Glutamate transporters bring competition to the synapse

Glutamate transporters (GluTs) prevent the accumulation of glutamate and influence the occupancy of receptors at synapses. The ability of extrasynaptic NMDA receptors and metabotropic glutamate receptors to participate in signaling is tightly regulated by GluT activity. Astrocytes express the highest density of GluTs and dominate clearance away from these receptors; synapses that are not associated with astrocyte processes experience greater mGluR activation and can be exposed to glutamate released at adjacent synapses. Although less abundant, neuronal transporters residing in the postsynaptic membrane can also shield receptors from the glutamate that is released. The diversity in synaptic morphology suggests a correspondingly rich diversity of GluT function in excitatory transmission.     

Quantal glutamate release is essential for reliable neuronal encodings in cerebral networks

Background

The neurons and synapses work coordinately to program the brain codes of controlling cognition and behaviors. Spike patterns at the presynaptic neurons regulate synaptic transmission. The quantitative regulations of synapse dynamics in spike encoding at the postsynaptic neurons remain unclear.

Methodology/Principal Findings

With dual whole-cell recordings at synapse-paired cells in mouse cortical slices, we have investigated the regulation of synapse dynamics to neuronal spike encoding at cerebral circuits assembled by pyramidal neurons and GABAergic ones. Our studies at unitary synapses show that postsynaptic responses are constant over time, such as glutamate receptor-channel currents at GABAergic neurons and glutamate transport currents at astrocytes, indicating quantal glutamate release. In terms of its physiological impact, our results demonstrate that the signals integrated from quantal glutamatergic synapses drive spike encoding at GABAergic neurons reliably, which in turn precisely set spike encoding at pyramidal neurons through feedback inhibition.

Conclusion/Significance

Our studies provide the evidences for the quantal glutamate release to drive the spike encodings precisely in cortical circuits, which may be essential for programming the reliable codes in the brain to manage well-organized behaviors.     

The Protective Effects of Riluzole on Manganese-Induced Disruption of Glutamate Transporters and Glutamine Synthetase in the Cultured Astrocytes

Chronic exposure to excessive manganese (Mn) can lead to manganism, a type of neurotoxicity accomplished with extracellular glutamate (Glu) accumulation. To investigate this accumulation, this study focused on the role of astrocyte glutamate transporters (GluTs) and glutamine synthetase (GS), which have roles in Glu transport and metabolism, respectively. And the possible protective effects of riluzole (a glutamatergic modulator) were studied in relation to Mn exposure. At first, the astrocytes were exposed to 0, 125, 250, and 500 μM MnCl(2) for 24 h, and 100 μM riluzole was pretreated to astrocytes for 6 h before 500 μM MnCl(2) exposure. Then, [(3)H]-glutamate uptake was measured by liquid scintillation counting; Na(+)-K(+) ATPase and GS activities were determined by a colorimetric method; glutamate/aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), and GS mRNA expression were determined by RT-PCR and protein levels were measured by western blotting. The results showed that Mn inhibited Glu uptake, Na(+)-K(+) ATPase and GS activities, GLAST, GLT-1, and GS mRNA, and protein in a concentration-dependent manner. And they were significantly higher for astrocytes pretreated with 100 μM riluzole than the group exposed to 500 μM MnCl(2). The results suggested that Mn disrupted Glu transport and metabolism by inhibiting GluTs and GS. Riluzole activated protective effects on enhancing GluTs and GS to reverse Glu accumulation. In conclusion, Mn exposure results in the disruption of GLAST, GLT-1, and GS expression and function. Furthermore, riluzole attenuates this Mn toxicity.     

Modulation of synaptic transmission by astrocytes in the rat supraoptic nucleus.

One of the functions of astroglial cells in the central nervous system is to clear synaptically-released glutamate from the extracellular space. This is performed thanks to specific transporters of the excitatory amino acid expressed on their surface. The way by which astrocytic glutamate uptake contributes to synaptic transmission has been investigated via numerous experimental approaches but has never been addressed under conditions where neuroglial interactions are physiologically modified. Recently, we took advantage of the neuroglial plastic properties of the hypothalamo-neurohypophysial system to examine the consequences of a physiological reduction in the astrocytic coverage of neurons on glutamatergic synaptic transmission. This experimental model has brought some insights on the physiological interactions between glial cells and neurons at the level of the synapse. In particular, it has revealed that the degree of glial coverage of neurons influences glutamate concentration at the vicinity of excitatory synapses and, as a consequence, affects the level of activation of presynaptic glutamate receptors. Astrocytes, therefore, appear to contribute to the regulation of neuronal excitability by modulating synaptic efficacy at glutamatergic nerve terminals.     

