The endosomal Q-SNARE, Syntaxin 7, defines a rapidly replenishing synaptic vesicle recycling pool in hippocampal neurons

AbstractUpon the arrival of repetitive stimulation at the presynaptic terminals of neurons, replenishment of readily releasable synaptic vesicles (SVs) with vesicles in the recycling pool is important for sustained neurotransmitter release. Kinetics of replenishment and the available pool size define synaptic performance. However, whether all SVs in the recycling pool are recruited for release with equal probability and speed is unknown. Here, based on comprehensive optical imaging of various presynaptic endosomal SNARE proteins in cultured hippocampal neurons, all of which are implicated in organellar membrane fusion in non-neuronal cells, we show that part of the recycling pool bearing the endosomal Q-SNARE, syntaxin 7 (Stx7), is preferentially mobilized for release during high-frequency repetitive stimulation. Recruitment of the SV pool marked with an Stx7-reporter requires actin polymerization, as well as activation of the Ca2+/calmodulin signaling pathway, reminiscent of rapidly replenishing SVs characterized previously in calyx of Held synapses. Furthermore, disruption of Stx7 function by overexpressing its N-terminal domain selectively abolished this pool. Thus, our data indicate that endosomal membrane fusion involving Stx7 forms rapidly replenishing vesicles essential for synaptic responses to high-frequency repetitive stimulation, and also highlight functional diversities of endosomal SNAREs in generating distinct exocytic vesicles in the presynaptic terminals.

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Q-SNARE Syntaxin 7 confers actin-dependent rapidly replenishing synaptic vesicles upon high activity

10.1101/2020.08.13.249672 ◽

2020 ◽

Author(s):

Yasunori Mori ◽

Koichiro Takenaka ◽

Yugo Fukazawa ◽

Shigeo Takamori

Keyword(s):

High Frequency ◽

Transmitter Release ◽

Synaptic Vesicles ◽

Hippocampal Neurons ◽

Actin Polymerization ◽

Repetitive Stimulation ◽

Snare Proteins ◽

Endosomal Membrane ◽

Kinetics Of ◽

Cultured Hippocampal Neurons

AbstractReplenishment of readily releasable synaptic vesicles (SVs) with vesicles in the recycling pool is important for sustained transmitter release during repetitive stimulation. Kinetics of replenishment and available pool size define synaptic performance. However, whether all SVs in the recycling pool are recruited for release with equal probability is unknown. Here, using comprehensive optical imaging for various presynaptic endosomal SNARE proteins in cultured hippocampal neurons, we demonstrate that part of the recycling pool bearing the endosomal Q–SNARE Syntaxin 7 (Stx7) is preferentially mobilized for release during high–frequency repetitive stimulation. Recruitment of the SV pool marked with the Stx7–reporter requires high intra–terminal Ca2+ concentrations and actin polymerization. Furthermore, disruption of Stx7 function by overexpressing the N–terminal domain selectively abolished this pool. Thus, our data indicate that endosomal membrane fusion involving Stx7 is essential for adaptation of synapses to respond high-frequency repetitive stimulation.

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Faculty Opinions recommendation of Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13408987.14779101 ◽

2011 ◽

Author(s):

Stephen Lockett

Keyword(s):

Fluorescence Microscopy ◽

Membrane Fusion ◽

Actin Polymerization ◽

Computer Assisted

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Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion

The Journal of Lipid Research ◽

10.1194/jlr.m003822 ◽

2010 ◽

Vol 51 (7) ◽

pp. 1747-1760 ◽

Cited By ~ 54

Author(s):

Misbaudeen Abdul-Hammed ◽

Bernadette Breiden ◽

Matthew A. Adebayo ◽

Jonathan O. Babalola ◽

Günter Schwarzmann ◽

...

Keyword(s):

Membrane Fusion ◽

Membrane Lipids ◽

Endosomal Membrane

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The Core Membrane Fusion Complex Governs the Probability of Synaptic Vesicle Fusion But Not Transmitter Release Kinetics

Journal of Neuroscience ◽

10.1523/jneurosci.22-04-01266.2002 ◽

2002 ◽

Vol 22 (4) ◽

pp. 1266-1272 ◽

Cited By ~ 21

Author(s):

Michael F. A. Finley ◽

Sejal M. Patel ◽

Daniel V. Madison ◽

Richard H. Scheller

Keyword(s):

Membrane Fusion ◽

Synaptic Vesicle ◽

Transmitter Release ◽

Release Kinetics ◽

Vesicle Fusion ◽

The Core ◽

Synaptic Vesicle Fusion

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Dynamics of Excitatory Transmitter Release: Analysis of Synaptic Responses in CA3 Hippocampal Neurons After Repetitive Stimulation of Afferent Fibers

Journal of Neurophysiology ◽

10.1152/jn.1998.79.4.1977 ◽

1998 ◽

Vol 79 (4) ◽

pp. 1977-1988 ◽

Cited By ~ 15

Author(s):

Marco Canepari ◽

Enrico Cherubini

Keyword(s):

