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Sample records for evoked glutamate transporter

  1. Activation of glutamate transport evokes rapid glutamine release from perisynaptic astrocytes

    PubMed Central

    Uwechue, Nneka M; Marx, Mari-Carmen; Chevy, Quentin; Billups, Brian

    2012-01-01

    Stimulation of astrocytes by neuronal activity and the subsequent release of neuromodulators is thought to be an important regulator of synaptic communication. In this study we show that astrocytes juxtaposed to the glutamatergic calyx of Held synapse in the rat medial nucleus of the trapezoid body (MNTB) are stimulated by the activation of glutamate transporters and consequently release glutamine on a very rapid timescale. MNTB principal neurones express electrogenic system A glutamine transporters, and were exploited as glutamine sensors in this study. By simultaneous whole-cell voltage clamping astrocytes and neighbouring MNTB neurones in brainstem slices, we show that application of the excitatory amino acid transporter (EAAT) substrate d-aspartate stimulates astrocytes to rapidly release glutamine, which is detected by nearby MNTB neurones. This release is significantly reduced by the toxins l-methionine sulfoximine and fluoroacetate, which reduce glutamine concentrations specifically in glial cells. Similarly, glutamine release was also inhibited by localised inactivation of EAATs in individual astrocytes, using internal dl-threo-β-benzyloxyaspartic acid (TBOA) or dissipating the driving force by modifying the patch-pipette solution. These results demonstrate that astrocytes adjacent to glutamatergic synapses can release glutamine in a temporally precise, controlled manner in response to glial glutamate transporter activation. Since glutamine can be used by neurones as a precursor for glutamate and GABA synthesis, this represents a potential feedback mechanism by which astrocytes can respond to synaptic activation and react in a way that sustains or enhances further communication. This would therefore represent an additional manifestation of the tripartite relationship between synapses and astrocytes. PMID:22411007

  2. Glutamate Transporter-Mediated Glutamate Secretion in the Mammalian Pineal Gland

    PubMed Central

    Kim, Mean-Hwan; Uehara, Shunsuke; Muroyama, Akiko; Hille, Bertil; Moriyama, Yoshinori; Koh, Duk-Su

    2008-01-01

    Glutamate transporters are expressed throughout the central nervous system where their major role is to clear released glutamate from presynaptic terminals. Here we report a novel function of the transporter in rat pinealocytes. This electrogenic transporter conducted inward current in response to L-glutamate and L- or D-aspartate and depolarized the membrane in patch clamp experiments. Ca2+ imaging demonstrated that the transporter-mediated depolarization induced a significant Ca2+ influx through voltage-gated Ca2+ channels. The Ca2+ rise finally evoked glutamate exocytosis as detected by carbon-fiber amperometry and by high-performance liquid chromatography. In pineal slices with densely packed pinealocytes, glutamate released from the cells effectively activated glutamate transporters in neighboring cells. The Ca2+ signal generated by KCl depolarization or acetylcholine propagated through several cell layers by virtue of the regenerative ‘glutamate-induced glutamate release’. Therefore we suggest that glutamate transporters mediate synchronized elevation of L-glutamate and thereby efficiently down-regulate melatonin secretion via previously identified inhibitory metabotropic glutamate receptors in the pineal gland. PMID:18945893

  3. Computational Studies of Glutamate Transporters

    PubMed Central

    Setiadi, Jeffry; Heinzelmann, Germano; Kuyucak, Serdar

    2015-01-01

    Glutamate is the major excitatory neurotransmitter in the human brain whose binding to receptors on neurons excites them while excess glutamate are removed from synapses via transporter proteins. Determination of the crystal structures of bacterial aspartate transporters has paved the way for computational investigation of their function and dynamics at the molecular level. Here, we review molecular dynamics and free energy calculation methods used in these computational studies and discuss the recent applications to glutamate transporters. The focus of the review is on the insights gained on the transport mechanism through computational methods, which otherwise is not directly accessible by experimental probes. Recent efforts to model the mammalian glutamate and other amino acid transporters, whose crystal structures have not been solved yet, are included in the review. PMID:26569328

  4. Metabolic Control of Vesicular Glutamate Transport and Release

    PubMed Central

    Juge, Narinobu; Gray, John A.; Omote, Hiroshi; Miyaji, Takaaki; Inoue, Tsuyoshi; Hara, Chiaki; Uneyama, Hisayuki; Edwards, Robert H.; Nicoll, Roger A.; Moriyama, Yoshinori

    2010-01-01

    Fasting has been used to control epilepsy since antiquity, but the mechanism of coupling between metabolic state and excitatory neurotransmission remains unknown. Previous work has shown that the vesicular glutamate transporters (VGLUTs) required for exocytotic release of glutamate undergo an unusual form of regulation by Cl−. Using functional reconstitution of the purified VGLUTs into proteoliposomes, we now show that Cl− acts as an allosteric activator, and the ketone bodies that increase with fasting inhibit glutamate release by competing with Cl− at the site of allosteric regulation. Consistent with these observations, acetoacetate reduced quantal size at hippocampal synapses, and suppresses glutamate release and seizures evoked with 4-aminopyridine in the brain. The results indicate an unsuspected link between metabolic state and excitatory neurotransmission through anion-dependent regulation of VGLUT activity. PMID:20920794

  5. Local glutamate release in the rat ventral lateral thalamus evoked by high-frequency stimulation

    NASA Astrophysics Data System (ADS)

    Agnesi, Filippo; Blaha, Charles D.; Lin, Jessica; Lee, Kendall H.

    2010-04-01

    Thalamic deep brain stimulation (DBS) is proven therapy for essential tremor, Parkinson's disease and Tourette's syndrome. We tested the hypothesis that high-frequency electrical stimulation results in local thalamic glutamate release. Enzyme-linked glutamate amperometric biosensors were implanted in anesthetized rat thalamus adjacent to the stimulating electrode. Electrical stimulation was delivered to investigate the effect of frequency, pulse width, voltage-controlled or current-controlled stimulation, and charge balancing. Monophasic electrical stimulation-induced glutamate release was linearly dependent on stimulation frequency, intensity and pulse width. Prolonged stimulation evoked glutamate release to a plateau that subsequently decayed back to baseline after stimulation. Glutamate release was less pronounced with voltage-controlled stimulation and not present with charge balanced current-controlled stimulation. Using fixed potential amperometry in combination with a glutamate bioprobe and adjacent microstimulating electrode, the present study has shown that monophasic current-controlled stimulation of the thalamus in the anesthetized rat evoked linear increases in local extracellular glutamate concentrations that were dependent on stimulation duration, frequency, intensity and pulse width. However, the efficacy of monophasic voltage-controlled stimulation, in terms of evoking glutamate release in the thalamus, was substantially lower compared to monophasic current-controlled stimulation and entirely absent with biphasic (charge balanced) current-controlled stimulation. It remains to be determined whether similar glutamate release occurs with human DBS electrodes and similar charge balanced stimulation. As such, the present results indicate the importance of evaluating local neurotransmitter dynamics in studying the mechanism of action of DBS.

  6. Structural Features of the Glutamate Transporter Family

    PubMed Central

    Slotboom, Dirk Jan; Konings, Wil N.; Lolkema, Juke S.

    1999-01-01

    Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C4-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C4-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning α-helices and a loop-pore structure which is unique among secondary

  7. Transport mechanism of a glutamate transporter homologue GltPh

    PubMed Central

    Ji, Yurui; Postis, Vincent L.G.; Wang, Yingying; Bartlam, Mark; Goldman, Adrian

    2016-01-01

    Glutamate transporters are responsible for uptake of the neurotransmitter glutamate in mammalian central nervous systems. Their archaeal homologue GltPh, an aspartate transporter isolated from Pyrococcus horikoshii, has been the focus of extensive studies through crystallography, MD simulations and single-molecule FRET (smFRET). Here, we summarize the recent research progress on GltPh, in the hope of gaining some insights into the transport mechanism of this aspartate transporter. PMID:27284058

  8. GLT-1: The elusive presynaptic glutamate transporter.

    PubMed

    Rimmele, Theresa S; Rosenberg, Paul A

    2016-09-01

    Historically, glutamate uptake in the CNS was mainly attributed to glial cells for three reasons: 1) none of the glutamate transporters were found to be located in presynaptic terminals of excitatory synapses; 2) the putative glial transporters, GLT-1 and GLAST are expressed at high levels in astrocytes; 3) studies of the constitutive GLT-1 knockout as well as pharmacological studies demonstrated that >90% of glutamate uptake into forebrain synaptosomes is mediated by the operation of GLT-1. Here we summarize the history leading up to the recognition of GLT-1a as a presynaptic glutamate transporter. A major issue now is understanding the physiological and pathophysiological significance of the expression of GLT-1 in presynaptic terminals. To elucidate the cell-type specific functions of GLT-1, a conditional knockout was generated with which to inactivate the GLT-1 gene in different cell types using Cre/lox technology. Astrocytic knockout led to an 80% reduction of GLT-1 expression, resulting in intractable seizures and early mortality as seen also in the constitutive knockout. Neuronal knockout was associated with no obvious phenotype. Surprisingly, synaptosomal uptake capacity (Vmax) was found to be significantly reduced, by 40%, in the neuronal knockout, indicating that the contribution of neuronal GLT-1 to synaptosomal uptake is disproportionate to its protein expression (5-10%). Conversely, the contribution of astrocytic GLT-1 to synaptosomal uptake was much lower than expected. In contrast, the loss of uptake into liposomes prepared from brain protein from astrocyte and neuronal knockouts was proportionate with the loss of GLT-1 protein, suggesting that a large portion of GLT-1 in astrocytic membranes in synaptosomal preparations is not functional, possibly because of a failure to reseal. These results suggest the need to reinterpret many previous studies using synaptosomal uptake to investigate glutamate transport itself as well as changes in glutamate

