scholarly journals Counting the Number of Glutamate Molecules in Single Synaptic Vesicles

2019 ◽  
Author(s):  
Yuanmo Wang ◽  
Hoda fathali ◽  
devesh mishra ◽  
Thomas Olsson ◽  
Jacqueline Keighron ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
High Speed ◽  
Glutamate Release ◽  
Dependent Manner ◽  
Excitatory Neurotransmitter ◽  
Quantitative Measurements ◽  
Sensor Technique ◽  
Drug Treatments ◽  
Analytical Tools

<div><p>Analytical tools for direct quantitative measurements of glutamate, the principal excitatory neurotransmitter in brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for direct counting of the number of glutamate molecules stored inside single synaptic vesicles. An ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential dependent manner by applying a constant negative potential. High-speed (10 kHz) amperometry is used to record sub-millisecond current spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various concentrations of glutamate. Our measurements show that a single synaptic vesicle encapsulates about 8000 glutamate molecules, which is comparable to the measured exocytotic quantal glutamate release in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis and drug treatments for neuronal disorders where glutamate is involved.</p></div>

scholarly journals Counting the Number of Glutamate Molecules in Single Synaptic Vesicles

2019 ◽  
Author(s):  
Yuanmo Wang ◽  
Hoda fathali ◽  
devesh mishra ◽  
Thomas Olsson ◽  
Jacqueline Keighron ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
High Speed ◽  
Glutamate Release ◽  
Dependent Manner ◽  
Excitatory Neurotransmitter ◽  
Quantitative Measurements ◽  
Sensor Technique ◽  
Drug Treatments ◽  
Analytical Tools

<div><p>Analytical tools for direct quantitative measurements of glutamate, the principal excitatory neurotransmitter in brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for direct counting of the number of glutamate molecules stored inside single synaptic vesicles. An ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential dependent manner by applying a constant negative potential. High-speed (10 kHz) amperometry is used to record sub-millisecond current spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various concentrations of glutamate. Our measurements show that a single synaptic vesicle encapsulates about 8000 glutamate molecules, which is comparable to the measured exocytotic quantal glutamate release in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis and drug treatments for neuronal disorders where glutamate is involved.</p></div>

scholarly journals A Conditional Glutamatergic Synaptic Vesicle Marker for Drosophila

2022 ◽  
Author(s):  
Sarah J Certel ◽  
Evelyne Ruchti ◽  
Brian D McCabe ◽  
R Steven Stowers
Keyword(s):  
Nervous System ◽  
Synaptic Vesicles ◽  
Neural Circuit ◽  
Glutamate Release ◽  
Nervous System Development ◽  
Central Complex ◽  
Excitatory Neurotransmitter ◽  
Glutamatergic Neurons ◽  
Brain Functions ◽  
Conditional Expression

Abstract Glutamate is a principal neurotransmitter used extensively by the nervous systems of all vertebrate and invertebrate animals. It is primarily an excitatory neurotransmitter that has been implicated in nervous system development as well as a myriad of brain functions from the simple transmission of information between neurons to more complex aspects of nervous system function including synaptic plasticity, learning, and memory. Identification of glutamatergic neurons and their sites of glutamate release are thus essential for understanding the mechanisms of neural circuit function and how information is processed to generate behavior. Here we describe and characterize smFLAG-vGlut, a conditional marker of glutamatergic synaptic vesicles for the Drosophila model system. smFLAG-vGlut is validated for functionality, conditional expression, and specificity for glutamatergic neurons and synaptic vesicles. The utility of smFLAG-vGlut is demonstrated by glutamatergic neurotransmitter phenotyping of 26 different central complex neuron types of which nine were established to be glutamatergic. This illumination of glutamate neurotransmitter usage will enhance the modeling of central complex neural circuitry and thereby our understanding of information processing by this region of the fly brain. The use of smFLAG for glutamatergic neurotransmitter phenotyping and identification of glutamate release sites can be extended to any Drosophila neuron(s) represented by a binary transcription system driver.

