Activation of presynaptic ionotropic glutamate receptors stimulates GABA release from hippocampal and cortical nerve terminals in rats

One of the pathways implicated in a fine-tuning control of neurosecretory process is the activation of presynaptic receptors. The present study was focused on the role of presynaptic glutamate receptor activation in the regulation of inhibitory synaptic transmission in the rat hippocampus and cortex. We aimed to clarify what types of ionotropic glutamate receptors are involved in the modulation of GABA secretion, and what mechanism underlies this modulation. We have revealed that specific agonists of kainate and NMDA receptors, kainate and NMDA, like glutamate, induced the release of [3H]GABA from hippocampal and cortical nerve terminals suggesting the involvement of both types in the regulation of GABAergic transmission. Our results indicate preferential involvement of vesicular, but not cytosolic, pool in response to glutamate receptor activation. This is based on the finding that NO-711 (a specific inhibitor of plasma membrane GABA transporters), fails to attenuate [3H]GABA release. We have concluded that presynaptic glutamate receptor-induced modulation of the strength of synaptic response is due to increasing the release probability of synaptic vesicles.     

The synaptic vesicle protein SV2 is a novel type of transmembrane transporter.

The primary function of synaptic vesicles is to store and release neurotransmitter. Synaptic vesicles are locally recycled following exocytosis and rapidly refilled with neurotransmitter from the cytoplasm by a process that depends on the electrochemical gradient generated by a proton pump. Little is known about the molecules that import neurotransmitter into synaptic vesicles. We report here that the sequence of the synaptic vesicle protein SV2 identifies this protein as a novel type of transmembrane transporter. The deduced amino acid sequence of SV2 contains two sets of six predicted transmembrane domains: the six most N-terminal transmembrane domains are highly homologous to a subfamily of transporters that includes the human glucose transporter, while the six most C-terminal domains are homologous to the plasma membrane transporters for neurotransmitters. We propose that SV2 mediates transport of neurotransmitters into synaptic vesicles.     

Long-term potentiation of neuronal glutamate transporters

Persistent, use-dependent modulation of synaptic strength has been demonstrated for fast synaptic transmission mediated by glutamate and has been hypothesized to underlie persistent behavioral changes ranging from memory to addiction. Glutamate released at synapses is sequestered by the action of excitatory amino acid transporters (EAATs) in glia and postsynaptic neurons. So, the efficacy of glutamate transporter function is crucial for regulating glutamate spillover to adjacent presynaptic and postsynaptic receptors and the consequent induction of plastic or excitotoxic processes. Here, we report that tetanic stimulation of cerebellar climbing fiber-Purkinje cell synapses results in long-term potentiation (LTP) of a climbing fiber-evoked glutamate transporter current recorded in Purkinje cells. This LTP is postsynaptically expressed and requires activation of an mGluR1/PKC cascade. Together with a simultaneously induced long-term depression (LTD) of postsynaptic AMPA receptors, this might reflect an integrated antiexcitotoxic cellular response to strong climbing fiber synaptic activation, as occurs following an ischemic episode.     

Neurogenetics of vesicular transporters in C. elegans.

The nematode Caenorhabditis elegans has a number of advantages for the analysis of synaptic molecules. These include a simple nervous system in which all cells are identified and synaptic connectivity is known and reproducible, a large collection of mutants and powerful methods of genetic analysis, simple methods for the generation and analysis of transgenic animals, and a number of relatively simple quantifiable behaviors. Studies in C. elegans have made major contributions to our understanding of vesicular transmitter transporters. Two of the four classes of vesicular transporters so far identified (VAChT and VGAT) were first described and cloned in C. elegans; in both cases, the genes were first identified and cloned by means of mutations causing a suggestive phenotype (1, 2). The phenotypes of eat-4 mutants and the cell biology of the EAT-4 protein were critical in the identification of this protein as the vesicular glutamate transporter (3, 4). In addition, the unusual gene structure associated with the cholinergic locus was first described in C. elegans (5). The biochemical properties of the nematode transporters are surprisingly similar to their vertebrate counterparts, and they can be assayed under similar conditions using the same types of mammalian cells (6, 7). In addition, mild and severe mutants (including knockouts) are available for each of the four C. elegans vesicular transporters, which has permitted a careful evaluation of the role(s) of vesicular transport in transmitter-specific behaviors. Accordingly, it seems appropriate at this time to present the current status of the field. In this review, we will first discuss the properties of C. elegans vesicular transporters and transporter mutants, and then explore some of the lessons and insights C. elegans research has provided to the field of vesicular transport.     

Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft.     

Neuronal glutamate transporters control activation of postsynaptic metabotropic glutamate receptors and influence cerebellar long-term depression.

