Vesicular release of glutamate utilizes the proton gradient between the vesicle and synaptic cleft (original) (raw)
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Glutamate uptake occurs at an early stage of synaptic vesicle recycling
Current Biology, 1997
Rapid membrane recycling in nerve terminals is required to maintain rapid synaptic transmission. Following the fusion of synaptic vesicles with synaptic plasma membranes, recycling can occur via clathrincoated vesicles (CCVs) [1-3]. The fate of these vesicles is uncertain: they could simply uncoat and acquire other proteins from the cytosol to regenerate synaptic vesicles or they may fuse with endosomal structures from which synaptic vesicles could then bud. We have purified both CCVs and synaptic vesicles from rat brain, and measured the ability of these vesicle fractions to take up the excitatory neurotransmitter glutamic acid. We found that the normalized levels of glutamate uptake by the two types of vesicle were very similar. For each vesicle fraction, uptake required ATP and Cland could be fully inhibited by the specific vacuolar proton pump (v-ATPase) inhibitor concanamycin. We suggest that this ability to refill vesicles with neurotransmitter at the earliest intermediate on the recycling pathwaythe CCV -may allow uncoated vesicles to immediately enter the releasable pool without sacrificing the quantal nature of neurotransmitter release.
Characterization of Glutamate Uptake into Synaptic Vesicles
Journal of Neurochemistry, 1985
Recent evidence indicates that L-glutamate is taken up into synaptic vesicles in an ATP-dependent manner, supporting the notion that synaptic vesicles may be involved in glutamate synaptic transmission. In this study, we further characterized the ATP-dependent vesicular uptake of glutamate. Evidence is provided that a Mg-ATPase, not Ca-ATPase, is responsible for the ATP hydrolysis coupled to the glutamate uptake. The ATPdependent glutamate uptake was inhibited by agents known to dissipate the electrochemical proton gradient across the membrane of chromaffin granules. Hence, it is suggested that the vesicular uptake of glutamate is driven by electrochemical proton gradients generated by the Mg-ATPase. Of particular interest is the finding that the ATP-dependent glutamate uptake is markedly stimulated by chloride over a physiologically relevant, millimolar concentration range, suggesting an important role of intranerve terminal chloride in the accumulation of glutamate in synaptic vesicles. The vesicular glutamate translocator is highly specific for L-glutamate, and failed to interact with aspartate, its related agents, and most of the glutamate analogs tested. It is proposed that this vesicular translocator plays a crucial role in determining the fate of glutamate as a neurotransmitter. Key Words: Glutamate-Synaptic vesicle-Uptake-Mg-ATPase-Excitatory neurotransmitter. Naito S. and Ueda T. Characterization of glutamate uptake into synaptic vesicles.
Neuroscience Letters, 1991
We have studied glutamate release from synaptic vesicles in permeabilized synaptosomes, which were preloaded with [3H]glutamate in an ATPdependent manner. The release was found to be calcium-dependent and to require a heat-labile cytosolic macromolecule factor for maximum activity. Maximal release occurred at 5/zM free Ca 2+ and within 5 min. Of the other divalent cations tested, only barium stimulated release of vesicular glutamate. The release was inhibited by N-ethylmaleimide. These results are characteristic of exocytotic release of monoamines and peptides observed in endocrine systems, and constitute direct evidence for the notion that calcium-dependent release of glutamate originates from the vesicular pool.
Differential Control of Synaptic and Ectopic Vesicular Release of Glutamate
Journal of Neuroscience, 2004
Exocytosis of synaptic vesicles occurs not only at synaptic active zones but also at ectopic sites. Ectopic exocytosis provides a direct and rapid mechanism for neurons to communicate with glia that does not rely on transmitter spillover from the synaptic cleft. In the cerebellar cortex the processes of Bergmann glia cells encase synapses between presynaptic climbing fiber varicosities and postsynaptic Purkinje cell spines and express both AMPA receptors and electrogenic glutamate transporters. AMPA receptors expressed by Purkinje cells and Bergmann glia cells are activated predominantly by synaptic and ectopic release, respectively, and therefore can be used to compare the properties of the two release mechanisms. We report that vesicular release differs at synaptic and ectopic sites in the magnitude of short-term plasticity and the proportions of Ca 2ϩ channel subtypes that trigger glutamate release. High-affinity glutamate transporter-mediated currents in Bergmann glia cells follow the rules of synaptic release more closely than the rules of ectopic release, indicating that the majority of glutamate is released from conventional synapses. On the other hand, ectopic release produces highconcentration glutamate transients at Bergmann glia cell membranes that are necessary to activate low-affinity AMPA receptors rapidly. Ectopic release may provide a geographical cue to guide Bergmann glia cell membranes to surround active synapses and ensure efficient uptake of glutamate that diffuses out of the synaptic cleft.