  9. GLT-1: The elusive presynaptic glutamate transporter.

    PubMed

    Rimmele, Theresa S; Rosenberg, Paul A

    2016-09-01

    Historically, glutamate uptake in the CNS was mainly attributed to glial cells for three reasons: 1) none of the glutamate transporters were found to be located in presynaptic terminals of excitatory synapses; 2) the putative glial transporters, GLT-1 and GLAST are expressed at high levels in astrocytes; 3) studies of the constitutive GLT-1 knockout as well as pharmacological studies demonstrated that >90% of glutamate uptake into forebrain synaptosomes is mediated by the operation of GLT-1. Here we summarize the history leading up to the recognition of GLT-1a as a presynaptic glutamate transporter. A major issue now is understanding the physiological and pathophysiological significance of the expression of GLT-1 in presynaptic terminals. To elucidate the cell-type specific functions of GLT-1, a conditional knockout was generated with which to inactivate the GLT-1 gene in different cell types using Cre/lox technology. Astrocytic knockout led to an 80% reduction of GLT-1 expression, resulting in intractable seizures and early mortality as seen also in the constitutive knockout. Neuronal knockout was associated with no obvious phenotype. Surprisingly, synaptosomal uptake capacity (Vmax) was found to be significantly reduced, by 40%, in the neuronal knockout, indicating that the contribution of neuronal GLT-1 to synaptosomal uptake is disproportionate to its protein expression (5-10%). Conversely, the contribution of astrocytic GLT-1 to synaptosomal uptake was much lower than expected. In contrast, the loss of uptake into liposomes prepared from brain protein from astrocyte and neuronal knockouts was proportionate with the loss of GLT-1 protein, suggesting that a large portion of GLT-1 in astrocytic membranes in synaptosomal preparations is not functional, possibly because of a failure to reseal. These results suggest the need to reinterpret many previous studies using synaptosomal uptake to investigate glutamate transport itself as well as changes in glutamate

  10. Transport Mechanism of a Bacterial Homologue of Glutamate Transporters

    SciTech Connect

    Reyes, N.; Ginter, C; Boudker, O

    2009-01-01

    Glutamate transporters are integral membrane proteins that catalyse a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glia and neuronal cells by harnessing the energy of pre-existing electrochemical gradients of ions. Crucial to the reaction is the conformational transition of the transporters between outward and inward facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm, respectively. Here we describe the crystal structure of a double cysteine mutant of a glutamate transporter homologue from Pyrococcus horikoshii, GltPh, which is trapped in the inward facing state by cysteine crosslinking. Together with the previously determined crystal structures of Glt{sub Ph} in the outward facing state, the structure of the crosslinked mutant allows us to propose a molecular mechanism by which Glt{sub Ph} and, by analogy, mammalian glutamate transporters mediate sodium-coupled substrate uptake.

  11. Haloperidol abolished glutamate release evoked by photic stimulation of the visual cortex in rats.

    PubMed

    Reyes, Elbert; Rossell, Sergio; Paredes, Daniel; Rada, Pedro; Tucci, Sonia; Gonzalez, Luis E; Hernández, Luis

    2002-07-26

    There is evidence that systemic administration of haloperidol, a dopamine receptor blocker, attenuates visual cortex evoked potentials. However, there is scarce information on cortical neurochemical changes associated with haloperidol effects on visual function. The present experiment was designed to investigate: (1) the effect of photic stimulation on glutamate release in the visual cortex; and (2) whether systemic administration of haloperidol would affect those neurochemical changes. Microdialysis probes were implanted in the occipital cortex. Glutamate levels were measured every 30 s using capillary zone electrophoresis. Extracellular glutamate levels increased to about 282% 30 s after photic stimulation started and remain elevated for the 3 min that the photic stimulation lasted. Haloperidol (1.5 and 5 mg/kg, i.p.) completely suppressed the increased of glutamate efflux during photic stimulation. Finally, it was also found that the highest dose of haloperidol (5 mg/kg) did not change glutamate basal levels. The results are discussed with reference to possible dopaminergic actions on the visual system function.

  12. Modulation of glutamate transport and receptor binding by glutamate receptor antagonists in EAE rat brain.

    PubMed

    Sulkowski, Grzegorz; Dąbrowska-Bouta, Beata; Salińska, Elżbieta; Strużyńska, Lidia

    2014-01-01

    The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.

  13. Neuronal glutamate transporters regulate synaptic transmission in single synapses on CA1 hippocampal neurons.

    PubMed

    Kondratskaya, Elena; Shin, Min-Chul; Akaike, Norio

    2010-01-15

    Glutamate is the major excitatory transmitter in CNS although it causes severe brain damage by pathologic excitotoxicity. Efficient neurotransmission is controlled by powerful protection and support afforded by specific high-affinity glutamate transporters in neurons and glia, clearing synaptic glutamate. While the role of glial cells in glutamate uptake is well defined, the role of neuronal transporters remains poorly understood. The evaluation of impact of neuronal transporters on spontaneous and evoked EPSC in hippocampal CA1 neurons within a model 'single bouton preparation' by pre- and postsynaptic uptake was addressed. In whole-cell patch clamp experiments the influence of blocking, pre- or both pre- and postsynaptic glutamate transporters (GluT) on spontaneous and evoked postsynaptic currents (sEPSC and eEPSC), was examined by manipulating the content of intracellular solution. Suppressing GluT by non-transportable inhibitor TBOA (10 microM) led to remarkable alteration of glutamate uptake process and was reflected in measurable changes of general properties of synaptic currents. Elimination of intracellular K(+) concentration required for glutamate transporter operation by using Cs(+)-based internal solution (postsynaptic GluTs are non-functional apriori), causes the deficient of presynaptic glutamate transporters. Applied in such conditions glutamate transporter inhibitor TBOA (10 microM) affected the occurrence of synaptic event and thus unregulated the transmitter release. eEPSCs were generally suppressed both in amplitude (to 48.73+/-7.03% vs. control) and in success rate (R(suc)) by TBOA (from 91.1+/-7.5% in control to 79.57+/-13.2%). In contrast, with K(+)-based solution in patch pipette (pre- and postsynaptic GluT are intact), amplitude of eEPSC was substantially potentiated by pre-treatment with TBOA (152.1+/-11%), whereas (R(suc)) was reduced to 79.8+/-8.3% in average. The identical reduction of event success rate as well as increased pair

  14. Astroglial glutamate transporters coordinate excitatory signaling and brain energetics.

    PubMed

    Robinson, Michael B; Jackson, Joshua G

    2016-09-01

    In the mammalian brain, a family of sodium-dependent transporters maintains low extracellular glutamate and shapes excitatory signaling. The bulk of this activity is mediated by the astroglial glutamate transporters GLT-1 and GLAST (also called EAAT2 and EAAT1). In this review, we will discuss evidence that these transporters co-localize with, form physical (co-immunoprecipitable) interactions with, and functionally couple to various 'energy-generating' systems, including the Na(+)/K(+)-ATPase, the Na(+)/Ca(2+) exchanger, glycogen metabolizing enzymes, glycolytic enzymes, and mitochondria/mitochondrial proteins. This functional coupling is bi-directional with many of these systems both being regulated by glutamate transport and providing the 'fuel' to support glutamate uptake. Given the importance of glutamate uptake to maintaining synaptic signaling and preventing excitotoxicity, it should not be surprising that some of these systems appear to 'redundantly' support the energetic costs of glutamate uptake. Although the glutamate-glutamine cycle contributes to recycling of neurotransmitter pools of glutamate, this is an over-simplification. The ramifications of co-compartmentalization of glutamate transporters with mitochondria for glutamate metabolism are discussed. Energy consumption in the brain accounts for ∼20% of the basal metabolic rate and relies almost exclusively on glucose for the production of ATP. However, the brain does not possess substantial reserves of glucose or other fuels. To ensure adequate energetic supply, increases in neuronal activity are matched by increases in cerebral blood flow via a process known as 'neurovascular coupling'. While the mechanisms for this coupling are not completely resolved, it is generally agreed that astrocytes, with processes that extend to synapses and endfeet that surround blood vessels, mediate at least some of the signal that causes vasodilation. Several studies have shown that either genetic deletion or

  15. EVALUATING THE NMDA-GLUTAMATE RECEPTOR AS A SITE OF ACTION FOR TOLUENE USING PATTERN ELICITED VISUAL EVOKED POTENTIALS.

    EPA Science Inventory

    In vitro studies have demonstrated that toluene disrupts the function of NMDA-glutamate receptors, as well as other channels. This has led to the hypothesis that effects on NMDA receptor function may contribute to toluene neurotoxicity, CNS depression, and altered visual evoked ...

  16. Glutamate Receptor Agonists and Glutamate Transporter Antagonists Regulate Differentiation of Osteoblast Lineage Cells.

