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Timothy A. Ryan - One of the best experts on this subject based on the ideXlab platform.
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The kinetics of synaptic vesicle reacidification at hippocampal nerve terminals.
Journal of Neuroscience, 2006Co-Authors: Pradeep P. Atluri, Timothy A. RyanAbstract:After exocytosis, synaptic vesicles are recycled locally in the synaptic terminal and are refilled with neurotransmitter via vesicular transporters. The biophysical mechanisms of refilling are poorly understood, but it is clear that the generation of a proton gradient across the vesicle membrane is crucial. To better understand the determinants of vesicle refilling, we developed a novel method to measure unambiguously the kinetics of synaptic vesicle reacidification at individual synaptic terminals. Hippocampal neurons transfected with Synapto-pHluorin (SpH), a synaptic vesicle-targeted lumenal GFP (green fluorescent protein), whose fluorescence is quenched when protonated (pKa ∼ 7.1), were rapidly surface-quenched immediately after trains of repetitive electrical stimulation. The recently endocytosed alkaline pool of SpH is protected from such surface quenching, and its fluorescence decay reflects reacidification kinetics. These measurements indicate that, after compensatory endocytosis, synaptic vesicles reacidify with first-order kinetics (τ ∼ 4–5 s) and that their rate of reacidification is subject to slowing by increased external buffer.
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The General Anesthetic Isoflurane Depresses Synaptic Vesicle Exocytosis
Molecular Pharmacology, 2005Co-Authors: Hugh C. Hemmings, Wayne Yan, Robert I. Westphalen, Timothy A. RyanAbstract:General anesthetics have marked effects on synaptic transmission, but the mechanisms of their presynaptic actions are unclear. We used quantitative laser-scanning fluorescence microscopy to analyze the effects of the volatile anesthetic isoflurane on synaptic vesicle cycling in cultured neonatal rat hippocampal neurons monitored using either transfection of a pH-sensitive form of green fluorescent protein fused to the luminal domain of VAMP (vesicle-associated membrane protein), (Synapto-pHluorin) or vesicle loading with the fluorescent dye FM 1–43. Isoflurane reversibly inhibited action potential-evoked exocytosis over a range of concentrations, with little effect on vesicle pool size. In contrast, exocytosis evoked by depolarization in response to an elevated extracellular concentration of KCl, which is insensitive to the selective Na+ channel blocker tetrodotoxin, was relatively insensitive to isoflurane. Inhibition of exocytosis by isoflurane was resistant to bicuculline, indicating that this presynaptic effect is not caused by the well known GABAA receptor modulation by volatile anesthetics. Depression of exocytosis was mimicked by a reduction in stimulus frequency, suggesting a reduction in action potential initiation, conduction, or coupling to Ca2+ channel activation. There was no evidence for a direct effect on endocytosis. The effects of isoflurane on synaptic transmission are thus caused primarily by inhibition of action potential-evoked synaptic vesicle exocytosis at a site upstream of Ca2+ entry and exocytosis, possibly as a result of Na+ channel blockade and/or K+ channel activation, with the possibility of lesser contributions from Ca2+ channel blockade and/or soluble N -ethylmaleimide-sensitive factor attachment protein receptor-mediated vesicle fusion.
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kinetic efficiency of endocytosis at mammalian cns synapses requires synaptotagmin i
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Karin Nicholsontomishima, Timothy A. RyanAbstract:At nerve terminals, synaptic vesicle components are retrieved from the cell surface and recycled for local reuse soon after exocytosis. The kinetics of this coupling is critical for the proper functioning of synapses during repetitive action potential firing, because deficiencies in this process lead to abnormal depletion of the releasable vesicle pool. Although the molecular basis of this coupling is poorly understood, numerous biochemical data point to a role for synaptotagmin I (SytI), an essential synaptic vesicle protein required for fast calcium-dependent exocytosis. Here, using Synapto-pHluorin in an approach that allows the dissection of endocytosis and exocytosis into separate components during periods of stimulation, we examined exocytic–endocytic coupling in synapses from SytI knockout mice and their WT littermates. We show that endocytosis is significantly impaired in the absence of SytI with the relative rates of endocytosis compared with exocytosis reduced ≈3-fold with respect to WT. Thus, in addition to regulating exocytosis, SytI also controls the kinetic efficiency of endocytosis at nerve terminals.