Thematic Minireview Series: Molecular Mechanisms of Synaptic Plasticity

The human brain contains ∼86 billion neurons, which are precisely organized in specific brain regions and nuclei. High fidelity synaptic communication between subsets of neurons in specific circuits is required for most human behaviors, and is often disrupted in neuropsychiatric disorders. The presynaptic axon terminals of one neuron release neurotransmitters that activate receptors on multiple postsynaptic neuron targets to induce electrical and chemical responses. Typically, postsynaptic neurons integrate signals from multiple presynaptic neurons at thousands of synaptic inputs to control downstream communication to the next neuron in the circuit. Importantly, the strength (or efficiency) of signal transmission at each synapse can be modulated on time scales ranging up to the lifetime of the organism. This “synaptic plasticity” leads to changes in overall neuronal circuit activity, resulting in behavioral modifications. This series of minireviews will focus on recent advances in our understanding of the molecular and cellular mechanisms that control synaptic plasticity.     

Nerve growth factor collaborates with myocyte-derived factors to promote development of presynaptic sites in cultured sympathetic neurons

Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle-containing growth cones. In conjunction with previously published results showing that NGF has long-term effects on synaptic transmission between sympathetic neurons and myocytes, this work suggests that NGF acts to promote sympathetic neurotransmission by increasing the number of sympathetic fibers establishing target contact. Further, we found that developmental changes in cardiac myocytes led to an increase in the density of synaptic vesicle-containing variocosities along sympathetic fibers, a process regulated by NGF. Thus, as myocytes mature they produce factors that promote the formation of sympathetic presynaptic structures. These results argue that multiple target interactions regulate the extent of synapse formation between sympathetic neurons and cardiac cells and suggest that NGF promotes presynaptic development by increasing neuronal contact with myocyte-derived cell surface or matrix-associated factors.     

Two neuronal,nuclear-localized RNA binding proteins involved in synaptic transmission

While there is evidence that distinct protein isoforms resulting from alternative pre-mRNA splicing play critical roles in neuronal development and function, little is known about molecules regulating alternative splicing in the nervous system. Using Caenorhabditis elegans as a model for studying neuron/target communication, we report that unc-75 mutant animals display neuroanatomical and behavioral defects indicative of a role in modulating GABAergic and cholinergic neurotransmission but not neuronal development. We show that unc-75 encodes an RRM domain-containing RNA binding protein that is exclusively expressed in the nervous system and neurosecretory gland cells. UNC-75 protein, as well as a subset of related C. elegans RRM proteins, localizes to dynamic nuclear speckles; this localization pattern supports a role for the protein in pre-mRNA splicing. We found that human orthologs of UNC-75, whose splicing activity has recently been documented in vitro, are expressed nearly exclusively in brain and when expressed in C. elegans, rescue unc-75 mutant phenotypes and localize to subnuclear puncta. Furthermore, we report that the subnuclear-localized EXC-7 protein, the C. elegans ortholog of the neuron-restricted Drosophila ELAV splicing factor, acts in parallel to UNC-75 to also affect cholinergic synaptic transmission. In conclusion, we identified a new neuronal, putative pre-mRNA splicing factor, UNC-75, and show that UNC-75, as well as the C. elegans homolog of ELAV, is required for the fine tuning of synaptic transmission. These findings thus provide a novel molecular link between pre-mRNA splicing and presynaptic function.     

The SAD-1 kinase regulates presynaptic vesicle clustering and axon termination

During synapse formation, presynaptic axon outgrowth is terminated, presynaptic clusters of vesicles are associated with active zone proteins, and active zones are aligned with postsynaptic neurotransmitter receptors. We report here the identification of a novel serine/threonine kinase, SAD-1, that regulates several aspects of presynaptic differentiation in C. elegans. In sad-1 mutant animals presynaptic vesicle clusters in sensory neurons and motor neurons are diffuse and disorganized. Sensory axons fail to terminate in sad-1 mutants, whereas overexpression of SAD-1 causes sensory axons to terminate prematurely. SAD-1 protein is expressed in the nervous system and localizes to synapse-rich regions of the axons. SAD-1 is related to PAR-1, a kinase that regulates cell polarity during asymmetric cell division. Overexpression of SAD-1 causes mislocalization of vesicle proteins to dendrites, suggesting that sad-1 affects axonal-dendritic polarity as well as synaptic development.     