    PubMed

    Xie, Wenjie; Dolder, Silvia; Siegrist, Mark; Wetterwald, Antoinette; Hofstetter, Willy

    2016-08-01

    Development and function of osteoblast lineage cells are regulated by a complex microenvironment consisting of the bone extracellular matrix, cells, systemic hormones and cytokines, autocrine and paracrine factors, and mechanical load. Apart from receptors that transduce extracellular signals into the cell, molecular transporters play a crucial role in the cellular response to the microenvironment. Transporter molecules are responsible for cellular uptake of nutritional components, elimination of metabolites, ion transport, and cell-cell communication. In this report, the expression of molecular transporters in osteoblast lineage cells was investigated to assess their roles in cell development and activity. Low-density arrays, covering membrane and vesicular transport molecules, were used to assess gene expression in osteoblasts representing early and late differentiation states. Receptors and transporters for the amino acid glutamate were found to be differentially expressed during osteoblast development. Glutamate is a neurotransmitter in the central nervous system, and the mechanisms of its release, signal transduction, and cellular reabsorption in the synaptic cleft are well understood. Less clear, however, is the control of equivalent processes in peripheral tissues. In primary osteoblasts, inhibition of glutamate transporters with nonselective inhibitors leads to an increase in the concentration of extracellular glutamate. This change was accompanied by a decrease in osteoblast proliferation, stimulation of alkaline phosphatase, and the expression of transcripts encoding osteocalcin. Enzymatic removal of extracellular glutamate abolished these pro-differentiation effects, as did the inhibition of PKC- and Erk1/2-signaling pathways. These findings demonstrate that glutamate signaling promotes differentiation and activation of osteoblast lineage cells. Consequently, the glutamate system may represent a putative therapeutic target to induce an anabolic response

  17. Modulation of intestinal L-glutamate transport by luminal leptin.

    PubMed

    Fanjul, Carmen; Barrenetxe, Jaione; Lostao, María Pilar; Ducroc, Robert

    2015-06-01

    Leptin is secreted into the digestive tract and contributes to the absorption of dietary molecules by regulating transporters activity. Here, we studied the effect of luminal leptin on the intestinal transport of L-glutamate, an important component of human diet. We examined the effect of leptin on L-glutamate uptake in rat intestine in vitro measuring glutamate-induced short-circuit current (Isc) in Ussing chambers and L-[(3)H (U)]-glutamate uptake in jejunal everted rings. Glutamate-induced Isc was only observed in Na(+)-free conditions. This Isc was concentration (1-60 mmol L(-1)) and pH dependent. Luminal leptin increased glutamate Isc (∼100 %). Dose-response curve showed a biphasic pattern, with maximal stimulations observed at 10(-13) and 10(-10) mmol L(-1), that were sensitive to leptin receptor antagonist. In everted rings, two glutamate transport mechanisms were distinguished: a Na(+)-dependent, H(+)-independent, that was inhibited by leptin (∼20 %), and a Na(+)-independent but H(+)-dependent, that was enhanced by leptin (∼20 %), in line with data obtained in Ussing chambers. Altogether, these data reveal original non-monotonic effect of luminal leptin in the intestine and demonstrate a new role for this hormone in the modulation of L-glutamate transport, showing that luminal active gut peptides can influence absorption of amino acids.

  18. Presynaptic Na+-dependent transport and exocytose of GABA and glutamate in brain in hypergravity.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Pozdnyakova, N.; Krisanova, N.; Himmelreich, N.

    γ-Aminobutyric acid (GABA) and L-glutamate are the most widespread neurotransmitter amino acids in the mammalian central nervous system. GABA is now widely recognized as the major inhibitory neurotransmitter. L-glutamate mediates the most of excitatory synaptic neurotransmission in the brain. They involved in the main aspects of normal brain function. The nerve terminals (synaptosomes) offer several advantages as a model system for the study of general mechanisms of neurosecretion. Our data allowed to conclude that exposure of animals to hypergravity (centrifugation of rats at 10G for 1 hour) had a profound effect on synaptic processes in brain. Comparative analysis of uptake and release of GABA and glutamate have demonstrated that hypergravity loading evokes oppositely directed alterations in inhibitory and excitatory signal transmission. We studied the maximal velocities of [^3H]GABA reuptake and revealed more than twofold enhancement of GABA transporter activity (Vmax rises from 1.4 |pm 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for animals exposed to hypergravity (P ≤ 0.05)). Recently we have also demonstrated the significant lowering of glutamate transporter activity (Vmax of glutamate reuptake decreased from 12.5 ± 3.2 nmol/min/mg of protein in the control group to 5.6 ± 0.9 nmol/min/mg of protein in the group of animals, exposed to the hypergravity stress (P ≤ 0.05)). Significant changes occurred in release of neurotransmitters induced by stimulating exocytosis with the agents, which depolarized nerve terminal plasma membrane. Depolarization-evoked Ca2+-stimulated release was more abundant for GABA (7.2 ± 0.54% and 11,74 ±1,2 % of total accumulated label for control and hypergravity, respectively (P≤0.05)) and was essentially less for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%) after exposure of animals to centrifuge induced artificial gravity. Changes observed in depolarization-evoked exocytotic release

  19. Unveiling the secret lives of glutamate transporters: VGLUTs engage in multiple transport modes.

    PubMed

    Accardi, Alessio

    2014-12-17

    Accumulation of glutamate in synaptic vesicles is mediated by vesicular glutamate transporters called VGLUTs. In the current issue of Neuron, Preobraschenski et al. (2014) show that the VGLUTs, in addition to transporting glutamate, also provide the conductances necessary to maintain the appropriate voltage and pH inside these vesicles. PMID:25521371

  20. Functional changes in glutamate transporters and astrocyte biophysical properties in a rodent model of focal cortical dysplasia

    PubMed Central

    Campbell, Susan L.; Hablitz, John J.; Olsen, Michelle L.

    2014-01-01

    Cortical dysplasia is associated with intractable epilepsy and developmental delay in young children. Recent work with the rat freeze-induced focal cortical dysplasia (FCD) model has demonstrated that hyperexcitability in the dysplastic cortex is due in part to higher levels of extracellular glutamate. Astrocyte glutamate transporters play a pivotal role in cortical maintaining extracellular glutamate concentrations. Here we examined the function of astrocytic glutamate transporters in a FCD model in rats. Neocortical freeze lesions were made in postnatal day (PN) 1 rat pups and whole cell electrophysiological recordings and biochemical studies were performed at PN 21–28. Synaptically evoked glutamate transporter currents in astrocytes showed a near 10-fold reduction in amplitude compared to sham operated controls. Astrocyte glutamate transporter currents from lesioned animals were also significantly reduced when challenged exogenously applied glutamate. Reduced astrocytic glutamate transport clearance contributed to increased NMDA receptor-mediated current decay kinetics in lesioned animals. The electrophysiological profile of astrocytes in the lesion group was also markedly changed compared to sham operated animals. Control astrocytes demonstrate large-amplitude linear leak currents in response to voltage-steps whereas astrocytes in lesioned animals demonstrated significantly smaller voltage-activated inward and outward currents. Significant decreases in astrocyte resting membrane potential and increases in input resistance were observed in lesioned animals. However, Western blotting, immunohistochemistry and quantitative PCR demonstrated no differences in the expression of the astrocytic glutamate transporter GLT-1 in lesioned animals relative to controls. These data suggest that, in the absence of changes in protein or mRNA expression levels, functional changes in astrocytic glutamate transporters contribute to neuronal hyperexcitability in the FCD model

  1. Glial glutamate transporters: new actors in brain signaling.

    PubMed

    López-Bayghen, Esther; Ortega, Arturo

    2011-10-01

    Glutamate, the main excitatory amino acid in the vertebrate brain, is critically involved in most of the physiological functions of the central nervous system. It has traditionally been assumed that glutamate triggers a wide array of signaling cascades through the activation of specific membrane receptors. The extracellular levels are tightly regulated to prevent neurotoxic insults. Electrogenic Na(+)-dependent glial glutamate transporters remove the bulk of the neurotransmitter from the synaptic cleft. An exquisitely ordered coupling between glutamatergic neurons and surrounding glia cells is fundamental for excitatory transmission. The glutamate/glutamine and astrocyte/neuron lactate shuttles provide the biochemical framework of this compulsory association. In this context, recent advances show that glial glutamate transporters act as signal transducers that regulate the expression of proteins involved in their compartmentalization with neurons in the so-called tripartite synapse.

  2. Relationship between Increase in Astrocytic GLT-1 Glutamate Transport and Late-LTP

    ERIC Educational Resources Information Center

    Pita-Almenar, Juan D.; Zou, Shengwei; Colbert, Costa M.; Eskin, Arnold

    2012-01-01

    Na[superscript +]-dependent high-affinity glutamate transporters have important roles in the maintenance of basal levels of glutamate and clearance of glutamate during synaptic transmission. Interestingly, several studies have shown that basal glutamate transport displays plasticity. Glutamate uptake increases in hippocampal slices during early…

  3. Neuronal Activity and Glutamate Uptake Decrease Mitochondrial Mobility in Astrocytes and Position Mitochondria Near Glutamate Transporters

    PubMed Central

    Jackson, Joshua G.; O'Donnell, John C.; Takano, Hajime; Coulter, Douglas A.

