scholarly journals Unique pH dynamics in GABAergic synaptic vesicles illuminates the mechanism and kinetics of GABA loading

2016 ◽  
Vol 113 (38) ◽  
pp. 10702-10707 ◽  
Author(s):  
Yoshihiro Egashira ◽  
Miki Takase ◽  
Shoji Watanabe ◽  
Junji Ishida ◽  
Akiyoshi Fukamizu ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Glutamate Uptake ◽  
Ph Sensor ◽  
Mammalian Brain ◽  
Gaba Uptake ◽  
Inhibitory Neurotransmitter ◽  
Luminal Ph ◽  
Time Required ◽  
Kinetics Of

GABA acts as the major inhibitory neurotransmitter in the mammalian brain, shaping neuronal and circuit activity. For sustained synaptic transmission, synaptic vesicles (SVs) are required to be recycled and refilled with neurotransmitters using an H+ electrochemical gradient. However, neither the mechanism underlying vesicular GABA uptake nor the kinetics of GABA loading in living neurons have been fully elucidated. To characterize the process of GABA uptake into SVs in functional synapses, we monitored luminal pH of GABAergic SVs separately from that of excitatory glutamatergic SVs in cultured hippocampal neurons. By using a pH sensor optimal for the SV lumen, we found that GABAergic SVs exhibited an unexpectedly higher resting pH (∼6.4) than glutamatergic SVs (pH ∼5.8). Moreover, unlike glutamatergic SVs, GABAergic SVs displayed unique pH dynamics after endocytosis that involved initial overacidification and subsequent alkalization that restored their resting pH. GABAergic SVs that lacked the vesicular GABA transporter (VGAT) did not show the pH overshoot and acidified further to ∼6.0. Comparison of luminal pH dynamics in the presence or absence of VGAT showed that VGAT operates as a GABA/H+ exchanger, which is continuously required to offset GABA leakage. Furthermore, the kinetics of GABA transport was slower (τ > 20 s at physiological temperature) than that of glutamate uptake and may exceed the time required for reuse of exocytosed SVs, allowing reuse of incompletely filled vesicles in the presence of high demand for inhibitory transmission.

scholarly journals Co-localization of different Neurotransmitter Transporters on the same Synaptic Vesicle is Bona-fide yet Sparse

2021 ◽  
Author(s):  
Neha Upmanyu ◽  
Jialin Jin ◽  
Marcelo Ganzella ◽  
Leon Boesche ◽  
Viveka Nand Malviya ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Glutamate Uptake ◽  
Quantitative Imaging ◽  
Small Population ◽  
Mammalian Brain ◽  
Content Type ◽  
Quantal Size ◽  
Vesicle Pools ◽  
Bona Fide ◽  
Vesicular Transporters

Vesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain neurons in mammalian brain release multiple transmitters, the prevalence, physiology of such pluralism and if the release occurs from same or distinct vesicle pools is not clear. Using quantitative imaging and biochemical approaches, we show that only a small population of neuronal SVs contain different VTs to accomplish corelease. Surprisingly, a highly diverse SV population (27 types) exist that express dual transporters suggesting corelease of diverse combinations of dual neurotransmitters, which includes the vesicle type that contains glutamate and zinc accounting for ∼34% of all SVs. Importantly, we demonstrate that transporter colocalization influences vesicular glutamate uptake leading to enhanced synaptic quantal size. Thus, localization of diverse transporters on single vesicles is bona-fide and the mechanism may underlie regulation of transmitter content, type and release in space and time.

scholarly journals Transport of γ-aminobutyrate and l-glutamate into synaptic vesicles. Effect of different inhibitors on the vesicular uptake of neurotransmitters and on the Mg2+-ATPase

1991 ◽  
Vol 276 (2) ◽  
pp. 363-367 ◽  
Author(s):  
E M Fykse ◽  
F Fonnum
Keyword(s):  
Benzoic Acid ◽  
Rat Brain ◽  
Synaptic Vesicles ◽  
Glutamate Uptake ◽  
Gaba Uptake ◽  
Anion Channels ◽  
Low Concentrations ◽  
Lipophilic Anion ◽  
Vesicular Uptake ◽  
Disulphonic Acid

The uptakes of gamma-aminobutyrate (GABA) and L-glutamate into synaptic vesicles isolated from rat brain were compared with respect to the effects of 4-acetamido-4′-isothiocyanostilbene-2,2′- disulphonic acid (SITS), 4,4′-di-isothiocyanostilbene-2,2′-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (N144), agents known to block anion channels. The uptake of glutamate was inhibited by low micromolar concentrations of SITS, DIDS and N144. GABA uptake was much less sensitive to these agents than was glutamate uptake. SITS and N144 inhibited the vacuolar H(+)-ATPase of synaptic vesicles to a smaller extent than the glutamate uptake. The uptake of GABA was not affected by the permeant anions Cl- and Br-, whereas the uptake of glutamate was highly stimulated by low concentrations of these ions. The uptakes of both glutamate and GABA were inhibited by similar, but not identical, concentrations of the lipophilic anion SCN-.

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

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.