High-fat diets induce changes in hippocampal glutamate metabolism and neurotransmission

Obesity and high-fat (HF) diets have a deleterious impact on hippocampal function and lead to impaired synaptic plasticity and learning deficits. Because all of these processes need an adequate glutamatergic transmission, we have hypothesized that nutritional imbalance triggered by these diets might eventually concern glutamate (Glu) neural pathways within the hippocampus. Glu is withdrawn from excitatory synapses by specific uptake mechanisms involving neuronal (EAAT-3) and glial (GLT-1, GLAST) transporters, which regulate the time that synaptically released Glu remains in the extracellular space and, consequently, the duration and location of postsynaptic receptor activation. The goal of the present study was to evaluate in mouse hippocampus the effect of a short-term high-fat dietary treatment on 1) Glu uptake kinetics, 2) the density of Glu carriers and Glu-degrading enzymes, 3) the density of Glu receptor subunits, and 4) synaptic transmission and plasticity. Here, we show that HF diet triggers a 50% decrease of the Michaelis-Menten constant together with a 300% increase of the maximal velocity of the uptake process. Glial Glu carriers GLT-1 and GLAST were upregulated in HF mice (32 and 27%, respectively), whereas Glu-degrading enzymes glutamine synthase and GABA-decarboxilase appeared to be downregulated in these animals. In addition, HF diet hippocampus displayed diminished basal synaptic transmission and hindered NMDA-induced long-term depression (NMDA-LTD). This was coincident with a reduced density of the NR2B subunit of NMDA receptors. All of these results are compatible with the development of leptin resistance within the hippocampus. Our data show that HF diets upregulate mechanisms involved in Glu clearance and simultaneously impair Glu metabolism. Neurochemical changes occur concomitantly with impaired basal synaptic transmission and reduced NMDA-LTD. Taken together, our results suggest that HF diets trigger neurochemical changes, leading to a desensitization of NMDA receptors within the hippocampus, which might account for cognitive deficits.     

SAD: a presynaptic kinase associated with synaptic vesicles and the active zone cytomatrix that regulates neurotransmitter release

A serine/threonine kinase SAD-1 in C. elegans regulates synapse development. We report here the isolation and characterization of mammalian orthologs of SAD-1, named SAD-A and SAD-B, which are specifically expressed in the brain. SAD-B is associated with synaptic vesicles and, like the active zone proteins CAST and Bassoon, is tightly associated with the presynaptic cytomatrix in nerve terminals. A short conserved region (SCR) in the COOH-terminus is required for the synaptic localization of SAD-B. Overexpression of SAD-B in cultured rat hippocampal neurons significantly increases the frequency of miniature excitatory postsynaptic current but not its amplitude. Introduction of SCR into presynaptic superior cervical ganglion neurons in culture significantly inhibits evoked synaptic transmission. Moreover, SCR decreases the size of the readily releasable pool measured by applying hypertonic sucrose. Furthermore, SAD-B phosphorylates the active zone protein RIM1 but not Munc13-1. These results suggest that mammalian SAD kinase presynaptically regulates neurotransmitter release.     

Reduced SNAP‐25 alters short‐term plasticity at developing glutamatergic synapses

SNAP‐25 is a key component of the synaptic‐vesicle fusion machinery, involved in several psychiatric diseases including schizophrenia and ADHD. SNAP‐25 protein expression is lower in different brain areas of schizophrenic patients and in ADHD mouse models. How the reduced expression of SNAP‐25 alters the properties of synaptic transmission, leading to a pathological phenotype, is unknown. We show that, unexpectedly, halved SNAP‐25 levels at 13–14 DIV not only fail to impair synaptic transmission but instead enhance evoked glutamatergic neurotransmission. This effect is possibly dependent on presynaptic voltage‐gated calcium channel activity and is not accompanied by changes in spontaneous quantal events or in the pool of readily releasable synaptic vesicles. Notably, synapses of 13–14 DIV neurons with reduced SNAP‐25 expression show paired‐pulse depression as opposed to paired‐pulse facilitation occurring in their wild‐type counterparts. This phenotype disappears with synapse maturation. As alterations in short‐term plasticity represent a new mechanism contributing to cognitive impairments in intellectual disabilities, our data provide mechanistic clues for neuronal circuit alterations in psychiatric diseases characterized by reduced expression of SNAP‐25.     

