Synaptic Glutamate Concentration Research Articles

Astrocytic PAR1 and mGluR2/3 control synaptic glutamate time course at hippocampal CA1 synapses.

Astrocytes play an essential role in regulating synaptic transmission. This study describes a novel form of modulation of excitatory synaptic transmission in the mouse hippocampus by astrocytic G-protein-coupled receptors (GPCRs). We have previously described astrocytic glutamate release via protease-activated receptor-1 (PAR1) activation, although the regulatory mechanisms for this are complex. Through electrophysiological analysis and modeling, we discovered that PAR1 activation consistently increases the concentration and duration of glutamate in the synaptic cleft. This effect was not due to changes in the presynaptic glutamate release or alteration in glutamate transporter expression. However, blocking group II metabotropic glutamate receptors (mGluR2/3) abolished PAR1-mediated regulation of synaptic glutamate concentration, suggesting a role for this GPCR in mediating the effects of PAR1 activation on glutamate release. Furthermore, activation of mGluR2/3 causes glutamate release through the TREK-1 channel in hippocampal astrocytes. These data show that astrocytic GPCRs engage in a novel regulatory mechanism to shape the time course of synaptically-released glutamate in excitatory synapses of the hippocampus.

A glutamate concentration-biased allosteric modulator potentiates NMDA-induced ion influx in neurons.

Precisely controlled synaptic glutamate concentration is essential for the normal function of the N‐methyl D‐aspartate (NMDA) receptors. Atypical fluctuations in synaptic glutamate homeostasis lead to aberrant NMDA receptor activity that results in the pathogenesis of neurological and psychiatric disorders. Therefore, glutamate concentration‐dependent NMDA receptor modulators would be clinically useful agents with fewer on‐target adverse effects. In the present study, we have characterized a novel compound (CNS4) that potentiates NMDA receptor currents based on glutamate concentration. This compound alters glutamate potency and exhibits no voltage‐dependent effect. Patch‐clamp electrophysiology recordings confirmed agonist concentration‐dependent changes in maximum inducible currents. Dynamic Ca2+ and Na+ imaging assays using rat brain cortical, striatal and cerebellar neurons revealed CNS4 potentiated ion influx through native NMDA receptor activity. Overall, CNS4 is novel in chemical structure, mechanism of action and agonist concentration‐biased allosteric modulatory effect. This compound or its future analogs will serve as useful candidates to develop drug‐like compounds for the treatment of treatment‐resistant schizophrenia and major depression disorders associated with hypoglutamatergic neurotransmission.

If I do A, B will happen: Dissecting circuits detecting causal relations between actions and outcomes in marmoset prefrontal cortex

In this issue of Neuron, Duan etal. (2021) use pharmacological manipulation to reveal opposing influences of anterior cingulate and orbitofrontal cortex of marmosets on decisions that are based on action-outcome associations.

Amino Acid Transporters and Exchangers from the SLC1A Family: Structure, Mechanism and Roles in Physiology and Cancer.

The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems-the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues GltPh and GltTk have elucidated mechanisms of transport and inhibition common to the family. The recent determination of EAAT1 and ASCT2 structures may be of assistance in future efforts to design efficacious ASCT2 inhibitors. This review will focus on ASCT2, the present state of knowledge on its roles in tumour biology, and how structural biology is being used to progress the development of inhibitors.

Effects of 3-aminoglutarate, a “silent” false transmitter for glutamate neurons, on synaptic transmission and epileptiform activity

Pharmacological tools that interact with the mechanisms that regulate vesicular filling and release of the neurotransmitter l-glutamate would be of enormous value. In this study, we provide physiological evidence that the glutamate analog, 3-aminoglutarate (3-AG), acts as a false transmitter to reduce presynaptic glutamate release. 3-AG inhibits glutamate-mediated neurotransmission both in primary neuronal cultures and in brain slices with more intact neural circuits. When assayed with the low affinity glutamate receptor antagonist γ-DGG, we demonstrate that 3-AG significantly reduces the synaptic cleft glutamate concentration, suggesting that 3-AG may act as a false transmitter to compete with glutamate during vesicle filling. Furthermore, using three different epileptic models (Mg2+-free, 4-AP, and high K+), we demonstrate that 3-AG is capable of suppressing epileptiform activity both before and after its induction. Our studies, along with those of the companion paper by Foster et al. (2015) indicate that 3-AG is a “silent” false transmitter for glutamate neurons that is a useful pharmacological tool to probe the mechanisms governing vesicular storage and release of glutamate under both physiological and pathophysiological conditions. 3-AG may have potential therapeutic value in conditions where the glutamate neurotransmitter system is pathologically overactive.