Neuronal and glial isoforms of glutamate transporters show distinct distributions on membranes surrounding excitatory synapses, but specific roles for transporter subtypes remain unidentified. At parallel fiber (PF) synapses in cerebellum, neuronal glutamate transporters and metabotropic glutamate receptors (mGluRs) have overlapping postsynaptic distributions suggesting that postsynaptic transporters selectively regulate mGluR activation. We examined interactions between transporters and mGluRs by evoking mGluR-mediated excitatory postsynaptic currents (mGluR EPSCs) in slices of rat cerebellum. Selective inhibition of postsynaptic transporters enhanced mGluR EPSCs greater than 3-fold. Moreover, impairing glutamate uptake facilitated mGluR-dependent long-term depression at PF synapses. Our results demonstrate that uniquely positioned glutamate transporters strongly influence mGluR activation at cerebellar PF synapses. Postsynaptic glutamate uptake may serve as a general mechanism for regulating mGluR-initiated synaptic depression.     

The transport of neurotransmitters into synaptic vesicles.

As investigations identify additional plasma membrane neurotransmitter transporters, attention has focused on the molecular basis of neurotransmitter transport into synaptic vesicles. The transport of biogenic amines into chromaffin granules has served as the paradigm for understanding vesicular transport. Recent work now describes the vesicular transport of other classical neurotransmitters, which occur by distinct but related mechanisms. To determine their biochemical basis, several of the transporters have been functionally reconstituted in liposomes. The ability of vesicular amine transport to protect against the neurotoxin MPP+ has permitted the isolation of the first cDNA clone for a member of this family, and the sequence establishes a relationship with drug-resistance transporters in bacteria.     

Sodium-dependent neurotransmitter reuptake systems.

The recent cloning of the gamma-aminobutyric acid and norepinephrine Na(+)-dependent neurotransmitter transporters has led to new approaches for understanding the structure and function of these important synaptic proteins and provides a conceptual model in which to consider recent advances in the physiology, pharmacology and biochemistry of this gene family.     

Fast removal of synaptic glutamate by postsynaptic transporters

Glutamate transporters are believed to remove glutamate from the synaptic cleft only slowly because they cycle slowly. However, we show that when glutamate binds to postsynaptic transporters at the cerebellar climbing fiber synapse, it evokes a conformation change and inward current that reflect glutamate removal from the synaptic cleft within a few milliseconds, a time scale much faster than the overall cycle time. Contrary to present models, glutamate removal does not require binding of an extracellular proton, and the time course of transporter anion conductance activation differs from that of glutamate removal. The charge movement associated with glutamate removal is consistent with the majority of synaptically released glutamate being removed from the synaptic cleft by postsynaptic transporters.     

Glutamate uptake in synaptic plasticity: from mollusc to mammal

A great deal of research has been directed toward understanding the cellular mechanisms underlying synaptic plasticity and memory formation. To this point, most research has focused on the more "active" components of synaptic transmission: presynaptic transmitter release and postsynaptic transmitter receptors. Little work has been done characterizing the role neurotransmitter transporters might play during changes in synaptic efficacy. We review several new experiments that demonstrate glutamate transporters are regulated during changes in the efficacy of glutamatergic synapses. This regulation occurred during long-term facilitation of the sensorimotor synapse of Aplysia and long-term potentiation of the Schaffer-collateral synapse of the rat. We propose that glutamate transporters are "co-regulated" with other molecules/processes involved in synaptic plasticity, and that this process is phylogenetically conserved. These new findings indicate that glutamate transporters most likely play a more active role in neurotransmission than previously believed.     

Contribution of astrocytes to synaptic transmission in the rat supraoptic nucleus

Astrocytes, besides supporting metabolic and scaffolding functions, play a prominent role in the modulation of neuronal communication. In particular, they are responsible for clearing synaptically-released glutamate via highly specific transporters located on their plasma membrane. Since glutamate is the main excitatory neurotransmitter in the central nervous system (CNS), astrocytes are likely to play a central role in the regulation of synaptic processing and overall cellular excitability. We recently investigated the influence of astrocytes on glutamatergic and GABAergic transmission in the rat supraoptic nucleus (SON) of the hypothalamus. This nucleus is part of the hypothalamus-neurohypophysial system (HNS), which constitutes a conspicuous example of activity-dependent neuroglial plasticity, in which certains physiological conditions, such as parturition, lactation, and dehydration are accompanied by a structural remodeling of the neurones, their synaptic inputs and their surrounding glia. The use of pharmacological inhibitors of glutamate transporters on this model, in which a physiological change in the astrocyte environment occurs, has brought new insights on the contribution of astrocytes to both excitatory and inhibitory neurotransmissions. The astrocytic environment of neurons appears to control glutamate uptake and diffusion in the extracellular space. This has direct repercussions on the tonic level of activation of presynaptic glutamate receptors and, as a consequence, on the release of neurotransmitter. This short review summarizes data obtained so far, which clearly support the view that astrocytes are indeed a third partner in synaptic transmission, and which show that the supraoptic nucleus represents a remarkable model to study dynamic physiological interactions between astrocytes and neurons.     

