally known for its function in protein degradation, also appears to play an important role in regulating membrane trafficking. A role for the proteasome in regulating presynaptic release and vesicle trafficking has been proposed for invertebrates, but it remains to be tested in mammalian presynaptic terminals. We used the fluorescent styrylpyridinium dye FM4-64 to visualize changes in the recycling pool of vesicles in hippocampal culture under pharmacolog...

  10. Free D-aspartate regulates neuronal dendritic morphology, synaptic plasticity, gray matter volume and brain activity in mammals

    Errico, F; Nisticò, R; Di Giorgio, A; Squillace, M; Vitucci, D; Galbusera, A; Piccinin, S; Mango, D; Fazio, L; Middei, S; Trizio, S; Mercuri, N B; Teule, M A; Centonze, D; Gozzi, A; Blasi, G; Bertolino, A; Usiello, A

    2014-01-01

    D-aspartate (D-Asp) is an atypical amino acid, which is especially abundant in the developing mammalian brain, and can bind to and activate N-methyl-D-Aspartate receptors (NMDARs). In line with its pharmacological features, we find that mice chronically treated with D-Asp show enhanced NMDAR-mediated miniature excitatory postsynaptic currents and basal cerebral blood volume in fronto-hippocampal areas. In addition, we show that both chronic administration of D-Asp and deletion of the gene coding for the catabolic enzyme D-aspartate oxidase (DDO) trigger plastic modifications of neuronal cytoarchitecture in the prefrontal cortex and CA1 subfield of the hippocampus and promote a cytochalasin D-sensitive form of synaptic plasticity in adult mouse brains. To translate these findings in humans and consistent with the experiments using Ddo gene targeting in animals, we performed a hierarchical stepwise translational genetic approach. Specifically, we investigated the association of variation in the gene coding for DDO with complex human prefrontal phenotypes. We demonstrate that genetic variation predicting reduced expression of DDO in postmortem human prefrontal cortex is mapped on greater prefrontal gray matter and activity during working memory as measured with MRI. In conclusion our results identify novel NMDAR-dependent effects of D-Asp on plasticity and physiology in rodents, which also map to prefrontal phenotypes in humans. PMID:25072322

  11. Activation of the anti-inflammatory reflex blocks lipopolysaccharide-induced decrease in synaptic inhibition in the temporal cortex of the rat.

    Garcia-Oscos, Francisco; Peña, David; Housini, Mohammad; Cheng, Derek; Lopez, Diego; Cuevas-Olguin, Roberto; Saderi, Nadia; Salgado Delgado, Roberto; Galindo Charles, Luis; Salgado Burgos, Humberto; Rose-John, Stefan; Flores, Gonzalo; Kilgard, Michael P; Atzori, Marco

    2015-06-01

    Stress is a potential trigger for a number of neuropsychiatric conditions, including anxiety syndromes and schizophrenic psychoses. The temporal neocortex is a stress-sensitive area involved in the development of such conditions. We have recently shown that aseptic inflammation and mild electric shock shift the balance between synaptic excitation and synaptic inhibition in favor of the former in this brain area (Garcia-Oscos et al., 2012), as well as in the prefrontal cortex (Garcia-Oscos et al., 2014). Given the potential clinical importance of this phenomenon in the etiology of hyperexcitable neuropsychiatric illness, this study investigates whether inactivation of the peripheral immune system by the "anti-inflammatory reflex" would reduce the central response to aseptic inflammation. For a model of aseptic inflammation, this study used i.p. injections of the bacterial toxin lipopolysaccharide (LPS; 5 µM) and activated the anti-inflammatory reflex either pharmacologically by i.p. injections of the nicotinic α7 receptor agonist PHA543613 or physiologically through electrical stimulation of the left vagal nerve (VNS). Patch-clamp recording was used to monitor synaptic function. Recordings from LPS-injected Sprague Dawley rats show that activation of the anti-inflammatory reflex either pharmacologically or by VNS blocks or greatly reduces the LPS-induced decrease of the synaptic inhibitory-to-excitatory ratio and the saturation level of inhibitory current input-output curves. Given the ample variety of pharmacologically available α7 nicotinic receptor agonists as well as the relative safety of clinical VNS already approved by the FDA for the treatment of epilepsy and depression, our findings suggest a new therapeutic avenue in the treatment of stress-induced hyperexcitable conditions mediated by a decrease in synaptic inhibition in the temporal cortex. PMID:25626997