Effect of ceftriaxone and topiramate treatments on naltrexone-precipitated morphine withdrawal and glutamate receptor desensitization in the rat locus coeruleus.

Morphine withdrawal is associated with a hyperactivity of locus coeruleus (LC) neurons by an elevated glutamate neurotransmission in this nucleus. We postulate that reductions in the amount of glutamate in the LC by enhancing its reuptake or inhibiting its release could attenuate the behavioral and cellular consequences of morphine withdrawal. We investigated the effect of chronic treatment with ceftriaxone (CFT), an excitatory amino acid transporter (EAAT2) enhancer, and acute administration of topiramate (TPM), a glutamate release inhibitor, on morphine withdrawal syndrome and withdrawal-induced glutamate receptor (GluR) desensitization in LC neurons from morphine-dependent rats. Morphine withdrawal behavior was measured after naltrexone administration in rats implanted with a morphine (200 mg kg(-1)) emulsion for 3 days. GluR desensitization in the LC was assessed by performing concentration-effect curves for glutamate by extracellular electrophysiological recordings in vitro. Treatments with CFT or TPM reduced, in a dose-related manner, the total behavioral score of naltrexone-precipitated morphine withdrawal. CFT and TPM, at doses that were effective in behavioral tests, also reduced the induction of GluR desensitization normally occurring in LC neurons from morphine-dependent rats. Acute treatment with the specific EAAT2 inhibitor dihydrokainic acid (DHK) prevented the effect of CFT on withdrawal syndrome and GluR desensitization. Perfusion with TPM inhibited KCl-evoked but not glutamate-induced activation of LC neurons in vitro. Our results suggest that a reduction of synaptic concentrations of glutamate by enhancing EAAT2-mediated uptake or inhibiting glutamate release alleviates the behavioral response and the cellular changes in the LC during opiate withdrawal.

A complex relative motion between hairpin loop 2 and transmembrane domain 5 in the glutamate transporter GLT-1

Excitatory amino acid transporter 2, also known as glial glutamate transporter type 1 (GLT-1), plays an important role in maintaining suitable synaptic glutamate concentrations. Reentrant helical hairpin loop (HP) 2, as the extracellular gate, has been shown to participate in the binding of substrate and ions. Several residues in transmembrane domain (TM) 5 have been shown to be involved in the construction of the transport pathway. However, the spatial relationship between HP2 and TM5 during the recycling of glutamate has not yet been clarified. We introduced cysteine residue pairs in HP2 and TM5 of cysteine-less-GLT-1 by using site-directed mutagenesis in order to assess the proximity of HP2 and TM5. A significant decrease in substrate uptake was seen in the I283C/S443C and S287C/S443C mutants when the oxidative cross-linking agent copper(II) (1,10-phenanthroline)3 (CuPh) was used. The inhibitory effect of CuPh on the transport activity of the S287/S443C mutant was increased after the application of glutamate or potassium. In contrast, an apparent protection of the transport activity of the I283C/S443C mutant was observed after glutamate or potassium addition. The membrane-impermeable sulfhydryl reagent (2-trimethylammonium) methanethiosulfonate (MTSET) was used to detect the aqueous permeability of each single mutant. The aqueous permeability of the I283C mutant was identical to that of the S443C mutant. The sensitivity of I283C and S443C to MTSET was attenuated by glutamate and potassium. All these data indicate that there is a complex relative motion between TM5 and HP2 during the transport cycle.

Investigating substrate-induced motion between the scaffold and transport domains in the glutamate transporter EAAT1.