Fusion-related Release of Glutamate from Astrocytes
Journal of Biological Chemistry, 2004
Although cell culture studies have implicated the presence of vesicle proteins in mediating the release of glutamate from astrocytes, definitive proof requires the identification of the glutamate release mechanism and the localization of this mechanism in astrocytes at synaptic locales. In cultured murine astrocytes we show an array of vesicle proteins, including SNARE proteins, and vesicular glutamate transporters that are required to fill vesicles with glutamate. Using immunocytochemistry and single-cell multiplex reverse transcription-PCR we demonstrate the presence of these proteins and their transcripts within astrocytes freshly isolated from the hippocampus. Moreover, immunoelectron microscopy demonstrates the presence of VGLUT1 in processes of astrocytes of the hippocampus. To determine whether calcium-dependent glutamate release is mediated by exocytosis, we expressed the SNARE motif of synaptobrevin II to prevent the formation of SNARE complexes, which reduces glutamate release from astrocytes. To further determine whether vesicular exocytosis mediates calcium-dependent glutamate release from astrocytes, we performed whole cell capacitance measurements from individual astrocytes and demonstrate an increase in whole cell capacitance, coincident with glutamate release. Together, these data allow us to conclude that astrocytes in situ express vesicle proteins necessary for filling vesicles with the chemical transmitter glutamate and that astrocytes release glutamate through a vesicle-or fusion-related mechanism.
2021
The balance between fast synchronous and delayed asynchronous release of neurotransmitters has a major role in defining computational properties of neuronal synapses and regulation of neuronal network activity. However, how it is tuned at the single synapse level remains poorly understood. Here, using the fluorescent glutamate sensor SF-iGluSnFR, we image quantal vesicular release in tens to hundreds of individual synaptic outputs (presynaptic boutons) from single pyramidal cells in culture with 4 millisecond temporal resolution, and localise vesicular release sites with ~ 75 nm spatial resolution. We find that the ratio between synchronous and asynchronous synaptic vesicle exocytosis varies extensively among presynaptic boutons supplied by the same axon, and that asynchronous release fraction is elevated in parallel with short-term facilitation at synapses with low release probability. We further demonstrate that asynchronous exocytosis sites are more widely distributed within the presynaptic release area than synchronous sites. These findings are consistent with a model in which functional presynaptic properties are regulated via a synapsespecific adjustment of the coupling distance between presynaptic Ca 2+ channels and releaseready synaptic vesicles. Together our results reveal a universal relationship between the two major functional properties of synapses-the timing and the overall probability of neurotransmitter release. Main text Synaptic transmission provides the basis for neuronal communication. When an actionpotential propagates through the axonal arbour, it activates voltage-gated Ca 2+ channels (VGCCs) located in the vicinity of release-ready synaptic vesicles docked at the presynaptic active zone 1. Ca 2+ ions enter the presynaptic terminal and activate the vesicular Ca 2+ sensor Synaptotagmin 1 (Syt1, or its isoforms Syt2 and Syt9), thus triggering exocytosis of synaptic vesicles filled with neurotransmitter molecules. Neurotransmitter diffuses across the synaptic cleft, binds postsynaptic receptors and evokes further electrical or chemical signalling in the postsynaptic target cell. This whole process occurs on a time scale of a few milliseconds. Recent data demonstrate that such speed and precision are in large part achieved via the formation of nanocomplexes that include presynaptic VGCCs, vesicles belonging to a readily releasable pool (RRP) and postsynaptic neurotransmitter receptors 2-4. In addition to fast, synchronous release, which keeps pace with action potentials, many synapses also exhibit delayed asynchronous release that persists for tens to hundreds of milliseconds 1, 5. Asynchronous release is potentiated during repetitive presynaptic firing and is triggered via activation of multiple sensors with both low (e.g. Syt1) and high (e.g. Syt7) Ca 2+ affinity 6. Accumulating evidence demonstrates that the balance between synchronous and asynchronous release plays an important role in coordinating activity within neuronal networks, for example, by increasing the probability of postsynaptic cell firing and/or modulating action potential precision 7-10. It is well established that asynchronous release levels vary among different types of neurons 1, 10, 11. Interestingly, recent data show that the ratio between asynchronous and synchronous release can also be differentially regulated among presynaptic boutons supplied by the same axon and depends on the identity of the postsynaptic cell, which contributes to target cell-specific communication in the brain 7, 8. The mechanisms that control the relative contributions of synchronous and asynchronous release at the level of single synapses are however poorly understood. Variability in asynchronous release among different neuronal types has been attributed to differences in