  12. EphA4 Activation of c-Abl Mediates Synaptic Loss and LTP Blockade Caused by Amyloid-β Oligomers

    M. Vargas, Lina; Leal, Nancy; Estrada, Lisbell D.; González, Adrian; Serrano, Felipe; Araya, Katherine; Gysling, Katia; Inestrosa, Nibaldo C.; Pasquale, Elena B.; Alvarez, Alejandra R.

    2014-01-01

    The early stages of Alzheimer's disease are characterised by impaired synaptic plasticity and synapse loss. Here, we show that amyloid-β oligomers (AβOs) activate the c-Abl kinase in dendritic spines of cultured hippocampal neurons and that c-Abl kinase activity is required for AβOs-induced synaptic loss. We also show that the EphA4 receptor tyrosine kinase is upstream of c-Abl activation by AβOs. EphA4 tyrosine phosphorylation (activation) is increased in cultured neurons and synaptoneurosomes exposed to AβOs, and in Alzheimer-transgenic mice brain. We do not detect c-Abl activation in EphA4-knockout neurons exposed to AβOs. More interestingly, we demonstrate EphA4/c-Abl activation is a key-signalling event that mediates the synaptic damage induced by AβOs. According to this results, the EphA4 antagonistic peptide KYL and c-Abl inhibitor STI prevented i) dendritic spine reduction, ii) the blocking of LTP induction and iii) neuronal apoptosis caused by AβOs. Moreover, EphA4-/- neurons or sh-EphA4-transfected neurons showed reduced synaptotoxicity by AβOs. Our results are consistent with EphA4 being a novel receptor that mediates synaptic damage induced by AβOs. EphA4/c-Abl signalling could be a relevant pathway involved in the early cognitive decline observed in Alzheimer's disease patients. PMID:24658113

  13. ZD7288, a selective hyperpolarization-activated cyclic nucleotide-gated channel blocker, inhibits hippocampal synaptic plasticity

    Xiao-xue Zhang; Xiao-chun Min; Xu-lin Xu; Min Zheng; Lian-jun Guo

    2016-01-01

    The selective hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker 4-(N-ethyl-N-phenylamino)-1,2-dimeth-yl-6-(methylamino) pyrimidinium chloride (ZD7288) blocks the induction of long-term potentiation in the perforant path–CA3 region in rat hippocampusin vivo. To explore the mechanisms underlying the action of ZD7288, we recorded excitatory postsynaptic potentials in perforant path–CA3 synapses in male Sprague-Dawley rats. We measured glutamate content in the hippocampus and in cultured hip-pocampal neurons using high performance liquid chromatography, and determined intracellular Ca2+ concentration ([Ca2+]i) using Fura-2. ZD7288 inhibited the induction and maintenance of long-term potentiation, and these effects were mirrored by the nonspeciifc HCN channel blocker cesium. ZD7288 also decreased glutamate release in hippocampal tissue and in cultured hippocampal neurons. Further-more, ZD7288 attenuated glutamate-induced rises in [Ca2+]i in a concentration-dependent manner and reversed 8-Br-cAMP-mediated facilitation of these glutamate-induced [Ca2+]i rises. Our results suggest that ZD7288 inhibits hippocampal synaptic plasticity both gluta-mate release and resultant [Ca2+]i increases in rat hippocampal neurons.