Excitatory amino acid transporter 1 plays an important role in keeping the synaptic glutamate concentration below neurotoxic levels by translocating this neurotransmitter into the cell. Both reentrant hairpin loops, HP1 and -2, have been shown to take part in binding the substrate and the more deeply buried sodium ion, and might therefore be a part of the intra- or extracellular gate of the transporter. However, the shape of the motion of either loop relative to transmembrane domain (TM) 4 during the transport cycle has not yet been fully resolved. Using copper(II) (1,10-phenanthroline)3 (CuPh) for cross-linking cysteine pairs, we found strong inhibition of transport when A243C (TM4) was combined with S366C (HP1), I453C (HP2), or T456C (HP2). These findings were reinforced by the impact of cadmium on transport activity, and both approaches consistently showed that proximity was exclusively intramonomeric. Under conditions that promote the inward-facing state, inhibition by CuPh in A243C/S366C was reduced, while the opposite was seen when the outward-facing one was stabilized, suggesting that the two positions are farther apart in the former conformation than in the latter. Surprisingly, maximal cross-linking of A243C with I453C or T456C was not observed under conditions that promote the inward-facing state. Altogether, our data suggest that the transporter may undergo complex relative movement between these positions on TM4 and HP1/HP2 during the transport cycle.

Ephrin-A2 Un-Glu’s the Synapse

Ephrin-A2 increases glutamate reuptake transporter abundance to control synaptic glutamate concentrations and prevent synapse elimination.

Protease activated receptor‐1 (PAR1) control of glutamate time course in the synapse cleft

The protease activated receptor‐1 (PAR1) is prominently expressed in astrocytes of the central nervous system. Activation of PAR1 increases the ability of synaptically‐released glutamate to surmount block by the rapidly unbinding competitive antagonist kynurenic acid. A variety of functional and molecular experiments suggest that this effect does not involve changes in presynaptic glutamate release or postsynaptic AMPA receptor function. Kinetic analysis of native AMPA receptors in outside‐out patches allowed the development of a simplified kinetic model of the actions of kynurenic acid. We subsequently used this model to fit the EPSC time course in the absence and presence of kynurenic acid with variable synaptic glutamate concentration profile. The result of this analysis showed that astrocytic G‐protein coupled receptor‐activation increased the peak synaptic glutamate concentration from 0.96 to 1.72 mM and prolonged the duration of glutamate in the synaptic cleft following PAR1 activation by approximately two‐fold. This effect was blocked by selective expression of a PAR1 shRNA in astrocytes. These data suggest that astrocytic G‐protein coupled receptors can influence the glutamate concentration time course in the synaptic cleft, which has important implications for excitatory synaptic transmission, as well as synaptic plasticity.

The glutamate transporter, GLAST, participates in a macromolecular complex that supports glutamate metabolism

GLAST is the predominant glutamate transporter in the cerebellum and contributes substantially to glutamate transport in forebrain. This astroglial glutamate transporter quickly binds and clears synaptically released glutamate and is principally responsible for ensuring that synaptic glutamate concentrations remain low. This process is associated with a significant energetic cost. Compartmentalization of GLAST with mitochondria and proteins involved in energy metabolism could provide energetic support for glutamate transport. Therefore, we performed immunoprecipitation and co-localization experiments to determine if GLAST might co-compartmentalize with proteins involved in energy metabolism. GLAST was immunoprecipitated from rat cerebellum and subunits of the Na+/K+ ATPase, glycolytic enzymes, and mitochondrial proteins were detected. GLAST co-localized with mitochondria in cerebellar tissue. GLAST also co-localized with mitochondria in fine processes of astrocytes in organotypic hippocampal slice cultures. From these data, we hypothesized that mitochondria participate in a macromolecular complex with GLAST to support oxidative metabolism of transported glutamate. To determine the functional metabolic role of this complex, we measured CO2 production from radiolabeled glutamate in cultured astrocytes and compared it to overall glutamate uptake. Within 15min, 9% of transported glutamate was converted to CO2. This CO2 production was blocked by inhibitors of glutamate transport and glutamate dehydrogenase, but not by an inhibitor of glutamine synthetase. Our data support a model in which GLAST exists in a macromolecular complex that allows transported glutamate to be metabolized in mitochondria to support energy production.