scholarly journals Ion transport and regulation in a synaptic vesicle glutamate transporter

Science ◽  
2020 ◽  
Vol 368 (6493) ◽  
pp. 893-897 ◽  
Author(s):  
Fei Li ◽  
Jacob Eriksen ◽  
Janet Finer-Moore ◽  
Roger Chang ◽  
Phuong Nguyen ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Structural Information ◽  
Glutamate Transporter ◽  
Excitatory Neurotransmitter ◽  
Glutamate Binding ◽  
Cryo Electron Microscopy ◽  
Vesicle Cycle ◽  
Do So

Synaptic vesicles accumulate neurotransmitters, enabling the quantal release by exocytosis that underlies synaptic transmission. Specific neurotransmitter transporters are responsible for this activity and therefore are essential for brain function. The vesicular glutamate transporters (VGLUTs) concentrate the principal excitatory neurotransmitter glutamate into synaptic vesicles, driven by membrane potential. However, the mechanism by which they do so remains poorly understood owing to a lack of structural information. We report the cryo–electron microscopy structure of rat VGLUT2 at 3.8-angstrom resolution and propose structure-based mechanisms for substrate recognition and allosteric activation by low pH and chloride. A potential permeation pathway for chloride intersects with the glutamate binding site. These results demonstrate how the activity of VGLUTs can be coordinated with large shifts in proton and chloride concentrations during the synaptic vesicle cycle to ensure normal synaptic transmission.

scholarly journals Synaptobrevin-2 dependent regulation of single synaptic vesicle endocytosis

2021 ◽  
pp. mbc.E21-04-0213
Author(s):  
Natali L. Chanaday ◽  
Ege T. Kavalali
Keyword(s):  
Synaptic Vesicle ◽  
Molecular Mechanism ◽  
Synaptic Vesicles ◽  
Dependent Manner ◽  
Multiple Systems ◽  
Receptor Proteins ◽  
Kinetics Of

Evidence from multiple systems indicates that vesicle SNARE ( Soluble NSF Attachment REceptor) proteins are involved in synaptic vesicle endocytosis, although their exact action at the level of single vesicles are unknown. Here we interrogate the role of the main synaptic vesicle SNARE mediating fusion, synaptobrevin-2 (also called VAMP2), in modulation of single synaptic vesicle retrieval. We report that in the absence of synaptobrevin-2 fast and slow modes of single synaptic vesicle retrieval are impaired, indicating a role of the SNARE machinery in coupling exocytosis to endocytosis of single synaptic vesicles. Ultrafast endocytosis was impervious to changes in the levels of synaptobrevin-2, pointing to a separate molecular mechanism underlying this type of recycling. Taken together with earlier studies suggesting a role of synaptobrevin-2 in endocytosis, these results indicate that the machinery for fast synchronous release couples fusion to retrieval and regulates the kinetics of endocytosis in Ca2+ dependent manner.

scholarly journals Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jason D Vevea ◽  
Grant F Kusick ◽  
Kevin C Courtney ◽  
Erin Chen ◽  
Shigeki Watanabe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Glutamate Release ◽  
Vesicle Docking ◽  
Presynaptic Function ◽  
Asynchronous Release ◽  
Vesicle Cycle ◽  
Activity Dependent

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs.

scholarly journals BDNF mobilizes synaptic vesicles and enhances synapse formation by disrupting cadherin–β-catenin interactions

2006 ◽  
Vol 174 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Shernaz X. Bamji ◽  
Beatriz Rico ◽  
Nikole Kimes ◽  
Louis F. Reichardt
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Synapse Formation ◽  
Time Lapse ◽  
Synapse Density ◽  
Vesicle Mobility ◽  
Small Clusters ◽  
Vertebrate Central Nervous System