  14. Caffeine Modulates Vesicle Release and Recovery at Cerebellar Parallel Fibre Terminals, Independently of Calcium and Cyclic AMP Signalling.

    Katharine L Dobson

    Full Text Available Cerebellar parallel fibres release glutamate at both the synaptic active zone and at extrasynaptic sites-a process known as ectopic release. These sites exhibit different short-term and long-term plasticity, the basis of which is incompletely understood but depends on the efficiency of vesicle release and recycling. To investigate whether release of calcium from internal stores contributes to these differences in plasticity, we tested the effects of the ryanodine receptor agonist caffeine on both synaptic and ectopic transmission.Whole cell patch clamp recordings from Purkinje neurons and Bergmann glia were carried out in transverse cerebellar slices from juvenile (P16-20 Wistar rats.Caffeine caused complex changes in transmission at both synaptic and ectopic sites. The amplitude of postsynaptic currents in Purkinje neurons and extrasynaptic currents in Bergmann glia were increased 2-fold and 4-fold respectively, but paired pulse ratio was substantially reduced, reversing the short-term facilitation observed under control conditions. Caffeine treatment also caused synaptic sites to depress during 1 Hz stimulation, consistent with inhibition of the usual mechanisms for replenishing vesicles at the active zone. Unexpectedly, pharmacological intervention at known targets for caffeine--intracellular calcium release, and cAMP signalling--had no impact on these effects.We conclude that caffeine increases release probability and inhibits vesicle recovery at parallel fibre synapses, independently of known pharmacological targets. This complex effect would lead to potentiation of transmission at fibres firing at low frequencies, but depression of transmission at high frequency connections.

  15. Neuralized1 Activates CPEB3: A Novel Function of Ubiquitination in Synaptic Plasticity and Memory Storage

    Pavlopoulos, Elias; Trifilieff, Pierre; Chevaleyre, Vivien; Fioriti, Luana; Zairis, Sakellarios; Pagano, Andrew; Malleret, Gaël; Kandel, Eric R.

    2011-01-01

    The cytoplasmic polyadenylation element-binding protein 3 (CPEB3), a regulator of local protein synthesis, is the mouse homologue of ApCPEB, a functional prion protein in Aplysia. Here, we provide evidence that CPEB3 is activated by Neuralized1, an E3 ubiquitin ligase. In hippocampal cultures, CPEB3 activated by Neuralized1-mediated ubiquitination leads both to the growth of new dendritic spines and to an increase of the GluA1 and GluA2 subunits of AMPA receptors, two CPEB3 targets essential ...

  16. Regulation of synaptic connectivity by glia

    Eroglu, Cagla; Barres, Ben A

    2010-01-01

    The human brain contains more than 100 trillion (1014) synaptic connections, which form all of its neural circuits. Neuroscientists have long been interested in how this complex synaptic web is weaved during development and remodelled during learning and disease. Recent studies have uncovered that glial cells are important regulators of synaptic connectivity. These cells are far more active than was previously thought and are powerful controllers of synapse formation, function, plasticity and...

  17. Synaptically Released Matrix Metalloproteinase Activity in Control of Structural Plasticity and the Cell Surface Distribution of GluA1-AMPA Receptors

    Zsuzsanna Szepesi; Eric Hosy; Blazej Ruszczycki; Monika Bijata; Marta Pyskaty; Arthur Bikbaev; Martin Heine; Daniel Choquet; Leszek Kaczmarek; Jakub Wlodarczyk

    2014-01-01

    Synapses are particularly prone to dynamic alterations and thus play a major role in neuronal plasticity. Dynamic excitatory synapses are located at the membranous neuronal protrusions called dendritic spines. The ability to change synaptic connections involves both alterations at the morphological level and changes in postsynaptic receptor composition. We report that endogenous matrix metalloproteinase (MMP) activity promotes the structural and functional plasticity of local synapses by its ...