Co-compartmentalization of the Astroglial Glutamate Transporter, GLT-1, with Glycolytic Enzymes and Mitochondria

Efficient excitatory transmission depends on a family of transporters that use the Na(+)-electrochemical gradient to maintain low synaptic concentrations of glutamate. These transporters consume substantial energy in the spatially restricted space of fine astrocytic processes. GLT-1 (EAAT2) mediates the bulk of this activity in forebrain. To date, relatively few proteins have been identified that associate with GLT-1. In the present study, GLT-1 immunoaffinity isolates were prepared from rat cortex using three strategies and analyzed by liquid chromatography-coupled tandem mass spectrometry. In addition to known interacting proteins, the analysis identified glycolytic enzymes and outer mitochondrial proteins. Using double-label immunofluorescence, GLT-1 was shown to colocalize with the mitochondrial matrix protein, ubiquinol-cytochrome c reductase core protein 2 or the inner mitochondrial membrane protein, ADP/ATP translocase, in rat cortex. In biolistically transduced hippocampal slices, fluorescently tagged GLT-1 puncta overlapped with fluorescently tagged mitochondria along fine astrocytic processes. In a Monte Carlo-type computer simulation, this overlap was significantly more frequent than would occur by chance. Furthermore, fluorescently tagged hexokinase-1 overlapped with mitochondria or GLT-1, strongly suggesting that GLT-1, mitochondria, and the first step in glycolysis are cocompartmentalized in astrocytic processes. Acute inhibition of glycolysis or oxidative phosphorylation had no effect on glutamate uptake in hippocampal slices, but simultaneous inhibition of both processes significantly reduced transport. Together with previous results, these studies show that GLT-1 cocompartmentalizes with Na(+)/K(+) ATPase, glycolytic enzymes, and mitochondria, providing a mechanism to spatially match energy and buffering capacity to the demands imposed by transport.

Distinct modes of modulation of GABAergic transmission by Group I metabotropic glutamate receptors in rat entorhinal cortex

Activation of metabotropic glutamate receptors (mGluRs) modulates synaptic transmission, whereas the roles of mGluRs in GABAergic transmission in the entorhinal cortex (EC) are elusive. Here, we examined the effects of mGluRs on GABAergic transmission onto the principal neurons in the superficial layers of the EC. Bath application of DHPG, a selective Group I mGluR agonist, increased the frequency and amplitude of spontaneous IPSCs (sIPSCs) whereas application of DCG-IV, an agonist for Group II mGluRs or L-AP4, an agonist for Group III mGluRs failed to change significantly sIPSC frequency and amplitude. Bath application of DHPG failed to change significantly the frequency and amplitude of miniature IPSCs (mIPSCs) recorded in the presence of tetradotoxin but significantly reduced the amplitude of IPSCs evoked by extracellular field stimulation or in synaptically connected interneuron-pyramidal neuron pairs in layer III of the EC. DHPG increased the frequency but reduced the amplitude of APs recorded from entorhinal interneurons. Bath application of DHPG generated membrane depolarization and increased the input resistance of GABAergic interneurons. DHPG-mediated depolarization of GABAergic interneurons was mediated by inhibition of background K(+) channels which are insensitive to extracellular Cs(+), TEA, 4-AP, and Ba(2+). DHPG-induced facilitation of sIPSCs was mediated by mGluR(5) and required the function of Galphaq but was independent of phospholipase C activity. Elevation of synaptic glutamate concentration by bath application of glutamate transporter inhibitors significantly increased sIPSC frequency and amplitude demonstrating a physiological role of mGluRs in GABAergic transmission. Our results provide a cellular and molecular mechanism to explain the physiological and pathological roles of mGluRs in the EC.

How do soluble oligomers of amyloid β-protein impair hippocampal synaptic plasticity?

How do soluble oligomers of amyloid β-protein impair hippocampal synaptic plasticity?