    2014-01-01

    Within neurons, mitochondria are nonuniformly distributed and are retained at sites of high activity and metabolic demand. Glutamate transport and the concomitant activation of the Na+/K+-ATPase represent a substantial energetic demand on astrocytes. We hypothesized that mitochondrial mobility within astrocytic processes might be regulated by neuronal activity and glutamate transport. We imaged organotypic hippocampal slice cultures of rat, in which astrocytes maintain their highly branched morphologies and express glutamate transporters. Using time-lapse confocal microscopy, the mobility of mitochondria within individual astrocytic processes and neuronal dendrites was tracked. Within neurons, a greater percentage of mitochondria were mobile than in astrocytes. Furthermore, they moved faster and farther than in astrocytes. Inhibiting neuronal activity with tetrodotoxin (TTX) increased the percentage of mobile mitochondria in astrocytes. Mitochondrial movement in astrocytes was inhibited by vinblastine and cytochalasin D, demonstrating that this mobility depends on both the microtubule and actin cytoskeletons. Inhibition of glutamate transport tripled the percentage of mobile mitochondria in astrocytes. Conversely, application of the transporter substrate d-aspartate reversed the TTX-induced increase in the percentage of mobile mitochondria. Inhibition of reversed Na+/Ca2+ exchange also increased the percentage of mitochondria that were mobile. Last, we demonstrated that neuronal activity increases the probability that mitochondria appose GLT-1 particles within astrocyte processes, without changing the proximity of GLT-1 particles to VGLUT1. These results imply that neuronal activity and the resulting clearance of glutamate by astrocytes regulate the movement of astrocytic mitochondria and suggest a mechanism by which glutamate transporters might retain mitochondria at sites of glutamate uptake. PMID:24478345

  4. Glutamate transporters in brain ischemia: to modulate or not?

    PubMed Central

    Krzyżanowska, Weronika; Pomierny, Bartosz; Filip, Małgorzata; Pera, Joanna

    2014-01-01

    In this review, we briefly describe glutamate (Glu) metabolism and its specific transports and receptors in the central nervous system (CNS). Thereafter, we focus on excitatory amino acid transporters, cystine/glutamate antiporters (system xc-) and vesicular glutamate transporters, specifically addressing their location and roles in CNS and the molecular mechanisms underlying the regulation of Glu transporters. We provide evidence from in vitro or in vivo studies concerning alterations in Glu transporter expression in response to hypoxia or ischemia, including limited human data that supports the role of Glu transporters in stroke patients. Moreover, the potential to induce brain tolerance to ischemia through modulation of the expression and/or activities of Glu transporters is also discussed. Finally we present strategies involving the application of ischemic preconditioning and pharmacological agents, eg β-lactam antibiotics, amitriptyline, riluzole and N-acetylcysteine, which result in the significant protection of nervous tissues against ischemia. PMID:24681894

  5. [Glutamate transporter dysfunction and major mental illnesses].

    PubMed

    Tanaka, Kohichi

    2016-01-01

    Glutamate is the main excitatory neurotransmitter in the central nervous system and plays an important role in most aspects of normal brain function. In spite of its importance as a neurotransmitter, excess glutamate is toxic to neurons. Clearance of extracellular glutamate is critical for maintenance of low extracellular glutamate concentration, and occurs in large part through the activity of GLT1 (EAAT2) and GLAST (EAAT1), which are primarily expressed by astrocytes. Rare variants and down-regulation of GLT1 and GLAST, in psychiatric disorders have been reported. In this review, we demonstrate that various kinds of GLT1 and/or GLAST knockout mice replicate many aspects of the behavioral abnormalities seen in major mental illnesses including schizophrenia, depression, obsessive -compulsive disorders, autism, epilepsy and addiction. PMID:26793898

  6. Diffuse Brain Injury Elevates Tonic Glutamate Levels and Potassium-Evoked Glutamate Release in Discrete Brain Regions at Two Days Post-Injury: An Enzyme-Based Microelectrode Array Study

    PubMed Central

    Hinzman, Jason M.; Currier Thomas, Theresa; Burmeister, Jason J.; Quintero, Jorge E.; Huettl, Peter; Pomerleau, Francois; Gerhardt, Greg A.

    2010-01-01

    Abstract Traumatic brain injury (TBI) survivors often suffer from a wide range of post-traumatic deficits, including impairments in behavioral, cognitive, and motor function. Regulation of glutamate signaling is vital for proper neuronal excitation in the central nervous system. Without proper regulation, increases in extracellular glutamate can contribute to the pathophysiology and neurological dysfunction seen in TBI. In the present studies, enzyme-based microelectrode arrays (MEAs) that selectively measure extracellular glutamate at 2 Hz enabled the examination of tonic glutamate levels and potassium chloride (KCl)-evoked glutamate release in the prefrontal cortex, dentate gyrus, and striatum of adult male rats 2 days after mild or moderate midline fluid percussion brain injury. Moderate brain injury significantly increased tonic extracellular glutamate levels by 256% in the dentate gyrus and 178% in the dorsal striatum. In the dorsal striatum, mild brain injury significantly increased tonic glutamate levels by 200%. Tonic glutamate levels were significantly correlated with injury severity in the dentate gyrus and striatum. The amplitudes of KCl-evoked glutamate release were increased significantly only in the striatum after moderate injury, with a 249% increase seen in the dorsal striatum. Thus, with the MEAs, we measured discrete regional changes in both tonic and KCl-evoked glutamate signaling, which were dependent on injury severity. Future studies may reveal the specific mechanisms responsible for glutamate dysregulation in the post-traumatic period, and may provide novel therapeutic means to improve outcomes after TBI. PMID:20233041

  7. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L.; Frago, Laura M.; Dickson, Suzanne L.; Argente, Jesús; Chowen, Julie A.

    2016-01-01

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons. PMID:27026049

  8. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes.

    PubMed

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L; Frago, Laura M; Dickson, Suzanne L; Argente, Jesús; Chowen, Julie A

    2016-03-30

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons.

  9. Current approaches to enhance glutamate transporter function and expression.

    PubMed

    Fontana, Andréia C K

    2015-09-01

    L-glutamate is the predominant excitatory neurotransmitter in the CNS and has a central role in a variety of brain functions. The termination of glutamate neurotransmission by excitatory amino acid transporters (EAATs) is essential to maintain glutamate concentration low in extracellular space and avoid excitotoxicity. EAAT2/GLT-1, being the most abundant subtype of glutamate transporter in the CNS, plays a key role in regulation of glutamate transmission. Dysfunction of EAAT2 has been correlated with various pathologies such as traumatic brain injury, stroke, amyotrophic lateral sclerosis, Alzheimer's disease, among others. Therefore, activators of the function or enhancers of the expression of EAAT2/GLT-1 could serve as a potential therapy for these conditions. Translational activators of EAAT2/GLT-1, such as ceftriaxone and LDN/OSU-0212320, have been described to have significant protective effects in animal models of amyotrophic lateral sclerosis and epilepsy. In addition, pharmacological activators of the activity of EAAT2/GLT-1 have been explored for decades and are currently emerging as promising tools for neuroprotection, having potential advantages over expression activators. This review describes the current status of the search for EAAT2/GLT-1 activators and addresses challenges and limitations that this approach might encounter. Termination of glutamate neurotransmission by glutamate transporter EAAT2 is essential to maintain homeostasis in the brain and to avoid excitotoxicity. Dysfunction of EAAT2 has been correlated with various neurological pathologies. Therefore, activators of the function or enhancers of the expression of EAAT2 (green arrows) could serve as a potential therapy for these conditions. This review describes the current status of the search for EAAT2 activators and addresses challenges and limitations of this approach. PMID:26096891

  10. 4-Hydroxyhexenal (HHE) impairs glutamate transport in astrocyte cultures.

    PubMed

    Lovell, Mark A; Bradley, Melissa A; Fister, Shuling X

    2012-01-01

    Multiple studies show elevations of α,β-unsaturated aldehydic by-products of lipid peroxidation including 4-hydroxynonenal and acrolein in vulnerable brain regions of subjects throughout the progression of Alzheimer's disease (AD). More recently 4-hydroxyhexenal (HHE), a diffusible α,β-unsaturated aldehyde resulting from peroxidation of ω-3 polyunsaturated fatty acids, was shown to be elevated in the hippocampus/parahippocampal gyrus (HPG) of subjects with preclinical AD (PCAD) and in late stage AD (LAD). HHE treatment of primary rat cortical neuron cultures led to a time- and concentration-dependent decrease in survival and glucose uptake. To determine if HHE also impairs glutamate uptake, primary rat astrocyte cultures were exposed to HHE for 4 hours and glutamate transport measured. Results show subtoxic (2.5 μM) HHE concentrations significantly (p < 0.05) impair glutamate uptake in primary astrocytes. Immunoprecipitation of excitatory amino acid transporter-2 (EAAT-2), the primary glutamate transporter in brain, from normal control, mild cognitive impairment (MCI), PCAD, and LAD HPG followed by quantification of HHE immunolabeling showed a significant increase in HHE positive EAAT-2 in MCI and LAD HPG. Together these data suggest HHE can significantly impair glutamate uptake and may play a role in the pathogenesis of AD. PMID:22766736

  11. Vesicular Monoamine and Glutamate Transporters Select Distinct Synaptic Vesicle Recycling Pathways

    PubMed Central

    Onoa, Bibiana; Li, Haiyan; Gagnon-Bartsch, Johann A.; Elias, Laura A. B.; Edwards, Robert H.