Neurons of the vertebrate central nervous system have the capacity to modify synapse number, morphology, and efficacy in response to activity. Some of these functions can be attributed to activity-induced synthesis and secretion of the neurotrophin brain-derived neurotrophic factor (BDNF); however, the molecular mechanisms by which BDNF mediates these events are still not well understood. Using time-lapse confocal analysis, we show that BDNF mobilizes synaptic vesicles at existing synapses, resulting in small clusters of synaptic vesicles “splitting” away from synaptic sites. We demonstrate that BDNF's ability to mobilize synaptic vesicle clusters depends on the dissociation of cadherin–β-catenin adhesion complexes that occurs after tyrosine phosphorylation of β-catenin. Artificially maintaining cadherin–β-catenin complexes in the presence of BDNF abolishes the BDNF-mediated enhancement of synaptic vesicle mobility, as well as the longer-term BDNF-mediated increase in synapse number. Together, this data demonstrates that the disruption of cadherin–β-catenin complexes is an important molecular event through which BDNF increases synapse density in cultured hippocampal neurons.

Microwave On-Line Measurements on Conveyed Products Application to Paper and Wood Industries

1994 ◽  
Vol 347 ◽  
Author(s):  
J.Ch. Bolomey ◽  
G. Cottard ◽  
P. Berthaud ◽  
A. Lemaitre ◽  
J. F. Portala
Keyword(s):  
High Speed ◽  
Transformation Process ◽  
Wood Material ◽  
Quantitative Measurements ◽  
On Line ◽  
Phase Data ◽  
Polynomial Expansions ◽  
Physical Quantities ◽  
Data Calibration

ABSTRACTMicrowave multiport sensors have been shown to provide some unique capabilities to achieve real-time testing of products conveyed at high speed. In many applications, quantitative measurements of physical quantities such as moisture content, density, etc… are required, either to guarantee reliable production or to optimally control a fabrication/transformation process. In this paper, different ways of extracting such physical quantities from microwave measurements performed by multiport sensors are presented. Model approaches are used, based on polynomial expansions of the physical quantities to be measured as a function of the microwave amplitude and phase data. Calibration procedures have been investigated for both paper and wood material samples. Comparisons between in-situ, microwave and conventional, measurements are analysed.

scholarly journals Region-Specific Microtubule Transport in Motile Cells

2000 ◽  
Vol 151 (5) ◽  
pp. 1003-1012 ◽  
Author(s):  
Anne-Marie C. Yvon ◽  
Patricia Wadsworth
Keyword(s):  
Growth Factor ◽  
Cell Motility ◽  
Cell Body ◽  
Dependent Manner ◽  
Retrograde Flow ◽  
Cell Locomotion ◽  
Quantitative Measurements ◽  
Motile Cells ◽  
Microtubule Transport ◽  
Hepatocyte Growth

Photoactivation and photobleaching of fluorescence were used to determine the mechanism by which microtubules (MTs) are remodeled in PtK2 cells during fibroblast-like motility in response to hepatocyte growth factor (HGF). The data show that MTs are transported during cell motility in an actomyosin-dependent manner, and that the direction of transport depends on the dominant force in the region examined. MTs in the leading lamella move rearward relative to the substrate, as has been reported in newt cells (Waterman-Storer, C.M., and E.D. Salmon. 1997. J. Cell Biol. 139:417–434), whereas MTs in the cell body and in the retraction tail move forward, in the direction of cell locomotion. In the transition zone between the peripheral lamella and the cell body, a subset of MTs remains stationary with respect to the substrate, whereas neighboring MTs are transported either forward, with the cell body, or rearward, with actomyosin retrograde flow. In addition to transport, the photoactivated region frequently broadens, indicating that individual marked MTs are moved either at different rates or in different directions. Mark broadening is also observed in nonmotile cells, indicating that this aspect of transport is independent of cell locomotion. Quantitative measurements of the dissipation of photoactivated fluorescence show that, compared with MTs in control nonmotile cells, MT turnover is increased twofold in the lamella of HGF-treated cells but unchanged in the retraction tail, demonstrating that microtubule turnover is regionally regulated.

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