Prothrombin Evaluation as Obtained by Kinetics Studies of Antigen-Antibody Reaction in a Laser Nephelometer

1984 ◽  
Vol 52 (01) ◽  
pp. 015-018 ◽  
Author(s):  
A Girolami ◽  
A Sticchi ◽  
R Melizzi ◽  
L Saggin ◽  
G Ruzza
Keyword(s):  
Liver Cirrhosis ◽  
Cirrhotic Patient ◽  
Maximum Rate ◽  
Serum Proteins ◽  
Clotting Factors ◽  
Kinetics Studies ◽  
Maximum Value ◽  
Time Required ◽  
Antigen Antibody ◽  
Kinetics Of

SummaryLaser nephelometry is a technique which allows the evaluation of the concentration of several serum proteins and clotting factors. By means of this technique it is also possible to study the kinetics of the reaction between antigen and antibody. We studied the kinetics of the reaction between prothrombin and an antiprothrombin antiserum using several prothrombins namely: Prothrombin Padua, prothrombin Molise, which are two congenital dysprothrombinemias, cirrhotic, coumarin or normal prothrombins. Different behaviors in the kinetics of the reactions were shown even when the concentration of prothrombins was about the same in all plasma tested. These differences were analyzed by means of a computer (Apple II 48 RAM) programmed to solve four unknown equations (Rodbard’s equation). From the data so obtained one can see that when voltages at the beginning and at the end of the reaction are in all cases about the same, a clear difference in the time required to reach half the maximum value of the voltage can still be demonstrated. This parameter, which is expressed in minutes, is longer in coumarin and prothrombin Molise than in controls. On the contrary it is shorter in prothrombin Padua and has about the same value of controls in the cirrhotic patient. Moreover the time at which the maximum rate is obtained is longer in coumarin and prothrombin Molise than in controls and shorter in liver cirrhosis and prothrombin Padua. In conclusion data obtained show that coumarin prothrombin behaves in a different way from cirrhotic prothrombin and also that there is a different behaviour between the two congenital dysprothrombinemias.

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.

scholarly journals Control of AMPA receptor activity by the extracellular loops of auxiliary proteins

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Irene Riva ◽  
Clarissa Eibl ◽  
Rudolf Volkmer ◽  
Anna L Carbone ◽  
Andrew JR Plested
Keyword(s):  
Ampa Receptor ◽  
Ampa Receptors ◽  
De Novo ◽  
Dominant Role ◽  
Mammalian Brain ◽  
Binding Assays ◽  
Receptor Kinetics ◽  
Receptor Complexes ◽  
Extracellular Loops ◽  
Kinetics Of

At synapses throughout the mammalian brain, AMPA receptors form complexes with auxiliary proteins, including TARPs. However, how TARPs modulate AMPA receptor gating remains poorly understood. We built structural models of TARP-AMPA receptor complexes for TARPs γ2 and γ8, combining recent structural studies and de novo structure predictions. These models, combined with peptide binding assays, provide evidence for multiple interactions between GluA2 and variable extracellular loops of TARPs. Substitutions and deletions of these loops had surprisingly rich effects on the kinetics of glutamate-activated currents, without any effect on assembly. Critically, by altering the two interacting loops of γ2 and γ8, we could entirely remove all allosteric modulation of GluA2, without affecting formation of AMPA receptor-TARP complexes. Likewise, substitutions in the linker domains of GluA2 completely removed any effect of γ2 on receptor kinetics, indicating a dominant role for this previously overlooked site proximal to the AMPA receptor channel gate.

Cooling of Fruit with Arbitrary Shape: Simulation Using Galerkin-Based Integral Method

2017 ◽  
Vol 10 ◽  
pp. 1-15
Author(s):  
P. Morais Pessôa ◽  
A.G. Barbosa de Lima ◽  
R. Swarnakar ◽  
J.P. Gomes ◽  
W.M.P. Barbosa de Lima
Keyword(s):  
Integral Method ◽  
Arbitrary Shape ◽  
Heat Conduction Equation ◽  
Low Cost ◽  
Experimental Studies ◽  
Volume Ratio ◽  
Transient Heat Conduction ◽  
Cooling Time ◽  
Time Required ◽  
Kinetics Of

Cooling has been used for the preservation of fresh produce such as fruit and vegetables due to its low cost and high effectiveness in maintaining the product quality. Recently, several researchers have conducted theoretical and experimental studies for obtaining the kinetics of cooling and cooling time for fruits with different geometries. Present work, therefore, aims to simulate the cooling of fruits with particular reference to banana, orange, strawberry and Tahiti lemon. The transient heat conduction equation and its analytical solution using Galerkin based integral method are presented. It has been found that the strawberry has lower dimensionless cooling time compared with time required to cool other fruits, which is due to its higher surface area/volume ratio value. In orange and lemon the temperature distribution was found to be homogeneous in the angular direction, while in banana and strawberry it was two-dimensional due to shape of the fruits.

A Simple Depletion Model of the Readily Releasable Pool of Synaptic Vesicles Cannot Account for Paired-Pulse Depression

2007 ◽  
Vol 97 (1) ◽  
pp. 948-950 ◽  
Author(s):  
Jane M. Sullivan
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Synaptic Activity ◽  
Excitatory Synapses ◽  
Short Term ◽  
Paired Pulse ◽  
Short Term Plasticity ◽  
Releasable Pool ◽  
Readily Releasable Pool ◽  
Paired Pulse Depression

Paired-pulse depression (PPD) is a form of short-term plasticity that plays a central role in processing of synaptic activity and is manifest as a decrease in the size of the response to the second of two closely timed stimuli. Despite mounting evidence to the contrary, PPD is still commonly thought to reflect depletion of the pool of synaptic vesicles available for release in response to the second stimulus. Here it is shown that PPD cannot be accounted for by depletion at excitatory synapses made by hippocampal neurons because PPD is unaffected by changes in the fraction of the readily releasable pool (RRP) released by the first of a pair of pulses.

scholarly journals Activity and cytosolic Na+ regulates synaptic vesicle endocytosis

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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