  18. Hypocretinergic facilitation of synaptic activity of neurons in the nucleus pontis oralis of the cat.

    Xi, Ming Chu; Fung, Simon J; Yamuy, Jack; Morales, Francisco R; Chase, Michael H

    2003-06-27

    The present study was undertaken to explore the neuronal mechanisms of hypocretin actions on neurons in the nucleus pontis oralis (NPO), a nucleus which plays a key role in the generation of active (REM) sleep. Specifically, we sought to determine whether excitatory postsynaptic potentials (EPSPs) evoked by stimulation of the laterodorsal tegmental nucleus (LDT) and spontaneous EPSPs in NPO neurons are modulated by hypocretin. Accordingly, recordings were obtained from NPO neurons in the cat in conjunction with the juxtacellular microinjection of hypocretin-1 onto intracellularly recorded cells. The application of hypocretin-1 significantly increased the mean amplitude of LDT-evoked EPSPs of NPO neurons. In addition, the frequency and the amplitude of spontaneous EPSPs in NPO neurons increased following hypocretin-1 administration. These data suggest that hypocretinergic processes in the NPO are capable of modulating the activity of NPO neurons that receive excitatory cholinergic inputs from neurons in the LDT. PMID:12763260

  19. Synaptic GABA release prevents GABA transporter type-1 reversal during excessive network activity

    Savtchenko, L.; Megalogeni, M.; Rusakov, D. A.; Walker, M. C.; Pavlov, I.

    2015-01-01

    GABA transporters control extracellular GABA, which regulates the key aspects of neuronal and network behaviour. A prevailing view is that modest neuronal depolarization results in GABA transporter type-1 (GAT-1) reversal causing non-vesicular GABA release into the extracellular space during intense network activity. This has important implications for GABA uptake-targeting therapies. Here we combined a realistic kinetic model of GAT-1 with experimental measurements of tonic GABAA receptor cu...

  20. Enhanced synaptic activity and epileptiform events in the embryonic Kcc2 deficient hippocampus

    Thomas J Jentsch

    2011-11-01

    Full Text Available The neuronal potassium-chloride co-transporter Kcc2 is thought to play an important role in the post natal excitatory to inhibitory switch of GABA actions in the rodent hippocampus. Here, by studying hippocampi of wild-type (Kcc2+/+ and Kcc2 deficient (Kcc2-/- mouse embryos, we unexpectedly found increased spontaneous neuronal network activity at E18.5, a developmental stage when Kcc2 is thought not to be functional in the hippocampus. Embryonic Kcc2-/- hippocampi have also an augmented synapse density and a higher frequency of spontaneous glutamatergic and GABAergic postsynaptic currents (PSCs than naïve age matched neurons. However, intracellular chloride concentration ([Cl-]i and the reversal potential of GABA-mediated currents (EGABA were similar in embryonic Kcc2+/+ and Kcc2-/- CA3 neurons. In addition, Kcc2 immuno-labelling was cytoplasmic in the majority of neurons suggesting that the molecule is not functional as a plasma membrane chloride co-transporter. Collectively, our results show that already at an embryonic stage, Kcc2 controls the formation of synapses and, when deleted, the hippocampus has a higher density of GABAergic and glutamatergic synapses and generates spontaneous and evoked epileptiform activities. These results may be explained either by a small population of orchestrating neurons in which Kcc2 operates early as a chloride exporter or by transporter independent actions of Kcc2 that are instrumental in synapses formation and networks construction.