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Calcium accelerates endocytosis of vSNAREs at hippocampal synapses
Nature Neuroscience, 2001Co-Authors: Suresh Sankaranarayanan, Timothy A. RyanAbstract:A pH-sensitive form of green-fluorescent protein (GFP) fused to the lumenal domain of VAMP (Synapto-pHluorin) provides a sensitive optical probe to track the net balance between exocytosis and endocytosis of this protein at small synaptic terminals of the central nervous system. Here we used a reversible proton-pump blocker that prevents vesicle re-acidification upon endocytosis to trap vesicles in the alkaline state during recycling. In combination with optical measurements of Synapto-pHluorin, we used alkaline trapping to examine the kinetic components of exocytosis and endocytosis separately at synaptic terminals. Using this approach, we show that, in addition to controlling exocytosis, intracellular calcium levels tightly regulate the speed of endocytosis, increasing it to a maximal speed of approximately one vesicle per second.
Gero Miesenböck - One of the best experts on this subject based on the ideXlab platform.
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Synapto-pHluorins: Genetically Encoded Reporters of Synaptic Transmission
Cold Spring Harbor protocols, 2012Co-Authors: Gero MiesenböckAbstract:pHluorins are pH-sensitive mutants of green fluorescent protein (GFP). Attached to proteins with defined cellular locations or itineraries, pHluorins report subcellular pH as well as protein transport between compartments of differing pH. Key applications in neurobiology include the optical detection of neurotransmitter release with Synapto-pHluorins and their derivatives, as well as measurements of neurotransmitter receptor trafficking. This article describes the properties and uses of Synapto-pHluorins, as well as their advantages and limitations.
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Video-Rate Nonlinear Microscopy of Neuronal Membrane Dynamics With Genetically Encoded Probes
Journal of Neurophysiology, 2004Co-Authors: Robert D. Roorda, Tobias M. Hohl, Ricardo Toledo-crow, Gero MiesenböckAbstract:Biological membranes decorated with suitable contrast agents give rise to nonlinear optical signals such as two-photon fluorescence and harmonic up-conversion when illuminated with ultra-short, high-intensity pulses of infrared laser light. Microscopic images based on these nonlinear contrasts were acquired at video or higher frame rates by scanning a focused illuminating spot rapidly across neural tissues. The scan engine relied on an acousto-optic deflector (AOD) to produce a fast horizontal raster and on corrective prisms to offset the AOD-induced dispersion of the ultra-short excitation light pulses in space and time. Two membrane-bound derivatives of the green fluorescent protein (GFP) were tested as nonlinear contrast agents. Synapto-pHluorin, a pH-sensitive GFP variant fused to a synaptic vesicle membrane protein, provided a time-resolved fluorescent read-out of neurotransmitter release at genetically specified synaptic terminals in the intact brain. Arrays of dually lipidated GFP molecules at the plasma membrane generated intense two-photon fluorescence but no detectable second-harmonic power. Comparison with second-harmonic generation by membranes stained with a synthetic styryl dye suggested that the genetically encoded chromophore arrangement lacked the orientational anisotropy and/or dipole density required for efficient coherent scattering of the incident optical field.
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Synapto-pHluorins: chimeras between pH-sensitive mutants of green fluorescent protein and synaptic vesicle membrane proteins as reporters of neurotransmitter release.