Reduced expression of glutamate transporters vGluT1, EAAT2 and EAAT4 in learned helpless rats, an animal model of depression

It has been widely accepted that glial pathology and disturbed synaptic transmission contribute to the neurobiology of depression. Apart from monoaminergic alterations, an influence of glutamatergic signal transduction has been reported. Therefore, gene expression of glutamate transporters that strictly control synaptic glutamate concentrations have to be assessed in animal models of stress and depression. We performed in situ-hybridizations in learned helplessness rats, a well established animal model of depression, to assess vGluT1 and EAAT1-4. Sprague-Dawley rats of two inbred lines were tested for helpless behavior and grouped into three cohorts according to the number of failures to stop foot shock currents by lever pressing. Helpless animals showed a significantly suppressed expression of the glial glutamate transporter EAAT2 (rodent nomenclature GLT1) in hippocampus and cerebral cortex compared to littermates with low failure rate and not helpless animals. This finding was validated on protein level using immunohistochemistry. Additionally, expression levels of EAAT4 and the vesicular transporter vGluT1 were reduced in helpless animals. In contrast, the transcript levels of EAAT1 (GLAST) and EAAT3 (EAAC1) were not significantly altered. These results strongly suggest reduced astroglial glutamate uptake and implicate increased glutamate levels in learned helplessness. The findings are in concert with antidepressant effects of NMDA-receptor antagonists and the hypotheses that impaired astroglial functions contribute to the pathogenesis of affective disorders.

Presynaptic G-Protein-Coupled Receptors Dynamically Modify Vesicle Fusion, Synaptic Cleft Glutamate Concentrations, and Motor Behavior

Understanding how neuromodulators regulate behavior requires investigating their effects on functional neural systems, but also their underlying cellular mechanisms. Utilizing extensively characterized lamprey motor circuits, and the unique access to reticulospinal presynaptic terminals in the intact spinal cord that initiate these behaviors, we investigated effects of presynaptic G-protein-coupled receptors on locomotion from the systems level, to the molecular control of vesicle fusion. 5-HT inhibits neurotransmitter release via a Gbetagamma interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that promotes kiss-and-run vesicle fusion. In the lamprey spinal cord, we demonstrate that, although presynaptic 5-HT receptors inhibit evoked neurotransmitter release from reticulospinal command neurons, their activation does not abolish locomotion but rather modulates locomotor rhythms. Liberation of presynaptic Gbetagamma causes substantial inhibition of AMPA receptor-mediated synaptic responses but leaves NMDA receptor-mediated components of neurotransmission mostly intact. Because Gbetagamma binding to the SNARE complex is displaced by Ca(2+)-synaptotagmin binding, 5-HT-mediated inhibition displays Ca(2+) sensitivity. We show that, as Ca(2+) accumulates presynaptically during physiological bouts of activity, 5-HT/Gbetagamma-mediated presynaptic inhibition is relieved, leading to a frequency-dependent increase in synaptic concentrations of glutamate. This frequency-dependent phenomenon mirrors a shift in the vesicle fusion mode and a recovery of AMPA receptor-mediated EPSCs from inhibition without a modification of NMDA receptor EPSCs. We conclude that activation of presynaptic 5-HT G-protein-coupled receptors state-dependently alters vesicle fusion properties to shift the weight of NMDA versus AMPA receptor-mediated responses at excitatory synapses. We have therefore identified a novel mechanism in which modification of vesicle fusion modes may profoundly alter locomotor behavior.

Evidence for change in current–flux coupling of GLT1 at high glutamate concentrations in rat primary forebrain neurons and GLT1a‐expressing COS‐7 cells

Glutamate is the major excitatory neurotransmitter of the central nervous system and is toxic to neurons even at low concentrations. GLT1, the rodent analog of human EAAT2, is primarily responsible for glutamate clearance in the cerebrum. GLT1 was thought to be expressed exclusively in astrocytes in the mature brain. Recently, however, GLT1a was demonstrated in excitatory axon terminals where synaptic glutamate concentration rises above 1 mm during excitatory transmission. GLT1 function in neurons with accurate control of both intracellular and extracellular solutions mimicking synaptic concentration gradients has never been studied. Here we characterized the kinetics of coupled glutamate transporter current in whole-cell configuration and [(3)H]-l-glutamate uptake in cultured rat cerebral neurons across the entire range of synaptic glutamate concentrations. In both neurons and GLT1a-transfected COS-7 cells, the kinetics were similar and revealed two specific components: a high-affinity component with glutamate k(D) value around 15 mum and a low-affinity component with k(D) value around 0.2 mm. The specific low-affinity component was discovered as a result of significant deviation of the transporter current from Michaelis-Menten kinetics in the 100-300 mum concentration range. Activation of the specific low-affinity component led to a two-fold decrease in the current/flux ratio, implying a change in the transport coupling. Our data indicate that GLT1 endogenously expressed in cultured rat forebrain neurons displays high and low glutamate affinity uptake components that are different in current-flux coupling ratios. This property is intrinsic to the protein because it was also observed in GLT1a-transfected COS-7 cells.