Interactions between glutamate transporters and metabotropic glutamate receptors at excitatory synapses in the cerebellar cortex

Five glutamate transporter genes have been identified; two of these (EAAT3 and EAAT4) are expressed in neurons and are predominantly confined to the membranes of cell bodies and dendrites. At an ultrastructural level, glutamate transporters have been shown to surround excitatory synapses in hippocampus and cerebellum [J. Neurosci. 18 (1998) 3606; J. Comp. Neurol. 418 (2000) 255]. This pattern of localization overlaps the well-described perisynaptic distribution of Group I metabotropic glutamate receptors or mGluRs [Neuron 11 (1993) 771; J. Chem. Neuroanat. 13 (1997) 77]. Both of the principal excitatory synaptic inputs to cerebellar Purkinje neurons, the parallel fiber (PF) and climbing fiber (CF) synapses, express mGluR-dependent forms of synaptic plasticity [Nat. Neurosci. 4 (2001) 467]. Prompted by the colocalization of postsynaptic glutamate transporters and mGluRs, we have examined whether glutamate uptake limits mGluR-mediated signals and mGluR-dependent forms of plasticity at PF and CF synapses in cerebellar slices. We find that, at PF and, surprisingly also at CF synapses, mGluR activation generates a slow synaptic current and triggers intracellular calcium release. At both PF and CF synapses, mGluR responses are strongly limited by glutamate transporters under resting conditions and are facilitated by short trains of stimuli. Nearly every Purkinje neuron expresses an mGluR-mediated synaptic current upon inhibition of glutamate transport. Global applications of glutamate achieved by photolysis of chemically caged glutamate yield similar results and argue that the colocalized transporters can effectively limit glutamate access to the mGluRs even in the face of such a large amount of transmitter. We hypothesize that neuronal glutamate transporters and Group I mGluRs located in the perisynaptic space interact to sense and then regulate the amount of glutamate escaping excitatory synapses. This hypothesis is currently being tested using electrophysiological methods and the introduction of optically tagged glutamate transporter proteins. In the brain, synaptic signals are terminated mainly by neurotransmitter transporters. Families of genes encoding transporters for the major neurotransmitters (dopamine, GABA, glutamate, glycine, norepinephrine and 5-HT) have been identified. Although transporters serve as targets for important classes of therapeutic drugs (e.g. selective serotonin reuptake inhibitors) and drugs of abuse (amphetamine, cocaine), little is known about how they operate at a molecular level or contribute to synaptic transmission.     

中枢神经系统谷氨酸转运体的研究进展

在中枢神经系统,谷氨酸转运体在谷氨酸一谷氨酰胺循环中发挥着重要作用。谷氨酸转运体有高亲和力转运体,即兴奋性氨基酸转运体（excitatory amino acid transporters,EAATs）和低亲和力转运体,即囊泡谷氨酸转运体（vesicular glutamate transporters,VGLUTs）两种类型。其中,VGLUTs的功能是特异地将突触囊泡外的谷氨酸转运进入突触囊泡内,它包括三个成员,分别是VGLUT1、VGLUT2和VGLUT3。一方面,VGLUT1和VGLUT2标记了所有的谷氨酸能神经元,是谷氦酸能神经元和它们轴突末端高度特异的标志;另一方面,VGLUT1标志着皮质一皮质投射,而VGLUT2则标志着丘脑一皮层投射,VGLUT3则位于抑制性突触末端。     

Synaptic modulation by substance P.

The phrase "synaptic modulation," to describe a role of neurotropic peptides, has been used in a number of different ways by a number of different investigators. Using the phrase in its original context, i.e. altered (increased or decreased) synaptic excitability without reference to site or mode of action, evidence is presented that substance P modulates synaptic transmission of cat alpha-motoneurons. The effect appears to be biphasic, with low doses inhibiting, and high doses facilitating synaptic transmission.     

On the Role of a Conserved Methionine in the Na+-Coupling Mechanism of a Neurotransmitter Transporter Homolog

Neurochemical Research - Excitatory amino acid transporters (EAAT) play a key role in glutamatergic synaptic communication. Driven by transmembrane cation gradients, these transporters catalyze the...     