    2011-01-01

    Previous work has characterized the properties of neurotransmitter release at excitatory and inhibitory synapses, but we know remarkably little about the properties of monoamine release because these neuromodulators do not generally produce a fast ionotropic response. Since dopamine and serotonin neurons can also release glutamate in vitro and in vivo, we have used the vesicular monoamine transporter VMAT2 and the vesicular glutamate transporter VGLUT1 to compare the localization and recycling of synaptic vesicles that store, respectively, monoamines and glutamate. First, VMAT2 segregates partially from VGLUT1 in the boutons of midbrain dopamine neurons, indicating the potential for distinct release sites. Second, endocytosis after stimulation is slower for VMAT2 than VGLUT1. During the stimulus, however, the endocytosis of VMAT2 (but not VGLUT1) accelerates dramatically in midbrain dopamine but not hippocampal neurons, indicating a novel, cell-specific mechanism to sustain high rates of release. On the other hand, we find that in both midbrain dopamine and hippocampal neurons, a substantially smaller proportion of VMAT2 than VGLUT1 is available for evoked release, and VMAT2 shows considerably more dispersion along the axon after exocytosis than VGLUT1. Even when expressed in the same neuron, the two vesicular transporters thus target to distinct populations of synaptic vesicles, presumably due to their selection of distinct recycling pathways. PMID:20534840

  12. The role of dorsomedial hypotalamus ionotropic glutamate receptors in the hypertensive and tachycardic responses evoked by Tityustoxin intracerebroventricular injection.

    PubMed

    Silva, F C; Guidine, Patrícia Alves Maia; Machado, Natalia Lima; Xavier, Carlos Henrique; de Menezes, R C; Moraes-Santos, Tasso; Moraes, Márcio Flávio; Chianca, Deoclécio Alves

    2015-03-01

    The scorpion envenoming syndrome is an important worldwide public health problem due to its high incidence and potential severity of symptoms. Some studies address the high sensitivity of the central nervous system to this toxin action. It is known that cardiorespiratory manifestations involve the activation of the autonomic nervous system. However, the origin of this modulation remains unclear. Considering the important participation of the dorsomedial hypotalamus (DMH) in the cardiovascular responses during emergencial situations, the aim of this work is to investigate the involvement of the DMH on cardiovascular responses induced by intracerebroventricular (icv) injection of Tityustoxin (TsTX, a α-type toxin extracted from the Tityus serrulatus scorpion venom). Urethane-anaesthetized male Wistar rats (n=30) were treated with PBS, muscimol or ionotropic glutamate receptor antagonists, bilaterally in DMH and later, with an icv injection of TsTX, or treated only with PBS in both regions. TsTX evoked a marked increase in mean arterial pressure and heart rate in all control rats. Interestingly, injection of muscimol, a GABAA receptor agonist, did not change the pressor and tachycardic responses evoked by TsTX. Remarkably, the injection ionotropic glutamate receptors antagonists in DMH abolished the pressor and the tachycardic response evoked by TsTX. Our data suggest that the central circuit recruited by TsTX, whose activation results in an array of physiological and behavioral alterations, depend on the activation of DMH ionotropic glutamate receptors. Moreover, our data provide new insights on the central mechanisms involved in the development of symptoms in the severe scorpion envenomation syndrome. PMID:25616225

  13. Glutamate transporter dysfunction associated with nerve injury-induced pain in mice.

    PubMed

    Napier, Ian A; Mohammadi, Sarasa A; Christie, MacDonald J

    2012-01-01

    Dysfunction at glutamatergic synapses has been proposed as a mechanism in the development of neuropathic pain. Here we sought to determine whether peripheral nerve injury-induced neuropathic pain results in functional changes to primary afferent synapses. Signs of neuropathic pain as well as an induction of glial fibrillary acidic protein in immunostained spinal cord sections 4 days after partial ligation of the sciatic nerve indicated the induction of neuropathic pain. We found that following nerve injury, no discernable change to kinetics of dl-α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) or N-methyl-d-aspartate receptor (NMDAR)-mediated evoked excitatory postsynaptic currents (eEPSCs) could be observed in dorsal horn (lamina I/II) neurons compared with those of naïve mice. However, we did find that nerve injury was accompanied by slowed decay of the early phase of eEPSCs in the presence of glutamate transporter inhibition by the competitive nontransportable inhibitor dl-threo-β-benzyloxyaspartic acid (TBOA). Concomitantly, expression patterns for the two major glutamate transporters in the spinal cord, excitatory amino acid transporters (EAAT) 1 and EAAT2, were found to be reduced at this time (4 days postinjury). We then sought to directly determine whether nerve injury results in glutamate spillover to NMDARs at dorsal horn synapses. By employing the use-dependent NMDAR blocker (±)MK-801 to block subsynaptic receptors, we found that although TBOA-induced spillover to extrasynaptic receptors trended to increased activation of these receptors after nerve injury, this was not significant compared with naïve mice. Together, these results suggest the development of neuropathic pain involves subtle changes to glutamate transporter expression and function that could contribute to neuropathic pain during excessive synaptic activity. PMID:22072505

  14. Relationship between increase in astrocytic GLT-1 glutamate transport and late-LTP

    PubMed Central

    Pita-Almenar, Juan D.; Zou, Shengwei; Colbert, Costa M.; Eskin, Arnold

    2012-01-01

    Na+-dependent high-affinity glutamate transporters have important roles in the maintenance of basal levels of glutamate and clearance of glutamate during synaptic transmission. Interestingly, several studies have shown that basal glutamate transport displays plasticity. Glutamate uptake increases in hippocampal slices during early long-term potentiation (E-LTP) and late long-term potentiation (L-LTP). Four issues were addressed in this research: Which glutamate transporter is responsible for the increase in glutamate uptake during L-LTP? In what cell type in the hippocampus does the increase in glutamate uptake occur? Does a single type of cell contain all the mechanisms to respond to an induction stimulus with a change in glutamate uptake? What role does the increase in glutamate uptake play during L-LTP? We have confirmed that GLT-1 is responsible for the increase in glutamate uptake during L-LTP. Also, we found that astrocytes were responsible for much, if not all, of the increase in glutamate uptake in hippocampal slices during L-LTP. Additionally, we found that cultured astrocytes alone were able to respond to an induction stimulus with an increase in glutamate uptake. Inhibition of basal glutamate uptake did not affect the induction of L-LTP, but inhibition of the increase in glutamate uptake did inhibit both the expression of L-LTP and induction of additional LTP. It seems likely that heightened glutamate transport plays an ongoing role in the ability of hippocampal circuitry to code and store information. PMID:23166293

  15. Interactions of MK-801 with glutamate-, glutamine- and methamphetamine-evoked release of ( sup 3 H)dopamine from striatal slices

    SciTech Connect

    Bowyer, J.F.; Scallet, A.C.; Holson, R.R.; Lipe, G.W.; Slikker, W. Jr.; Ali, S.F. )

    1991-04-01

    The interactions of MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d) cyclohepten-5,10-imine), glutamate and glutamine with methamphetamine (METH)-evoked release of ({sup 3}H)dopamine were assessed in vitro to determine whether MK-801 inhibition of METH neurotoxicity might be mediated presynaptically, and to evaluate the effects of glutamatergic stimulation on METH-evoked dopamine release. MK-801 inhibition of glutamate- or METH-evoked dopamine release might reduce synaptic dopamine levels during METH exposure and decrease the formation of 6-hydroxydopamine or other related neurotoxins. Without Mg{sup 2}{sup +} present, 40 microM and 1 mM glutamate evoked a N-methyl-D-aspartate receptor-mediated ({sup 3}H)dopamine and ({sup 3}H)metabolite (tritium) release of 3 to 6 and 12 to 16% of total tritium stores, respectively, from striatal slices. With 1.50 mM Mg{sup 2}{sup +} present, 10 mM glutamate alone or in combination with the dopamine uptake blocker nomifensine released only 2.1 or 4.2%, respectively, of total tritium stores, and release was only partially dependent on N-methyl-D-aspartate-type glutamate receptors. With or without 1.50 mM Mg{sup 2}{sup +} present, 0.5 or 5 microM METH evoked a substantial release of tritium (5-8 or 12-21% of total stores, respectively). METH-evoked dopamine release was not affected by 5 microM MK-801 but METH-evoked release was additive with glutamate-evoked release. Without Mg{sup 2}{sup +} present, 1 mM glutamine increased glutamate release and induced the release of ({sup 3}H)dopamine and metabolites. Both 0.5 and 5 microM METH also increased tritium release with 1 mM glutamine present. When striatal slices were exposed to 5 microM METH this glutamine-evoked release of glutamate was increased more than 50%.

  16. Age-related changes in the dynamics of potassium-evoked L-glutamate release in the striatum of Fischer 344 rats.

    PubMed

    Nickell, J; Pomerleau, F; Allen, J; Gerhardt, G A

    2005-01-01

    In the present studies we used a multisite ceramic-based microelectrode for rapid (800 ms) and low level measures of L-glutamate in vivo. We measured the amplitude and clearance rate of phasic changes in L-glutamate release produced by local application of potassium by a micropipette placed adjacent to the recording sites in the striatum of young (6 month), late middle aged (18 month) and aged (24 month) Fischer 344 rats. Our results showed that the amplitudes and clearance rates of potassium-evoked release of L-glutamate in the striatum were significantly decreased in aged rats as compared to the other age groups. In addition, the sensitivity of glutamate fibers to depolarization with potassium was significantly decreased in the aged rats as compared to young animals. Taken together, these data are consistent with age-related alterations in glutamate release dynamics, which may involve a compensatory mechanism for maintaining static glutamate concentrations within the striatum.