  1. INVOLVEMENT OF SYNAPTIC GENES IN THE PATHOGENESIS OF AUTISM SPECTRUM DISORDERS: THE CASE OF SYNAPSINS

    Silvia eGiovedi

    2014-09-01

    Full Text Available Autism spectrum disorders (ASDs are heterogeneous neurodevelopmental disorders characterized by deficits in social interaction and social communication, restricted interests and repetitive behaviors. Many synaptic protein genes are linked to the pathogenesis of ASDs, making them prototypical synaptopathies. An array of mutations in the synapsin (Syn genes in humans have been recently associated with ASD and epilepsy, diseases that display a frequent comorbidity. Synapsins are presynaptic proteins regulating synaptic vesicle traffic, neurotransmitter release and short-term synaptic plasticity. In doing so, Syn isoforms control the tone of activity of neural circuits and the balance between excitation and inhibition. As ASD pathogenesis is believed to result from dysfunctions in the balance between excitatory and inhibitory transmissions in neocortical areas, Syns are novel ASD candidate genes. Accordingly, deletion of single Syn genes in mice, in addition to epilepsy, causes core symptoms of ASD by affecting social behavior, social communication and repetitive behaviors. Thus, Syn knockout mice represent a good experimental model to define synaptic alterations involved in the pathogenesis of ASD and epilepsy.

  2. Engineered Asymmetric Synthetic Vesicles

    Lu, Li; Chiarot, Paul

    2013-11-01

    Synthetic vesicles are small, fluid-filled spheres that are enclosed by a bilayer of lipid molecules. They can be used as models for investigating membrane biology and as delivery vehicles for pharmaceuticals. In practice, it is difficult to simultaneously control membrane asymmetry, unilamellarity, vesicle size, vesicle-to-vesicle uniformity, and luminal content. Membrane asymmetry, where each leaflet of the bilayer is composed of different lipids, is of particular importance as it is a feature of most natural membranes. In this study, we leverage microfluidic technology to build asymmetric vesicles at high-throughput. We use the precise flow control offered by microfluidic devices to make highly uniform emulsions, with controlled internal content, that serve as templates to build the synthetic vesicles. Flow focusing, dielectrophoretic steering, and interfacial lipid self-assembly are critical procedures performed on-chip to produce the vesicles. Fluorescent and confocal microscopy are used to evaluate the vesicle characteristics.

  3. Enhanced Synaptic Connectivity in the Dentate Gyrus during Epileptiform Activity: Network Simulation

    Keite Lira de Almeida França

    2013-01-01

    Full Text Available Structural rearrangement of the dentate gyrus has been described as the underlying cause of many types of epilepsies, particularly temporal lobe epilepsy. It is said to occur when aberrant connections are established in the damaged hippocampus, as described in human epilepsy and experimental models. Computer modelling of the dentate gyrus circuitry and the corresponding structural changes has been used to understand how abnormal mossy fibre sprouting can subserve seizure generation observed in experimental models when epileptogenesis is induced by status epilepticus. The model follows the McCulloch-Pitts formalism including the representation of the nonsynaptic mechanisms. The neuronal network comprised granule cells, mossy cells, and interneurons. The compensation theory and the Hebbian and anti-Hebbian rules were used to describe the structural rearrangement including the effects of the nonsynaptic mechanisms on the neuronal activity. The simulations were based on neuroanatomic data and on the connectivity pattern between the cells represented. The results suggest that there is a joint action of the compensation theory and Hebbian rules during the inflammatory process that accompanies the status epilepticus. The structural rearrangement simulated for the dentate gyrus circuitry promotes speculation about the formation of the abnormal mossy fiber sprouting and its role in epileptic seizures.

  4. Melamine Alters Glutamatergic Synaptic Transmission of CA3-CA1 Synapses Presynaptically Through Autophagy Activation in the Rat Hippocampus.