Glutamate transporters: confining runaway excitation by shaping synaptic transmission

Traditionally, glutamate transporters have been viewed as membrane proteins that harness the electrochemical gradient to slowly transport glutamate from the extracellular space into glial cells. However, recent studies have shown that glutamate transporters on glial and neuronal membranes also rapidly bind released glutamate to shape synaptic transmission. In this Review, we summarize the properties of glutamate transporters that influence synaptic transmission and are subject to regulation and plasticity. We highlight how the diversity of glutamate-transporter function relates to transporter location, density and affinity.     

GABA and glycine in synaptic vesicles: storage and transport characteristics.

gamma-Aminobutyric acid (GABA) and glycine are major inhibitory neurotransmitters that are released from nerve terminals by exocytosis via synaptic vesicles. Here we report that synaptic vesicles immunoisolated from rat cerebral cortex contain high amounts of GABA in addition to glutamate. Synaptic vesicles from the rat medulla oblongata also contain glycine and exhibit a higher GABA and a lower glutamate concentration than cortical vesicles. No other amino acids were detected. In addition, the uptake activities of synaptic vesicles for GABA and glycine were compared. Both were very similar with respect to substrate affinity and specificity, bioenergetic properties, and regional distribution. We conclude that GABA, glycine, and glutamate are the only major amino acid neurotransmitters stored in synaptic vesicles and that GABA and glycine are transported by similar, if not identical, transporters.     

The structure of glutamate transporters shows channel-like features

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large family of secondary transporters, which includes transporters from a variety of bacterial, archaeal and eukaryotic organisms. The transporters consist of eight membrane-spanning alpha-helices and two pore-loop structures, which are unique among secondary transporters but may resemble pore-loops found in ion channels. Another distinctive structural feature is the presence of a highly amphipathic membrane-spanning alpha-helix that provides a hydrophilic path through the membrane. The unusual structural features of the transporters are discussed in relation to their function.     

Glutamine uptake by System A transporters maintains neurotransmitter GABA synthesis and inhibitory synaptic transmission

GABA synthesis is necessary to maintain synaptic vesicle filling, and key proteins in its biosynthetic pathways may play a role in regulating inhibitory synaptic stability and strength. GABAergic neurons require a source of precursor glutamate, possibly from glutamine, although it is controversial whether glutamine contributes to the synaptic pool of GABA. Here we report that inhibition of System A glutamine transporters with alpha-(methyl-amino) isobutyric acid rapidly reduced the amplitude of inhibitory post-synaptic currents and miniature inhibitory post-synaptic currents (mIPSCs) recorded in rat hippocampal area cornu ammonis 1 (CA1) pyramidal neurons, indicating that synaptic vesicle content of GABA was reduced. After inhibiting astrocytic glutamine synthesis by either blocking glutamate transporters or the glutamine synthetic enzyme, the effect of alpha-(methyl-amino) isobutyric acid on mIPSC amplitudes was abolished. Exogenous glutamine did not affect mIPSC amplitudes, suggesting that the neuronal transporters are normally saturated. Our findings demonstrate that a constitutive supply of glutamine is provided by astrocytes to inhibitory neurons to maintain vesicle filling. Therefore, glutamine transporters, like those for glutamate, are potential regulators of inhibitory synaptic strength. However, in contrast to glutamate, extracellular glutamine levels are normally high. Therefore, we propose a supportive role for glutamine, even under resting conditions, to maintain GABA vesicle filling.     

Electrogenic Glutamate Transporters in the CNS: Molecular Mechanism,Pre-steady-state Kinetics,and their Impact on Synaptic Signaling

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. The spatiotemporal profile of the glutamate concentration in the synapse is critical for excitatory synaptic signalling. The control of this spatiotemporal concentration profile requires the presence of large numbers of synaptically localized glutamate transporters that remove pre-synaptically released glutamate by uptake into neurons and adjacent glia cells. These glutamate transporters are electrogenic and utilize energy stored in the transmembrane potential and the Na⁺/K⁺-ion concentration gradients to accumulate glutamate in the cell. This review focuses on the kinetic and electrogenic properties of glutamate transporters, as well as on the molecular mechanism of transport. Recent results are discussed that demonstrate the multistep nature of the transporter reaction cycle. Results from pre-steady-state kinetic experiments suggest that at least four of the individual transporter reaction steps are electrogenic, including reactions associated with the glutamate-dependent transporter halfcycle. Furthermore, the kinetic similarities and differences between some of the glutamate transporter subtypes and splice variants are discussed. A molecular mechanism of glutamate transport is presented that accounts for most of the available kinetic data. Finally, we discuss how synaptic glutamate transporters impact on glutamate receptor activity and how transporters may shape excitatory synaptic transmission.