  17. Electrogenic Steps Associated with Substrate Binding to the Neuronal Glutamate Transporter EAAC1.

    PubMed

    Tanui, Rose; Tao, Zhen; Silverstein, Nechama; Kanner, Baruch; Grewer, Christof

    2016-05-27

    Glutamate transporters actively take up glutamate into the cell, driven by the co-transport of sodium ions down their transmembrane concentration gradient. It was proposed that glutamate binds to its binding site and is subsequently transported across the membrane in the negatively charged form. With the glutamate binding site being located partially within the membrane domain, the possibility has to be considered that glutamate binding is dependent on the transmembrane potential and, thus, is electrogenic. Experiments presented in this report test this possibility. Rapid application of glutamate to the wild-type glutamate transporter subtype EAAC1 (excitatory amino acid carrier 1) through photo-release from caged glutamate generated a transient inward current, as expected for the electrogenic inward movement of co-transported Na(+) In contrast, glutamate application to a transporter with the mutation A334E induced transient outward current, consistent with movement of negatively charged glutamate into its binding site within the dielectric of the membrane. These results are in agreement with electrostatic calculations, predicting a valence for glutamate binding of -0.27. Control experiments further validate and rule out other possible explanations for the transient outward current. Electrogenic glutamate binding can be isolated in the mutant glutamate transporter because reactions, such as glutamate translocation and/or Na(+) binding to the glutamate-bound state, are inhibited by the A334E substitution. Electrogenic glutamate binding has to be considered together with other voltage-dependent partial reactions to cooperatively determine the voltage dependence of steady-state glutamate uptake and glutamate buffering at the synapse. PMID:27044739

  18. Leptin regulates glutamate and glucose transporters in hypothalamic astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Granado, Miriam; de Ceballos, María L.; Sánchez-Garrido, Miguel Ángel; Sarman, Beatrix; Liu, Zhong-Wu; Dietrich, Marcelo O.; Tena-Sempere, Manuel; Argente-Arizón, Pilar; Díaz, Francisca; Argente, Jesús; Horvath, Tamas L.; Chowen, Julie A.

    2012-01-01

    Glial cells perform critical functions that alter the metabolism and activity of neurons, and there is increasing interest in their role in appetite and energy balance. Leptin, a key regulator of appetite and metabolism, has previously been reported to influence glial structural proteins and morphology. Here, we demonstrate that metabolic status and leptin also modify astrocyte-specific glutamate and glucose transporters, indicating that metabolic signals influence synaptic efficacy and glucose uptake and, ultimately, neuronal function. We found that basal and glucose-stimulated electrical activity of hypothalamic proopiomelanocortin (POMC) neurons in mice were altered in the offspring of mothers fed a high-fat diet. In adulthood, increased body weight and fasting also altered the expression of glucose and glutamate transporters. These results demonstrate that whole-organism metabolism alters hypothalamic glial cell activity and suggest that these cells play an important role in the pathology of obesity. PMID:23064363

  19. Prefrontal changes in the glutamate-glutamine cycle and neuronal/glial glutamate transporters in depression with and without suicide.

    PubMed

    Zhao, J; Verwer, R W H; van Wamelen, D J; Qi, X-R; Gao, S-F; Lucassen, P J; Swaab, D F

    2016-11-01

    There are indications for changes in glutamate metabolism in relation to depression or suicide. The glutamate-glutamine cycle and neuronal/glial glutamate transporters mediate the uptake of the glutamate and glutamine. The expression of various components of the glutamate-glutamine cycle and the neuronal/glial glutamate transporters was determined by qPCR in postmortem prefrontal cortex. The anterior cingulate cortex (ACC) and the dorsolateral prefrontal cortex (DLPFC) were selected from young MDD patients who had committed suicide (MDD-S; n = 17), from MDD patients who died of non-suicide related causes (MDD-NS; n = 7) and from matched control subjects (n = 12). We also compared elderly depressed patients who had not committed suicide (n = 14) with matched control subjects (n = 22). We found that neuronal located components (EAAT3, EAAT4, ASCT1, SNAT1, SNAT2) of the glutamate-glutamine cycle were increased in the ACC while the astroglia located components (EAAT1, EAAT2, GLUL) were decreased in the DLPFC of MDD-S patients. In contrast, most of the components in the cycle were increased in the DLPFC of MDD-NS patients. In conclusion, the glutamate-glutamine cycle - and thus glutamine transmission - is differentially affected in depressed suicide patients and depressed non-suicide patients in an area specific way.

  20. The glutamate and neutral amino acid transporter family: physiological and pharmacological implications.

    PubMed

    Kanai, Yoshikatsu; Hediger, Matthias A

    2003-10-31

    The solute carrier family 1 (SLC1) is composed of five high affinity glutamate transporters, which exhibit the properties of the previously described system XAG-, as well as two Na+-dependent neutral amino acid transporters with characteristics of the so-called "ASC" (alanine, serine and cysteine). The SLC1 family members are structurally similar, with almost identical hydropathy profiles and predicted membrane topologies. The transporters have eight transmembrane domains and a structure reminiscent of a pore loop between the seventh and eighth domains [Neuron 21 (1998) 623]. However, each of these transporters exhibits distinct functional properties. Glutamate transporters mediate transport of L-Glu, L-Asp and D-Asp, accompanied by the cotransport of 3 Na+ and one 1 H+, and the countertransport of 1 K+, whereas ASC transporters mediate Na+-dependent exchange of small neutral amino acids such as Ala, Ser, Cys and Thr. Given the high concentrating capacity provided by the unique ion coupling pattern of glutamate transporters, they play crucial roles in protecting neurons against glutamate excitotoxicity in the central nervous system (CNS). The regulation and manipulation of their function is a critical issue in the pathogenesis and treatment of CNS disorders involving glutamate excitotoxicity. Loss of function of the glial glutamate transporter GLT1 (SLC1A2) has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), resulting in damage of adjacent motor neurons. The importance of glial glutamate transporters in protecting neurons from extracellular glutamate was further demonstrated in studies of the slc1A2 glutamate transporter knockout mouse. The findings suggest that therapeutic upregulation of GLT1 may be beneficial in a variety of pathological conditions. Selective inhibition of the neuronal glutamate transporter EAAC1 (SLC1A1) but not the glial glutamate transporters may be of therapeutic interest, allowing blockage of glutamate exit from

  1. HIV protease inhibitors disrupt astrocytic glutamate transporter function and neurobehavioral performance

    PubMed Central

    Vivithanaporn, Pornpun; Asahchop, Eugene L.; Acharjee, Shaona; Baker, Glen B.; Power, Christopher

    2016-01-01

    Objective: The neurotoxic actions of the HIV protease inhibitors, amprenavir (APV) and lopinavir (LPV) were investigated. Design: With combination antiretroviral therapy (cART), HIV-infected persons exhibit neurocognitive impairments, raising the possibility that cART might exert adverse central nervous system (CNS) effects. We examined the effects of LPV and APV using in-vitro and in-vivo assays of CNS function. Methods: Gene expression, cell viability and amino-acid levels were measured in human astrocytes, following exposure to APV or LPV. Neurobehavioral performance, amino-acid levels and neuropathology were examined in HIV-1 Vpr transgenic mice after treatment with APV or LPV. Results: Excitatory amino-acid transporter-2 (EAAT2) expression was reduced in astrocytes treated with LPV or APV, especially LPV (P < 0.05), which was accompanied by reduced intracellular l-glutamate levels in LPV-treated cells (P < 0.05). Treatment of astrocytes with APV or LPV reduced the expression of proliferating cell nuclear antigen (PCNA) and Ki-67 (P < 0.05) although cell survival was unaffected. Exposure of LPV to astrocytes augmented glutamate-evoked transient rises in [Cai] (P < 0.05). Vpr mice treated with LPV showed lower concentrations of l-glutamate, l-aspartate and l-serine in cortex compared with vehicle-treated mice (P < 0.05). Total errors in T-maze assessment were increased in LPV and APV-treated animals (P < 0.05). EAAT2 expression was reduced in the brains of protease inhibitor-treated animals, which was associated with gliosis (P < 0.05). Conclusion: These results indicated that contemporary protease inhibitors disrupt astrocyte functions at therapeutic concentrations with enhanced sensitivity to glutamate, which can lead to neurobehavioral impairments. ART neurotoxicity should be considered in future therapeutic regimens for HIV/AIDS. PMID:26558720

  2. Role of astrocytic glutamate transporter in alcohol use disorder

    PubMed Central

    Ayers-Ringler, Jennifer R; Jia, Yun-Fang; Qiu, Yan-Yan; Choi, Doo-Sup

    2016-01-01

    Alcohol use disorder (AUD) is one of the most widespread neuropsychiatric conditions, having a significant health and socioeconomic impact. According to the 2014 World Health Organization global status report on alcohol and health, the harmful use of alcohol is responsible for 5.9% of all deaths worldwide. Additionally, 5.1% of the global burden of disease and injury is ascribed to alcohol (measured in disability adjusted life years, or disability adjusted life years). Although the neurobiological basis of AUD is highly complex, the corticostriatal circuit contributes significantly to the development of addictive behaviors. In-depth investigation into the changes of the neurotransmitters in this circuit, dopamine, gamma-aminobutyricacid, and glutamate, and their corresponding neuronal receptors in AUD and other addictions enable us to understand the molecular basis of AUD. However, these discoveries have also revealed a dearth of knowledge regarding contributions from non-neuronal sources. Astrocytes, though intimately involved in synaptic function, had until recently been noticeably overlooked in their potential role in AUD. One major function of the astrocyte is protecting neurons from excitotoxicity by removing glutamate from the synapse via excitatory amino acid transporter type 2. The importance of this key transporter in addiction, as well as ethanol withdrawal, has recently become evident, though its regulation is still under investigation. Historically, pharmacotherapy for AUD has been focused on altering the activity of neuronal glutamate receptors. However, recent clinical evidence has supported the animal-based findings, showing that regulating glutamate homeostasis contributes to successful management of recovery from AUD. PMID:27014596