The immunoglobulin superfamily protein SYG-1 determines the location of specific synapses in C. elegans

During nervous system development, neurons form reproducible synapses onto specific targets. Here, we analyze the development of stereotyped synapses of the C. elegans HSNL neuron in vivo. Postsynaptic neurons and muscles were not required for accurate synaptic vesicle clustering in HSNL. Instead, vulval epithelial cells that contact HSNL act as synaptic guidepost cells that direct HSNL presynaptic vesicles to adjacent regions. The mutant syg-1(ky652) has defects in synapse formation that resemble those in animals that lack vulval epithelial cells: HSNL synaptic vesicles fail to accumulate at normal synaptic locations and form ectopic anterior clusters. syg-1 encodes an immunoglobulin superfamily protein that acts in the presynaptic HSNL axon. SYG-1 protein is localized to the site of future synapses, where it initiates synapse formation and localizes synaptic connections in response to the epithelial signal. SYG-1 is related to Drosophila IrreC and vertebrate NEPH1 proteins, which mediate cell-cell recognition in diverse developmental contexts.     

Role of glutamate in locomotor rhythm generating neuronal circuitry

Central pattern generators (CPGs) are defined as neuronal circuits capable of producing a rhythmic and coordinated output without the influence of sensory input. The locomotor and respiratory neuronal circuits are two of the better-characterized CPGs, although much work remains to fully understand how these networks operate. Glutamatergic neurons are involved in most neuronal circuits of the nervous system and considerable efforts have been made to study glutamate receptors in nervous system signaling using a variety of approaches. Because of the complexity of glutamate-mediated signaling and the variety of receptors triggered by glutamate, it has been difficult to pinpoint the role of glutamatergic neurons in neuronal circuits. In addition, glutamate is an amino acid used by every cell, which has hampered identification of glutamatergic neurons. Glutamatergic excitatory neurotransmission is dependent on the release from glutamate-filled presynaptic vesicles loaded by three members of the solute carrier family, Slc17a6-8, which function as vesicular glutamate transporters (VGLUTs). Recent data describe that Vglut2 (Slc17a6) null mutant mice die immediately after birth due to a complete loss of the stable autonomous respiratory rhythm generated by the pre-B?tzinger complex. Surprisingly, we found that basal rhythmic locomotor activity is not affected in Vglut2 null mutant embryos. With this perspective, we discuss data regarding presence of VGLUT1, VGLUT2 and VGLUT3 positive neuronal populations in the spinal cord.     

Neurochemical aspects of interneuronal communication

It is suggested that the term neurotransmission, which is used to designate neuronal communication at synaptic level, be associated to the less restrictive term neuromodulation. These two types of intercellular communication seem in fact to be two basically different mechanisms, both of which contribute to neuronal integration. The integration of neuronal information at cellular level appears to be more complex than the simple addition of excitatory plus inhibitory influences eliciting postsynaptic responses. Evidence has been obtained that non synaptic transmission can alter the capacity of a given synapse to transfer neuronal information from the presynaptic element to the postsynaptic neuron. For instance, presynaptic mechanisms provide evidence for the functional independence of the nerve terminals, since the release of neuromediators by the latter is sometimes independent of the axonal firing rate. Similarly, the somato-dendritic part of some neurons exhibits intrinsic functions, such as a dendritic release of neuromediator, suggesting that the control of the axonal firing rate takes place partly at this somato-dendritic level and does not depend for the totality on afferent axonic information. The intercellular operations which organize individual neurons into neuronal networks will also occur either at somato-dendritic level or at the level of specific nerve terminals selected as the result of presynaptic interactions. This integration of neuronal information also seems to take place at postsynaptic level, where cooperative interactions have been shown to occur between various receptors. These mechanisms will function at the level of a single nerve terminal containing more than one neuromediator. Neuromodulation can therefore be said to involve very efficient adaptive processes, which help to account for the fact that such large behavioral responses are expressed by such a small number of neuronal elements.     

Nerve growth factor collaborates with myocyte‐derived factors to promote development of presynaptic sites in cultured sympathetic neurons

Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle‐containing growth cones. In conjunction with previously published results showing that NGF has long‐term effects on synaptic transmission between sympathetic neurons and myocytes, this work suggests that NGF acts to promote sympathetic neurotransmission by increasing the number of sympathetic fibers establishing target contact. Further, we found that developmental changes in cardiac myocytes led to an increase in the density of synaptic vesicle–containing variocosities along sympathetic fibers, a process regulated by NGF. Thus, as myocytes mature they produce factors that promote the formation of sympathetic presynaptic structures. These results argue that multiple target interactions regulate the extent of synapse formation between sympathetic neurons and cardiac cells and suggest that NGF promotes presynaptic development by increasing neuronal contact with myocyte‐derived cell surface or matrix‐associated factors. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 460–476, 2000     

The presynaptic active zone protein bassoon is essential for photoreceptor ribbon synapse formation in the retina

The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.     