    Zhang, Hui; Wang, Hui; Xiao, Xi; Zhang, Tao

    2016-01-01

    Melamine is an industrial chemical that can cause central nervous system disorders including excitotoxicity and cognitive impairment. Its illegal use in powdered baby formula was the focus of a milk scandal in China in 2008. One of our previous studies showed that melamine impaired glutamatergic transmission in rat hippocampal CA1 pyramidal cells. However, the underlying mechanism of action of melamine is unclear, and it is unknown if the CA3-CA1 pathway is directly involved. In the present study, a whole-cell patch-clamp technique was employed to investigate the effect of melamine on the hippocampal CA3-CA1 pathway in vitro. Both the evoked excitatory postsynaptic current (eEPSC) and the paired-pulse ratio (PPR) were recorded. Furthermore, we examined whether autophagy was involved in glutamatergic transmission alterations induced by melamine. Our data showed that melamine significantly increased the amplitude of eEPSCs in a dose-dependent manner. Inhibition of the N-methyl-D-aspartic acid receptor did not prevent the increase in eEPSC amplitude. In addition, the PPR was remarkably decreased by a melamine concentration of 5 × 10(-5) g/mL. It was found that autophagy could be activated by melamine and an autophagy inhibitor, 3-MA, prevented the melamine-induced increase in eEPSC amplitude. Overall, our results show that melamine presynaptically alters glutamatergic synaptic transmission of hippocampal CA3-CA1 synapses in vitro and this is likely associated with autophagy alteration. PMID:26530910

  5. Calcium-Activated Proteases Are Critical for Refilling Depleted Vesicle Stores in Cultured Sensory-Motor Synapses of "Aplysia"

    Khoutorsky, Arkady; Spira, Micha E.

    2005-01-01

    "Aplysia" motoneurons cocultured with a presynaptic sensory neuron exhibit homosynaptic depression when stimulated at low frequencies. A single bath application of serotonin (5HT) leads within seconds to facilitation of the depressed synapse. The facilitation is attributed to mobilization of neurotransmitter-containing vesicles from a feeding…

  6. POLYELEOSTEARIC ACID VESICLES

    LI Zichen; XIE Ximng; FAN Qinghua; FANG Yifei

    1992-01-01

    α-Eleostearic acid and β-eleostearic acid formed vesicles in aqueous medium when an ethanol solutionofeleostearic acid was injected rapidly into a vigorously vortexed aqueous phase. Formation of the vesicles was demonstrated by electron microscopic observation and bromothymol blue encapsulation experiments. Polymerizations of the eleostearic acids in the formed vesicles carried out by UV irradiation produced poly-α-eleostearic acid and poly-β-eleostearic acid vesicles.

  7. Dopamine modulates persistent synaptic activity and enhances the signal-to-noise ratio in the prefrontal cortex.

    Sven Kroener

    Full Text Available BACKGROUND: The importance of dopamine (DA for prefrontal cortical (PFC cognitive functions is widely recognized, but its mechanisms of action remain controversial. DA is thought to increase signal gain in active networks according to an inverted U dose-response curve, and these effects may depend on both tonic and phasic release of DA from midbrain ventral tegmental area (VTA neurons. METHODOLOGY/PRINCIPAL FINDINGS: We used patch-clamp recordings in organotypic co-cultures of the PFC, hippocampus and VTA to study DA modulation of spontaneous network activity in the form of Up-states and signals in the form of synchronous EPSP trains. These cultures possessed a tonic DA level and stimulation of the VTA evoked DA transients within the PFC. The addition of high (> or = 1 microM concentrations of exogenous DA to the cultures reduced Up-states and diminished excitatory synaptic inputs (EPSPs evoked during the Down-state. Increasing endogenous DA via bath application of cocaine also reduced Up-states. Lower concentrations of exogenous DA (0.1 microM had no effect on the up-state itself, but they selectively increased the efficiency of a train of EPSPs to evoke spikes during the Up-state. When the background DA was eliminated by depleting DA with reserpine and alpha-methyl-p-tyrosine, or by preparing corticolimbic co-cultures without the VTA slice, Up-states could be enhanced by low concentrations (0.1-1 microM of DA that had no effect in the VTA containing cultures. Finally, in spite of the concentration-dependent effects on Up-states, exogenous DA at all but the lowest concentrations increased intracellular current-pulse evoked firing in all cultures underlining the complexity of DA's effects in an active network. CONCLUSIONS/SIGNIFICANCE: Taken together, these data show concentration-dependent effects of DA on global PFC network activity and they demonstrate a mechanism through which optimal levels of DA can modulate signal gain to support

  8. Lung epithelial cell-derived extracellular vesicles activate macrophage-mediated inflammatory responses via ROCK1 pathway.