  3. Neutralizing Aspartate 83 Modifies Substrate Translocation of Excitatory Amino Acid Transporter 3 (EAAT3) Glutamate Transporters*

    PubMed Central

    Hotzy, Jasmin; Machtens, Jan-Philipp; Fahlke, Christoph

    2012-01-01

    Excitatory amino acid transporters (EAATs) terminate glutamatergic synaptic transmission by removing glutamate from the synaptic cleft into neuronal and glial cells. EAATs are not only secondary active glutamate transporters but also function as anion channels. Gating of EAAT anion channels is tightly coupled to transitions within the glutamate uptake cycle, resulting in Na+- and glutamate-dependent anion currents. A point mutation neutralizing a conserved aspartic acid within the intracellular loop close to the end of transmembrane domain 2 was recently shown to modify the substrate dependence of EAAT anion currents. To distinguish whether this mutation affects transitions within the uptake cycle or directly modifies the opening/closing of the anion channel, we used voltage clamp fluorometry. Using three different sites for fluorophore attachment, V120C, M205C, and A430C, we observed time-, voltage-, and substrate-dependent alterations of EAAT3 fluorescence intensities. The voltage and substrate dependence of fluorescence intensities can be described by a 15-state model of the transport cycle in which several states are connected to branching anion channel states. D83A-mediated changes of fluorescence intensities, anion currents, and secondary active transport can be explained by exclusive modifications of substrate translocation rates. In contrast, sole modification of anion channel opening and closing is insufficient to account for all experimental data. We conclude that D83A has direct effects on the glutamate transport cycle and that these effects result in changed anion channel function. PMID:22532568

  4. Pharmacology of Glutamate Transport in the CNS: Substrates and Inhibitors of Excitatory Amino Acid Transporters (EAATs) and the Glutamate/Cystine Exchanger System x c -

    NASA Astrophysics Data System (ADS)

    Bridges, Richard J.; Patel, Sarjubhai A.

    As the primary excitatory neurotransmitter in the mammalian CNS, l-glutamate participates not only in standard fast synaptic communication, but also contributes to higher order signal processing, as well as neuropathology. Given this variety of functional roles, interest has been growing as to how the extracellular concentrations of l-glutamate surrounding neurons are regulated by cellular transporter proteins. This review focuses on two prominent systems, each of which appears capable of influencing both the signaling and pathological actions of l-glutamate within the CNS: the sodium-dependent excitatory amino acid transporters (EAATs) and the glutamate/cystine exchanger, system x c - (Sx c -). While the family of EAAT subtypes limit access to glutamate receptors by rapidly and efficiently sequestering l-glutamate in neurons and glia, Sxc - provides a route for the export of glutamate from cells into the extracellular environment. The primary intent of this work is to provide an overview of the inhibitors and substrates that have been developed to delineate the pharmacological specificity of these transport systems, as well as be exploited as probes with which to selectively investigate function. Particular attention is paid to the development of small molecule templates that mimic the structural properties of the endogenous substrates, l-glutamate, l-aspartate and l-cystine and how strategic control of functional group position and/or the introduction of lipophilic R-groups can impact multiple aspects of the transport process, including: subtype selectivity, inhibitory potency, and substrate activity.

  5. Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells.

    PubMed

    Kobayashi, S; Millhorn, D E

    2001-03-01

    We investigated the effect of hypoxia on glutamate metabolism and uptake in rat pheochromocytoma (PC12) cells. Various key enzymes relevant to glutamate production, metabolism and transport were coordinately regulated by hypoxia. PC12 cells express two glutamate-metabolizing enzymes, glutamine synthetase (GS) and glutamate decarboxylase (GAD), as well as the glutamate-producing enzyme, phosphate-activated glutaminase (PAG). Exposure to hypoxia (1% O(2)) for 6 h or longer increased expression of GS mRNA and protein and enhanced GS enzymatic activity. In contrast, hypoxia caused a significant decrease in expression of PAG mRNA and protein, and also decreased PAG activity. In addition, hypoxia led to an increase in GAD65 and GAD67 protein levels and GAD enzymatic activity. PC12 cells express three Na(+)-dependent glutamate transporters; EAAC1, GLT-1 and GLAST. Hypoxia increased EAAC1 and GLT-1 protein levels, but had no effect on GLAST. Chronic hypoxia significantly enhanced the Na(+)-dependent component of glutamate transport. Furthermore, chronic hypoxia decreased cellular content of glutamate, but increased that of glutamine. Taken together, the hypoxia-induced changes in enzymes related to glutamate metabolism and transport are consistent with a decrease in the extracellular concentration of glutamate. This may have a role in protecting PC12 cells from the cytotoxic effects of glutamate during chronic hypoxia. PMID:11259512

  6. Effects of Ketamine and Ketamine Metabolites on Evoked Striatal Dopamine Release, Dopamine Receptors, and Monoamine Transporters.

    PubMed

    Can, Adem; Zanos, Panos; Moaddel, Ruin; Kang, Hye Jin; Dossou, Katinia S S; Wainer, Irving W; Cheer, Joseph F; Frost, Douglas O; Huang, Xi-Ping; Gould, Todd D

    2016-10-01

    Following administration at subanesthetic doses, (R,S)-ketamine (ketamine) induces rapid and robust relief from symptoms of depression in treatment-refractory depressed patients. Previous studies suggest that ketamine's antidepressant properties involve enhancement of dopamine (DA) neurotransmission. Ketamine is rapidly metabolized to (2S,6S)- and (2R,6R)-hydroxynorketamine (HNK), which have antidepressant actions independent of N-methyl-d-aspartate glutamate receptor inhibition. These antidepressant actions of (2S,6S;2R,6R)-HNK, or other metabolites, as well as ketamine's side effects, including abuse potential, may be related to direct effects on components of the dopaminergic (DAergic) system. Here, brain and blood distribution/clearance and pharmacodynamic analyses at DA receptors (D1-D5) and the DA, norepinephrine, and serotonin transporters were assessed for ketamine and its major metabolites (norketamine, dehydronorketamine, and HNKs). Additionally, we measured electrically evoked mesolimbic DA release and decay using fast-scan cyclic voltammetry following acute administration of subanesthetic doses of ketamine (2, 10, and 50 mg/kg, i.p.). Following ketamine injection, ketamine, norketamine, and multiple hydroxynorketamines were detected in the plasma and brain of mice. Dehydronorketamine was detectable in plasma, but concentrations were below detectable limits in the brain. Ketamine did not alter the magnitude or kinetics of evoked DA release in the nucleus accumbens in anesthetized mice. Neither ketamine's enantiomers nor its metabolites had affinity for DA receptors or the DA, noradrenaline, and serotonin transporters (up to 10 μM). These results suggest that neither the side effects nor antidepressant actions of ketamine or ketamine metabolites are associated with direct effects on mesolimbic DAergic neurotransmission. Previously observed in vivo changes in DAergic neurotransmission following ketamine administration are likely indirect. PMID:27469513

  7. Effects of Ketamine and Ketamine Metabolites on Evoked Striatal Dopamine Release, Dopamine Receptors, and Monoamine Transporters

    PubMed Central

    Can, Adem; Zanos, Panos; Moaddel, Ruin; Kang, Hye Jin; Dossou, Katinia S. S.; Wainer, Irving W.; Cheer, Joseph F.; Frost, Douglas O.; Huang, Xi-Ping

    2016-01-01

    Following administration at subanesthetic doses, (R,S)-ketamine (ketamine) induces rapid and robust relief from symptoms of depression in treatment-refractory depressed patients. Previous studies suggest that ketamine’s antidepressant properties involve enhancement of dopamine (DA) neurotransmission. Ketamine is rapidly metabolized to (2S,6S)- and (2R,6R)-hydroxynorketamine (HNK), which have antidepressant actions independent of N-methyl-d-aspartate glutamate receptor inhibition. These antidepressant actions of (2S,6S;2R,6R)-HNK, or other metabolites, as well as ketamine’s side effects, including abuse potential, may be related to direct effects on components of the dopaminergic (DAergic) system. Here, brain and blood distribution/clearance and pharmacodynamic analyses at DA receptors (D1–D5) and the DA, norepinephrine, and serotonin transporters were assessed for ketamine and its major metabolites (norketamine, dehydronorketamine, and HNKs). Additionally, we measured electrically evoked mesolimbic DA release and decay using fast-scan cyclic voltammetry following acute administration of subanesthetic doses of ketamine (2, 10, and 50 mg/kg, i.p.). Following ketamine injection, ketamine, norketamine, and multiple hydroxynorketamines were detected in the plasma and brain of mice. Dehydronorketamine was detectable in plasma, but concentrations were below detectable limits in the brain. Ketamine did not alter the magnitude or kinetics of evoked DA release in the nucleus accumbens in anesthetized mice. Neither ketamine’s enantiomers nor its metabolites had affinity for DA receptors or the DA, noradrenaline, and serotonin transporters (up to 10 μM). These results suggest that neither the side effects nor antidepressant actions of ketamine or ketamine metabolites are associated with direct effects on mesolimbic DAergic neurotransmission. Previously observed in vivo changes in DAergic neurotransmission following ketamine administration are likely indirect. PMID

  8. Oxaliplatin evokes P2X7-dependent glutamate release in the cerebral cortex: A pain mechanism mediated by Pannexin 1.

    PubMed

    Di Cesare Mannelli, Lorenzo; Marcoli, Manuela; Micheli, Laura; Zanardelli, Matteo; Maura, Guido; Ghelardini, Carla; Cervetto, Chiara

