Imaging Synaptic Vesicles Using VGLUT1-Venus Knock-In Mice: Insights into the Dynamic Nature of Intersynaptic Vesicle Exchange: Figure 1.

Padamsey, Zahid; Jeans, Alexander

doi:10.1523/jneurosci.6425-11.2012

Cited by 3 publications

(3 citation statements)

References 11 publications

(24 reference statements)

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“…These data show that the basic properties of axonal transport in adult neurons are not regulated by Syns, whereas the socalled "superpool" of SVs, which is spanning several en passant boutons of the same axon (Denker and Rizzoli, 2010;Staras et al, 2010), is increased in TKO neurons. Very recent findings render this result particularly relevant because SV redistribution within the same axon might be essential for the competitive strengthening/weakening of presynaptic efficacy and might improve pattern discrimination in dendritic networks (Herzog et al, 2011;Padamsey and Jeans, 2012). Through the mobilization/immobilization of SVs, Syns might locally regulate the plastic properties at single presynapses both acting as restrictors, by contributing with a resilient force to the tenacity of the terminal (Fisher-Lavie et al, 2011) and serving as integrators of presynaptic signaling in a phosphorylation-dependent manner .…”

Section: Discussionmentioning

confidence: 99%

Synapsins Contribute to the Dynamic Spatial Organization of Synaptic Vesicles in an Activity-Dependent Manner

et al. 2012

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The precise subcellular organization of synaptic vesicles (SVs) at presynaptic sites allows for rapid and spatially restricted exocytotic release of neurotransmitter. The synapsins (Syns) are a family of presynaptic proteins that control the availability of SVs for exocytosis by reversibly tethering them to each other and to the actin cytoskeleton in a phosphorylation-dependent manner. Syn ablation leads to reduction in the density of SV proteins in nerve terminals and increased synaptic fatigue under high-frequency stimulation, accompanied by the development of an epileptic phenotype. We analyzed cultured neurons from wild-type and Syn I,II,III Ϫ/Ϫ triple knock-out (TKO) mice and found that SVs were severely dispersed in the absence of Syns. Vesicle dispersion did not affect the readily releasable pool of SVs, whereas the total number of SVs was considerably reduced at synapses of TKO mice. Interestingly, dispersion apparently involved exocytosis-competent SVs as well; it was not affected by stimulation but was reversed by chronic neuronal activity blockade. Altogether, these findings indicate that Syns are essential to maintain the dynamic structural organization of synapses and the size of the reserve pool of SVs during intense SV recycling, whereas an additional Syn-independent mechanism, whose molecular substrate remains to be clarified, targets SVs to synaptic boutons at rest and might be outpaced by activity.

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Section: Discussionmentioning

confidence: 99%

Synapsins Contribute to the Dynamic Spatial Organization of Synaptic Vesicles in an Activity-Dependent Manner

et al. 2012

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“…Here we designed a biochemical assay measuring the amount of vesicular transporters exposed on the extracellular side of the presynaptic membrane during neurotransmitter release (Voglmaier et al, 2006; Padamsey and Jeans, 2012). Using non-membrane-permeable biotinylation reagents, we were able to simultaneously label vesicular transporters for glutamate and GABA expressed in glutamatergic and GABAergic synaptic vesicles, respectively (McIntire et al, 1997; Takamori et al, 2000; Fremeau et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Monitoring of Presynaptic Transmitter Release and Postsynaptic Receptor Trafficking Reveals an Enhancement of Presynaptic Activity in Metabotropic Glutamate Receptor-Mediated Long-Term Depression

Tse

Dobie

et al. 2013

J. Neurosci.

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Although the contribution of postsynaptic mechanisms to long-term synaptic plasticity has been studied extensively, understanding the contribution of presynaptic modifications to this process lags behind, primarily because of a lack of techniques with which to directly and quantifiably measure neurotransmitter release from synaptic terminals. Here, we developed a method to measure presynaptic activity through the biotinylation of vesicular transporters in vesicles fused with presynaptic membranes during neurotransmitter release. This method allowed us for the first time to selectively quantify the spontaneous or evoked release of glutamate or GABA at their respective synapses. Using this method to investigate presynaptic changes during the expression of group I metabotropic glutamate receptor (mGluR1/5)-mediated long-term depression (LTD) in cultured rat hippocampal neurons, we discovered that this form of LTD was associated with increased presynaptic release of glutamate, despite reduced miniature EPSCs measured with whole-cell recording. Moreover, we found that specific blockade of AMPA receptor (AMPAR) endocytosis with a membrane-permeable GluR2-derived peptide not only prevented the expression of LTD but also eliminated LTD-associated increase in presynaptic release. Thus, our work not only demonstrates that mGluR1/5-mediated LTD is associated with increased endocytosis of postsynaptic AMPARs but also reveals an unexpected homeostatic/compensatory increase in presynaptic release. In addition, this study indicates that biotinylation of vesicular transporters in live cultured neurons is a valuable tool for studying presynaptic function.

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“…A limitation of all FPs is that they suffer from photobleaching through repeated or prolonged illumination, thereby hindering their deployment in applications requiring long-term imaging of cellular activity (25) or high signal stability (26). We chose to improve mVenus (27,28), a bright YFP widely used as an individual tag (29,30) and within biosensors (31,32).…”

Section: Spotlight Screening Of ~3 Million Cells Identified the Most ...mentioning

confidence: 99%

Versatile phenotype-activated cell sorting

et al. 2020

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Unraveling the genetic and epigenetic determinants of phenotypes is critical for understanding and re-engineering biology and would benefit from improved methods to separate cells based on phenotypes. Here, we report SPOTlight, a versatile high-throughput technique to isolate individual yeast or human cells with unique spatiotemporal profiles from heterogeneous populations. SPOTlight relies on imaging visual phenotypes by microscopy, precise optical tagging of single target cells, and retrieval of tagged cells by fluorescence-activated cell sorting. To illustrate SPOTlight’s ability to screen cells based on temporal properties, we chose to develop a photostable yellow fluorescent protein for extended imaging experiments. We screened 3 million cells expressing mutagenesis libraries and identified a bright new variant, mGold, that is the most photostable yellow fluorescent protein reported to date. We anticipate that the versatility of SPOTlight will facilitate its deployment to decipher the rules of life, understand diseases, and engineer new molecules and cells.

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Imaging Synaptic Vesicles Using VGLUT1-Venus Knock-In Mice: Insights into the Dynamic Nature of Intersynaptic Vesicle Exchange: Figure 1.

Cited by 3 publications

References 11 publications

Synapsins Contribute to the Dynamic Spatial Organization of Synaptic Vesicles in an Activity-Dependent Manner

Synapsins Contribute to the Dynamic Spatial Organization of Synaptic Vesicles in an Activity-Dependent Manner

Simultaneous Monitoring of Presynaptic Transmitter Release and Postsynaptic Receptor Trafficking Reveals an Enhancement of Presynaptic Activity in Metabotropic Glutamate Receptor-Mediated Long-Term Depression

Versatile phenotype-activated cell sorting

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