Methods in Enzymology, 2000Co-Authors: Rafael Yuste, R B Miller, K Holthoff, S Zhang, Gero MiesenböckAbstract:Publisher Summary This chapter discusses chimeras between pH-sensitive mutants of green fluorescent protein (GFP) and synaptic vesicle membrane proteins as reporters of neurotransmitter release. Synapto-pHluorins use pH-sensitive mutants of GFP, termed “pHluorins,” to afford the distinction between resting and exocytosed vesicles. Because total fluorescence is the linear sum of emissions from all Synapto-pHluorins in a terminal, increases in the “on” content of this composite spectrum indicate neurotransmitter release. Synapto-pHluorins are chimeric membrane proteins composed of two modules: (1) a pHluorin module that reports local pH and (2) the synaptic vesicle membrane protein VAMP-2 that attaches the pHluorin to the inner vesicle surface. The pHluorin amino terminus is fused to the carboxy terminus of VAMP-2, which is located in the vesicle lumen. This results in type II topology, with a membrane anchor segment that also serves as a noncleavable signal peptide. pHluorins with enhanced brightness, Synapto-pHluorins carrying multiple fluorescent modules, and methods to fully substitute endogenous vesicle proteins with pHluorin-tagged derivatives help to maximize the fluorescent signal emitted per released quantum of neurotransmitter.
Oliver Welzel - One of the best experts on this subject based on the ideXlab platform.
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Dynamic properties of the alkaline vesicle population at hippocampal synapses.
PLoS ONE, 2014Co-Authors: Mareike Röther, Jasmin Jung, Jan M. Brauner, Johannes Kornhuber, Oliver Welzel, Katrin Ebert, Anna Bauereiss, Teja W. GroemerAbstract:In compensatory endocytosis, scission of vesicles from the plasma membrane to the cytoplasm is a prerequisite for intravesicular reacidification and accumulation of neurotransmitter molecules. Here, we provide time-resolved measurements of the dynamics of the alkaline vesicle population which appears upon endocytic retrieval. Using fast perfusion pH-cycling in live-cell microscopy, Synapto-pHluorin expressing rat hippocampal neurons were electrically stimulated. We found that the relative size of the alkaline vesicle population depended significantly on the electrical stimulus size: With increasing number of action potentials the relative size of the alkaline vesicle population expanded. In contrast to that, increasing the stimulus frequency reduced the relative size of the population of alkaline vesicles. Measurement of the time constant for reacification and calculation of the time constant for endocytosis revealed that both time constants were variable with regard to the stimulus condition. Furthermore, we show that the dynamics of the alkaline vesicle population can be predicted by a simple mathematical model. In conclusion, here a novel methodical approach to analyze dynamic properties of alkaline vesicles is presented and validated as a convenient method for the detection of intracellular events. Using this method we show that the population of alkaline vesicles is highly dynamic and depends both on stimulus strength and frequency. Our results implicate that determination of the alkaline vesicle population size may provide new insights into the kinetics of endocytic retrieval.
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The Antidepressant Fluoxetine Mobilizes Vesicles to the Recycling Pool of Rat Hippocampal Synapses During High Activity
Molecular Neurobiology, 2014Co-Authors: Jasmin Jung, Kristina Loy, Eva-maria Schilling, Mareike Röther, Jan M. Brauner, Tobias Huth, Ursula Schlötzer-schrehardt, Christian Alzheimer, Johannes Kornhuber, Oliver WelzelAbstract:Effects of the antidepressant fluoxetine in therapeutic concentration on stimulation-dependent synaptic vesicle recycling were examined in cultured rat hippocampal neurons using fluorescence microscopy. Short-term administration of fluoxetine neither inhibited exocytosis nor endocytosis of RRP vesicular membranes. On the contrary, acute application of the drug markedly increased the size of the recycling pool of hippocampal synapses. This increase in recycling pool size was corroborated using the styryl dye FM 1-43, antibody staining with αSyt1-CypHer™5E and overexpression of Synapto-pHluorin, and was accompanied by an increase in the frequency of miniature postsynaptic currents. Analysis of axonal transport and fluorescence recovery after photobleaching excluded vesicles originating from the synapse-spanning superpool as a source, indicating that these new release-competent vesicles derived from the resting pool. Super resolution microscopy and ultrastructural analysis by electron microscopy revealed that short-term incubation with fluoxetine had no influence on the number of active synapses and synaptic morphology compared to controls. These observations support the idea that therapeutic concentrations of fluoxetine enhance the recycling vesicle pool size and thus the recovery of neurotransmission from exhausting stimuli. The change in the recycling pool size is consistent with the plasticity hypothesis of the pathogenesis of major depressive disorder as stabilization of the vesicle recycling might be responsible for neural outgrowth and plasticity.