Localization of mineralocorticoid receptors at mammalian synapses

In the brain, membrane associated nongenomic steroid receptors can induce fast-acting responses to ion conductance and second messenger systems of neurons. Emerging data suggest that membrane associated glucocorticoid and mineralocorticoid receptors may directly regulate synaptic excitability during times of stress when adrenal hormones are elevated. As the key neuron signaling interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. The lateral amygdala is a key site for synaptic plasticity underlying conditioned fear, which can both trigger and be coincident with the stress response. A large body of electrophysiological data shows rapid regulation of neuronal excitability by steroid hormone receptors. Despite the importance of these receptors, to date, only the glucocorticoid receptor has been anatomically localized to the membrane. We investigated the subcellular sites of mineralocorticoid receptors in the lateral amygdala of the Sprague-Dawley rat. Immunoblot analysis revealed the presence of mineralocorticoid receptors in the amygdala. Using electron microscopy, we found mineralocorticoid receptors expressed at both nuclear including: glutamatergic and GABAergic neurons and extra nuclear sites including: presynaptic terminals, neuronal dendrites, and dendritic spines. Importantly we also observed mineralocorticoid receptors at postsynaptic membrane densities of excitatory synapses. These data provide direct anatomical evidence supporting the concept that, at some synapses, synaptic transmission is regulated by mineralocorticoid receptors. Thus part of the stress signaling response in the brain is a direct modulation of the synapse itself by adrenal steroids.     

CYY-1/cyclin Y and CDK-5 differentially regulate synapse elimination and formation for rewiring neural circuits

The assembly and maturation of neural circuits require a delicate balance between synapse formation and elimination. The cellular and molecular mechanisms that coordinate synaptogenesis and synapse elimination are poorly understood. In C. elegans, DD motoneurons respecify their synaptic connectivity during development by completely eliminating existing synapses and forming new synapses without changing cell morphology. Using loss- and gain-of-function genetic approaches, we demonstrate that CYY-1, a cyclin box-containing protein, drives synapse removal in this process. In addition, cyclin-dependent kinase-5 (CDK-5) facilitates new synapse formation by regulating the transport of synaptic vesicles to the sites of synaptogenesis. Furthermore, we show that coordinated activation of UNC-104/Kinesin3 and Dynein is required for patterning newly formed synapses. During the remodeling process, presynaptic components from eliminated synapses are recycled to new synapses, suggesting that signaling mechanisms and molecular motors link the deconstruction of existing synapses and the assembly of new synapses during structural synaptic plasticity.     

Presenilin/γ-secretase regulates neurexin processing at synapses

Neurexins are a large family of neuronal plasma membrane proteins, which function as trans-synaptic receptors during synaptic differentiation. The binding of presynaptic neurexins to postsynaptic partners, such as neuroligins, has been proposed to participate in a signaling pathway that regulates synapse formation/stabilization. The identification of mutations in neurexin genes associated with autism and mental retardation suggests that dysfunction of neurexins may underlie synaptic defects associated with brain disorders. However, the mechanisms that regulate neurexin function at synapses are still unclear. Here, we show that neurexins are proteolytically processed by presenilins (PS), the catalytic components of the γ-secretase complex that mediates the intramembraneous cleavage of several type I membrane proteins. Inhibition of PS/γ-secretase by using pharmacological and genetic approaches induces a drastic accumulation of neurexin C-terminal fragments (CTFs) in cultured rat hippocampal neurons and mouse brain. Neurexin-CTFs accumulate mainly at the presynaptic terminals of PS conditional double knockout (PS cDKO) mice lacking both PS genes in glutamatergic neurons of the forebrain. The fact that loss of PS function enhances neurexin accumulation at glutamatergic terminals mediated by neuroligin-1 suggests that PS regulate the processing of neurexins at glutamatergic synapses. Interestingly, presenilin 1 (PS1) is recruited to glutamatergic terminals mediated by neuroligin-1, thus concentrating PS1 at terminals containing β-neurexins. Furthermore, familial Alzheimer's disease (FAD)-linked PS1 mutations differentially affect β-neurexin-1 processing. Expression of PS1 M146L and PS1 H163R mutants in PS-/- cells rescues the processing of β-neurexin-1, whereas PS1 C410Y and PS1 ΔE9 fail to rescue the processing defect. These results suggest that PS regulate the synaptic function and processing of neurexins at glutamatergic synapses, and that impaired neurexin processing by PS may play a role in FAD.