Transmitter Release ◽

Hippocampal Neurons ◽

Repetitive Stimulation ◽

Patch Clamp Technique ◽

Afferent Fibers ◽

Presynaptic Mechanisms ◽

Release Analysis ◽

Excitatory Transmitter ◽

Stimulation Of ◽

Synaptic Responses

Canepari, Marco and Enrico Cherubini. Dynamics of excitatory transmitter release: analysis of synaptic responses in CA3 hippocampal neurons after repetitive stimulation of afferent fibers. J. Neurophysiol. 79: 1977–1988, 1998. The patch-clamp technique (whole cell configuration) was used to record excitatory postsynaptic currents (EPSCs) evoked by repetitive stimulation (4 pulses at 50-ms intervals) of afferent fibers in the stratum lucidum-radiatum. Different synaptic behaviors (EPSC patterns) were classified in terms of facilitation or depression of the mean amplitude of the second, third, and fourth EPSC with respect to the previous one. A large variety of EPSC patterns was observed by stimulating different afferent fibers. Experiments with the mGluR2/mGluR3 agonist 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) (1 μM), a compound that reduces release at mossy but not at associative commissural fibers and therefore allows to identify the origin of synaptic responses, showed that particular EPSC patterns could not be associated to the activation of a specific type of synaptic input. To investigate the role of the probability of release in the dynamics of synaptic activity, the extracellular calcium concentration was varied from 0.8 to 4 mM in several experiments. EPSC patterns dominated by depression, characteristics of high release probability conditions, could be observed in the majority of the cases in the presence of higher calcium concentrations. A quantitative model for dynamics of transmitter release has been developed. Experimental results were compared with data computed with the model taking into account the probability of release and the time course of reavailability. This work indicates that short-term changes of presynaptic conditions occurring during a train of action potentials can account for the high variability of EPSC responses. The model that is proposed also suggests a general method of experimental data analysis to investigate the possible presynaptic mechanisms underlying long-lasting changes in synaptic efficacy.

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Activity and cytosolic Na+ regulates synaptic vesicle endocytosis

10.1101/2019.12.27.889790 ◽

2019 ◽

Author(s):

Yun Zhu ◽

Dainan Li ◽

Hai Huang

Keyword(s):

Synaptic Transmission ◽

High Frequency ◽

Synaptic Vesicles ◽

Glutamate Uptake ◽

High Frequencies ◽

Vesicle Recycling ◽

Two Photon ◽

Readily Releasable Pool ◽

Content Change ◽

Intracellular Ca

ABSTRACTRetrieval of synaptic vesicles via endocytosis is essential for maintaining sustained synaptic transmission, especially for neurons that fire action potentials at high frequencies. However, how activity regulates synaptic vesicles recycling is largely unknown. Here we report that Na+ substantially accumulated in the mouse calyx of Held terminals during repetitive high-frequency spiking. Elevated presynaptic Na+ accelerated both slow and rapid forms of endocytosis and facilitated endocytosis overshoot but did not affect the readily releasable pool size, Ca2+ influx, or exocytosis. To examine whether this facilitation of endocytosis is related to the Na+-dependent vesicular content change, we dialyzed increasing concentrations of glutamate into the presynaptic cytosol or blocked the vesicular glutamate uptake with bafilomycin and found the rate of endocytosis was not affected by regulating the glutamate content in the presynaptic terminal. Endocytosis is critically dependent on intracellular Ca2+, and the activity of Na+/Ca2+ exchanger (NCX) may be altered when the Na+ gradient is changed. However, neither NCX blocker nor change of extracellular Na+ concentration affected the endocytosis rate. Moreover, two-photon Ca2+ imaging showed that presynaptic Na+ did not affect the action potential-evoked intracellular Ca2+ transient and decay. Therefore, we revealed a novel mechanism of cytosolic Na+ in accelerating vesicle endocytosis. During high-frequency synaptic transmission, when large amounts of synaptic vesicles are fused, Na+ accumulated in terminals, facilitated vesicle recycling and sustained reliable synaptic transmission.

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Tropomyosin Tpm3.1 is required to maintain the structure and function of the axon initial segment

10.1101/711614 ◽

2019 ◽

Cited By ~ 1

Author(s):

Amr Abouelezz ◽

Holly Stefen ◽

Mikael Segerstråle ◽

David Micinski ◽

Rimante Minkeviciene ◽

...

Keyword(s):

Actin Filaments ◽

Hippocampal Neurons ◽

Initial Segment ◽

Actin Polymerization ◽

Firing Frequency ◽

Axon Initial Segment ◽

Action Potential Initiation ◽

Sodium Ion Channels ◽

And Function ◽

Filament Network

ABSTRACTThe axon initial segment (AIS) is the site of action potential initiation and serves as a vesicular filter and diffusion barrier that help maintain neuronal polarity. Recent studies have revealed details about a specialized structural complex in the AIS. While an intact actin cytoskeleton is required for AIS formation, pharmacological disruption of actin polymerization compromises the AIS vesicle filter but does not affect overall AIS structure. In this study, we found that the tropomyosin isoform Tpm3.1 decorates a population of relatively stable actin filaments in the AIS. Inhibiting Tpm3.1 in cultured hippocampal neurons led to the loss of AIS structure, the AIS vesicle filter, the clustering of sodium ion channels, and reduced firing frequency. We propose that Tpm3.1-decorated actin filaments form a stable actin filament network under the AIS membrane which provides a scaffold for membrane organization and AIS proteins.

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Dual pools of actin at presynaptic terminals

Journal of Neurophysiology ◽

10.1152/jn.00789.2011 ◽

2012 ◽

Vol 107 (12) ◽

pp. 3479-3492 ◽

Cited By ~ 22

Author(s):

Adam Bleckert ◽

Huzefa Photowala ◽

Simon Alford

Keyword(s):

Synaptic Transmission ◽

Synaptic Vesicle ◽

Synaptic Vesicles ◽

Repetitive Stimulation ◽

Filamentous Actin ◽

Cortical Actin ◽

Vesicle Recycling ◽

Latrunculin A ◽

Kinetics Of

We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

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