    Moon, H-G; Cao, Y; Yang, J; Lee, J H; Choi, H S; Jin, Y

    2015-01-01

    Despite decades of research, the pathogenesis of acute respiratory distress syndrome (ARDS) remains poorly understood, thus impeding the development of effective treatment. Diffuse alveolar damage (DAD) and lung epithelial cell death are prominent features of ARDS. Lung epithelial cells are the first line of defense after inhaled stimuli, such as in the case of hyperoxia. We hypothesized that lung epithelial cells release 'messenger' or signaling molecules to adjacent or distant macrophages, thereby initiating or propagating inflammatory responses after noxious insult. We found that, after hyperoxia, a large amount of extracellular vesicles (EVs) were generated and released into bronchoalveolar lavage fluid (BALF). These hyperoxia-induced EVs were mainly derived from live lung epithelial cells as the result of hyperoxia-associated endoplasmic reticulum (ER) stress. These EVs were remarkably different from epithelial 'apoptotic bodies', as reflected by the significantly smaller size and differentially expressed protein markers. These EVs fall mainly in the size range of the exosomes and smaller microvesicles (MVs) (50-120 nm). The commonly featured protein markers of apoptotic bodies were not found in these EVs. Treating alveolar macrophages with hyperoxia-induced, epithelial cell-derived EVs led to an increased secretion of pro-inflammatory cytokines and macrophage inflammatory protein 2 (MIP-2). Robustly increased macrophage and neutrophil influx was found in the lung tissue of the mice intranasally treated with hyperoxia-induced EVs. It was determined that EV-encapsulated caspase-3 was largely responsible for the alveolar macrophage activation via the ROCK1 pathway. Caspase-3-deficient EVs induced less cytokine/MIP-2 release, reduced cell counts in BALF, less neutrophil infiltration and less inflammation in lung parenchyma, both in vitro and in vivo. Furthermore, the serum circulating EVs were increased and mainly derived from lung epithelial cells after

  9. Na(+)-I- symport activity is present in membrane vesicles from thyrotropin-deprived non-I(-)-transporting cultured thyroid cells.

    Kaminsky, S. M.; Levy, O.; Salvador, C.; Dai, G.; Carrasco, N.

    1994-01-01

    The active accumulation of I- in the thyroid gland is mediated by the Na(+)-I- symporter and driven by the Na+ gradient generated by the Na+/K(+)-ATPase. Thyrotropin (TSH) stimulates thyroidal I- accumulation. Rat thyroid-derived FRTL-5 cells require TSH to accumulate I-. TSH withdrawal for over 7 days results in complete loss of Na(+)-I-symport activity in these cells [Weiss, S. J., Philp, N. J. and Grollman, E. F. (1984) Endocrinology 114, 1090-1098]. Surprisingly, membrane vesicles prepare...

  10. Dynamic changes in cytosolic ATP levels in cultured glutamatergic neurons during NMDA-induced synaptic activity supported by glucose or lactate

    Lange, Sofie Cecilie; Winkler, Ulrike; Andresen, Lars;

    2015-01-01

    biosensor Ateam1.03YEMK. While inducing synaptic activity by subjecting cultured neurons to two 30 s pulses of NMDA (30 µM) with a 4 min interval, changes in relative ATP levels were measured in the presence of lactate (1 mM), glucose (2.5 mM) or the combination of the two. ATP levels reversibly declined...... following NMDA-induced neurotransmission activity, as indicated by a reversible 10-20 % decrease in the response of the biosensor. The responses were absent when the NMDA receptor antagonist memantine was present. In the presence of lactate alone, the ATP response dropped significantly more than in the...