Amyloid-beta peptide decreases glutamate uptake in cultured astrocytes: Involvement of oxidative stress and mitogen-activated protein kinase cascades

Alzheimer's disease (AD) is a progressive neurodegenerative disorder primarily characterized by excessive deposition of amyloid-β (Aβ) peptides in the brain. One of the earliest neuropathological changes in AD is the presence of a high number of reactive astrocytes at sites of Aβ deposition. Disturbance of glutamatergic neurotransmission and consequent excitotoxicity is also believed as implicated in the progression of this dementia. Therefore, the study of astrocyte responses to Aβ, the main cellular type involved in the maintenance of synaptic glutamate concentrations, is crucial for understanding the pathogenesis of AD. This study aims to investigate the effect of Aβ on the astrocytic glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate–aspartate transporter (GLAST), and their relative participation to glutamate clearance. In addition we have also investigated the involvement of mitogen-activated protein (MAP) kinases in the modulation of GLT-1 and GLAST levels and activity and the putative contribution of oxidative stress induced by Aβ to the astrocytic glutamate transport function. Therefore, we used primary cultures of rat brain astrocytes exposed to Aβ synthetic peptides. The data obtained show that Aβ 1-40 peptide decreased astroglial glutamate uptake capacity in a non-competitive mode of inhibition, assessed in terms of tritium radiolabeled d-aspartate ( d-[ 3H]aspartate) transport. The activity of GLT-1 seemed to be more affected than that of GLAST, and the levels of both transporters were decreased in Aβ 1-40-treated astrocytes. We demonstrated that MAP kinases, extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase, were activated in an early phase of Aβ 1-40 treatment and the whole pathways differentially modulated the glutamate transporters activity/levels. Moreover it was shown that oxidative stress induced by Aβ 1-40 may lead to the glutamate uptake impairment observed. Taken together, our results suggest that Aβ peptide downregulates the astrocytic glutamate uptake capacity and this effect may be in part mediated by oxidative stress and the differential activity and complex balance between the MAP kinase signaling pathways.

A Positive Correlation Between -Glutamate and Glutamine on Brain 1H-MR Spectroscopy and Neonatal Seizures in Moderate and Severe Hypoxic-Ischemic Encephalopathy

Cerebral metabolic disturbances during seizures and hypoxic-ischemic events in patients with hypoxic-ischemic encephalopathy (HIE) can lead to excessive synaptic and extracellular concentrations of glutamate with concomitant and subsequent neuronal cell injury or death.[1][1],[2][2] Multiple studies

Presynaptic G-Protein-Coupled Receptors Regulate Synaptic Cleft Glutamate via Transient Vesicle Fusion

When synaptic vesicles fuse with the plasma membrane, they may completely collapse or fuse transiently. Transiently fusing vesicles remain structurally intact and therefore have been proposed to represent a form of rapid vesicle recycling. However, the impact of a transient synaptic vesicle fusion event on neurotransmitter release, and therefore on synaptic transmission, has yet to be determined. Recently, the molecular mechanism by which a serotonergic presynaptic G-protein-coupled receptor (GPCR) regulates synaptic vesicle fusion and inhibits synaptic transmission was identified. By making paired electrophysiological recordings in the presence and absence of low-affinity antagonists, we now demonstrate that activation of this presynaptic GPCR lowers the peak synaptic cleft glutamate concentration independently of the probability of vesicle fusion. Furthermore, this change in cleft glutamate concentration differentially inhibits synaptic NMDA and AMPA receptor-mediated currents. We conclude that a presynaptic GPCR regulates the profile of glutamate in the synaptic cleft through altering the mechanism of vesicle fusion leading to qualitative as well as quantitative changes in neural signaling.