Glutamate-induced Exocytosis of Glutamate from Astrocytes
Journal of Biological Chemistry, 2007
Recent studies indicate that astrocytes can play a much more active role in neuronal circuits than previously believed, by releasing neurotransmitters such as glutamate and ATP. Here we report that local application of glutamate or glutamine synthetase inhibitors induces astrocytic release of glutamate, which activates a slowly decaying transient inward current (SIC) in CA1 pyramidal neurons and a transient inward current in astrocytes in hippocampal slices. The occurrence of SICs was accompanied by an appearance of large vesicles around the puffing pipette. The frequency of SICs was positively correlated with [glutamate] o. EM imaging of anti-glial fibrillary acid proteinlabeled astrocytes showed glutamate-induced large astrocytic vesicles. Imaging of FM 1-43 fluorescence using two-photon laser scanning microscopy detected glutamate-induced formation and fusion of large vesicles identified as FM 1-43-negative structures. Fusion of large vesicles, monitored by collapse of vesicles with a high intensity FM 1-43 stain in the vesicular membrane, coincided with SICs. Glutamate induced two types of large vesicles with high and low intravesicular [Ca 2؉ ]. The high [Ca 2؉ ] vesicle plays a major role in astrocytic release of glutamate. Vesicular fusion was blocked by infusing the Ca 2؉ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid, or the SNARE blocker, tetanus toxin, suggesting Ca 2؉-and SNARE-dependent fusion. Infusion of the vesicular glutamate transport inhibitor, Rose Bengal, reduced astrocytic glutamate release, suggesting the involvement of vesicular glutamate transports in vesicular transport of glutamate. Our results demonstrate that local [glutamate] o increases induce formation and exocytotic fusion of glutamate-containing large astrocytic vesicles. These large vesicles could play important roles in the feedback control of neuronal circuits and epileptic seizures.
Vesicular release of glutamate from hippocampal neurons in culture: an immunocytochemical assay
Experimental Brain Research, 2008
Glutamate, the main excitatory neurotransmitter in the brain, may cause excitotoxic damage through excessive release during a number of pathological conditions. We have developed an immunocytochemical assay to investigate the mechanisms and regulation of glutamate release from intact, cultured neurons. Our results indicate that cultured hippocampal neurons have a large surplus of glutamate available for release upon chemically induced depolarization. Long incubations with high K + -concentrations, and induction of repetitive action potentials with the K + -channel blocker 4-aminopyridine (4-AP), caused a significant reduction in glutamate labeling in a subset of boutons, demonstrating that transmitter release exceeded the capacity for replenishment. The number of boutons where release exceeded replenishment increased continuously with time of stimulation. This depletion was Ca 2+dependent and sensitive to bafilomycin A1 (baf), indicating that it was dominated by vesicular release mechanisms. The depletion of glutamate from cell bodies and dendrites was also Ca 2+ -dependent. Thus, under the present conditions, cytosolic glutamate is taken up in vesicles prior to release, and the main escape route for the amino acid is through vesicular exocytosis. Depolarization with lower concentrations of K + caused sustainable release of glutamate, i.e., without full depletion.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2002
Monte Carlo simulations were performed on a release model based on experimental data from single glutamatergic synapses containing a single release site in the hippocampal CA1 region of the neonatal rat. These simulations explored what can be learned about the release process by examining how the release probability in response to the second stimulus (P(2)) of a paired stimulus to a synapse depends on the release in response to the first stimulus. Comparisons between experimental data from a number of individual synapses and the simulated data support the notion that the immediately releasable vesicle pool is small (approximately one) and shows substantial intertrial variation. The simulations also show that the release dependence of P(2) is not necessarily an indicator of either intertrial variation in Ca(2+) influx, feedback effects of released transmitter, or activation failure of the axon.