Suresh Sankaranarayanan - One of the best experts on this subject based on the ideXlab platform.
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Calcium accelerates endocytosis of vSNAREs at hippocampal synapses
Nature Neuroscience, 2001Co-Authors: Suresh Sankaranarayanan, Timothy A. RyanAbstract:A pH-sensitive form of green-fluorescent protein (GFP) fused to the lumenal domain of VAMP (Synapto-pHluorin) provides a sensitive optical probe to track the net balance between exocytosis and endocytosis of this protein at small synaptic terminals of the central nervous system. Here we used a reversible proton-pump blocker that prevents vesicle re-acidification upon endocytosis to trap vesicles in the alkaline state during recycling. In combination with optical measurements of Synapto-pHluorin, we used alkaline trapping to examine the kinetic components of exocytosis and endocytosis separately at synaptic terminals. Using this approach, we show that, in addition to controlling exocytosis, intracellular calcium levels tightly regulate the speed of endocytosis, increasing it to a maximal speed of approximately one vesicle per second.
Graeme W Davis - One of the best experts on this subject based on the ideXlab platform.
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Mobilization and fusion of a non-recycling pool of synaptic vesicles under conditions of endocytic blockade
Neuropharmacology, 2004Co-Authors: Kira E Poskanzer, Graeme W DavisAbstract:Abstract At vertebrate central synapses, it has been demonstrated that a resting pool of synaptic vesicles (SVs) exists that normally does not participate in SV release and recycling. It remains unclear whether SVs within the resting pool are capable of mobilization and fusion. Here, we combine live imaging of SV exo- and endocytosis using pH-sensitive GFP (Synapto-pHluorins) with pharmacological and genetic manipulations of the SV cycle at the Drosophila NMJ. We demonstrate that a resting pool of SVs exists at this synapse that encompasses 30–41% of the total SV pool. Under conditions of endocytic blockade, using a temperature-sensitive dynamin mutation, the resting pool of SVs can be mobilized and released. We present a model for the presence of a resting pool of SVs that does not require molecular specification of a subpopulation of SVs.
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synaptotagmin i is necessary for compensatory synaptic vesicle endocytosis in vivo
Nature, 2003Co-Authors: Kira E Poskanzer, Kurt W Marek, Sean T Sweeney, Graeme W DavisAbstract:Neurotransmission requires a balance of synaptic vesicle exocytosis and endocytosis1. Synaptotagmin I (Syt I) is widely regarded as the primary calcium sensor for synaptic vesicle exocytosis2,3,4,5,6. Previous biochemical data suggest that Syt I may also function during synaptic vesicle endocytosis7,8,9,10,11,12,13,14,15,16; however, ultrastructural analyses at synapses with impaired Syt I function have provided an indirect and conflicting view of the role of Syt I during synaptic vesicle endocytosis3,8,9,10,14. Until now it has not been possible experimentally to separate the exocytic and endocytic functions of Syt I in vivo. Here, we test directly the role of Syt I during endocytosis in vivo. We use quantitative live imaging of a pH-sensitive green fluorescent protein fused to a synaptic vesicle protein (Synapto-pHluorin) to measure the kinetics of endocytosis in sytI-null Drosophila. We then combine live imaging of the Synapto-pHluorins with photoinactivation of Syt I, through fluorescein-assisted light inactivation, after normal Syt I-mediated vesicle exocytosis. By inactivating Syt I only during endocytosis, we demonstrate that Syt I is necessary for the endocytosis of synaptic vesicles that have undergone exocytosis using a functional Syt I protein.