    2015-10-01

    Anticancer therapy based on the repeated administration of oxaliplatin is limited by the development of a neuropathic syndrome difficult to treat. Oxaliplatin neurotoxicity is based on complex nervous mechanisms, the comprehension of the role of single neurotransmitters and the knowledge of the signal flow among cells is matter of importance to improve therapeutic chances. In a rat model of oxaliplatin-induced neuropathy, we report increased P2X7-evoked glutamate release from cerebrocortical synaptosomes. The release was abolished by the P2X7 receptor (P2X7R) antagonists Brilliant-Blue-G (BBG) and A-438079, and significantly reduced by Carbenoxolone and the Pannexin 1 (Panx1) selective inhibitors Erioglaucine and (10)Panx suggesting the recruitment of Panx1. Aimed to evaluate the significance of P2X7R-Panx1 system activation in pain generated by oxaliplatin, pharmacological modulators were spinally infused by intrathecal catheter in oxaliplatin-treated animals. BBG, Erioglaucine and (10)Panx reverted oxaliplatin-dependent pain. Finally, the influence of the P2X7R-Panx1 system blockade on oxaliplatin anticancer activity was evaluated on the human colon cancer cell line HT-29. Prevention of HT-29 apoptosis and mortality was dependent by kind and concentration of P2X7R antagonists. On the contrary, the inhibition of Panx1 did not alter oxaliplatin lethality in tumor cells. It is concluded that glutamate release dependent on P2X7R is increased in cerebrocortical nerve terminals from oxaliplatin-treated rats; the increase is mediated by functional recruitment of Panx1; P2X7R antagonists and Panx1 inhibitors revert oxaliplatin-induced neuropathic pain; Panx1 inhibitors do not alter the oxaliplatin-induced mortality of cancer cells HT-29. The inhibition of Panx1 channel is suggested as a new and safe pharmacological target.

  9. Loss of Astrocytic Glutamate Transporters in Wernicke Encephalopathy

    PubMed Central

    Hazell, Alan S.; Sheedy, Donna; Oanea, Raluca; Aghourian, Meghmik; Sun, Simon; Jung, Jee Yong; Wang, Dongmei; Wang, Chunlei

    2012-01-01

    Wernicke encephalopathy (WE), a neurological disorder caused by thiamine deficiency (TD), is characterized by structural damage in brain regions that include the thalamus and cerebral cortex. The basis for these lesions is unclear, but may involve a disturbance of glutamatergic neurotransmission. We have therefore investigated levels of the astrocytic glutamate transporters EAAT1 and EAAT2 in order to evaluate their role in the pathophysiology of this disorder. Histological assessment of the frontal cortex revealed a significant loss of neurons in neuropathologically confirmed cases of WE compared with age-matched controls, concomitant with decreases in α-internexin and synaptophysin protein content of 67 and 52% by immunoblotting. EAAT2 levels were diminished by 71% in WE, with levels of EAAT1 also reduced by 62%. Loss of both transporter sites was confirmed by immunohistochemical methods. Development of TD in rats caused a profound loss of EAAT1 and EAAT2 in the thalamus accompanied by decreases in other astrocyte-specific proteins. Treatment of TD rats with N-acetylcysteine prevented the downregulation of EAAT2 in the medial thalamus, and ameliorated the loss of several other astrocyte proteins, concomitant with increased neuronal survival. Our results suggest that (1) loss of EAAT1 and EAAT2 glutamate transporters is associated with structural damage to the frontal cortex in patients with WE, (2) oxidative stress plays an important role in this process, and (3) TD has a profound effect on the functional integrity of astrocytes. Based on these findings, we recommend that early treatment using a combination of thiamine AND antioxidant approaches should be an important consideration in cases of WE. PMID:19565658

  10. Designing Novel Nanoformulations Targeting Glutamate Transporter Excitatory Amino Acid Transporter 2: Implications in Treating Drug Addiction

    PubMed Central

    Rao, PSS; Yallapu, Murali M.; Sari, Youssef; Fisher, Paul B.; Kumar, Santosh

    2015-01-01

    Chronic drug abuse is associated with elevated extracellular glutamate concentration in the brain reward regions. Deficit of glutamate clearance has been identified as a contributing factor that leads to enhanced glutamate concentration following extended drug abuse. Importantly, normalization of glutamate level through induction of glutamate transporter 1 (GLT1)/ excitatory amino acid transporter 2 (EAAT2) expression has been described in several in vivo studies. GLT1 upregulators including ceftriaxone, a beta-lactam antibiotic, have been effective in attenuating drug-seeking and drug-consumption behavior in rodent models. However, potential obstacles toward clinical translation of GLT1 (EAAT2) upregulators as treatment for drug addiction might include poor gastrointestinal absorption, serious peripheral adverse effects, and/or suboptimal CNS concentrations. Given the growing success of nanotechnology in targeting CNS ailments, nanoformulating known GLT1 (EAAT2) upregulators for selective uptake across the blood brain barrier presents an ideal therapeutic approach for treating drug addiction. In this review, we summarize the results obtained with promising GLT1 (EAAT2) inducing compounds in animal models recapitulating drug addiction. Additionally, the various nanoformulations that can be employed for selectively increasing the CNS bioavailability of GLT1 (EAAT2) upregulators are discussed. Finally, the applicability of GLT1 (EAAT2) induction via central delivery of drug-loaded nanoformulations is described. PMID:26635971

  11. Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters.

    PubMed

    Silverstein, Nechama; Ewers, David; Forrest, Lucy R; Fahlke, Christoph; Kanner, Baruch I

    2015-11-27

    Crystal structures of the archaeal homologue GltPh have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, GltPh is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of GltPh are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by GltPh. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. GltPh-L130W/T352A exhibited an ~15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ~40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that GltPh-T352A not only transports aspartate but also l-glutamate. Remarkably, GltPh-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of GltPh-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation.

  12. Pharmacological inhibitions of glutamate transporters EAAT1 and EAAT2 compromise glutamate transport in photoreceptor to ON- bipolar cell synapses

    PubMed Central

    Tse, Dennis Y.; Chung, Inyoung; Wu, Samuel M.

    2015-01-01

    To maintain reliable signal transmission across a synapse, free synaptic neurotransmitters must be removed from the cleft in a timely manner. In the first visual synapse, this critical task is mainly undertaken by glutamate transporters (EAATs). Here we study the differential roles of the EAAT1, EAAT2 and EAAT5 subtypes in glutamate (GLU) uptake at the photoreceptor-to-depolarizing bipolar cell synapse in intact dark-adapted retina. Various doses of EAAT blockers and/or GLU were injected into the eye before the electroretinogram (ERG) was measured. Their effectiveness and potency in inhibiting the ERG b-wave were studied to determine their relative contributions to the GLU clearing activity at the synapse. The results showed that EAAT1 and EAAT2 plays different roles. Selectively blocking glial EAAT1 alone using UCPH101 inhibited the b-wave 2–24 hours following injection, suggesting a dominating role of EAAT1 in the overall GLU clearing capacity in the synaptic cleft. Selectively blocking EAAT2 on photoreceptor terminals had no significant effect on the b-wave, but increased the potency of exogenous GLU in inhibiting the b-wave. These suggest that EAAT2 play a secondary yet significant role in the GLU reuptake activity at the rod and the cone output synapses. Additionally, we have verified our electrophysiological findings with double-label immunohistochemistry, and extend the literature on the spatial distribution of EAAT2 splice variants in the mouse retina. PMID:25152321

  13. Coupled ion Binding and Structural Transitions Along the Transport Cycle of Glutamate Transporters

    SciTech Connect

    Verdon, Gregory; Oh, SeCheol; Serio, Ryan N.; Boudker, Olga

    2014-05-19

    Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. We report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations. We then show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Moreover, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues.

  14. Glutamate cycling may drive organic anion transport on the basal membrane of human placental syncytiotrophoblast.

    PubMed

    Lofthouse, Emma M; Brooks, Suzanne; Cleal, Jane K; Hanson, Mark A; Poore, Kirsten R; O'Kelly, Ita M; Lewis, Rohan M

    2015-10-15

    The organic anion transporter OAT4 (SLC22A11) and organic anion transporting polypeptide OATP2B1 (SLCO2B1) are expressed in the basal membrane of the placental syncytiotrophoblast. These transporters mediate exchange whereby uptake of one organic anion is coupled to efflux of a counter-ion. In placenta, these exchangers mediate placental uptake of substrates for oestrogen synthesis as well as clearing waste products and xenobiotics from the fetal circulation. However, the identity of the counter-ion driving this transport in the placenta, and in other tissues, is unclear. While glutamate is not a known OAT4 or OATP2B1 substrate, we propose that its high intracellular concentration has the potential to drive accumulation of substrates from the fetal circulation. In the isolated perfused placenta, glutamate exchange was observed between the placenta and the fetal circulation. This exchange could not be explained by known glutamate exchangers. However, glutamate efflux was trans-stimulated by an OAT4 and OATP2B1 substrate (bromosulphothalein). Exchange of glutamate for bromosulphothalein was only observed when glutamate reuptake was inhibited (by addition of aspartate). To determine if OAT4 and/or OATP2B1 mediate glutamate exchange, uptake and efflux of glutamate were investigated in Xenopus laevis oocytes. Our data demonstrate that in Xenopus oocytes expressing either OAT4 or OATP2B1 efflux of intracellular [(14)C]glutamate could be stimulated by conditions including extracellular glutamate (OAT4), estrone-sulphate and bromosulphothalein (both OAT4 and OATP2B1) or pravastatin (OATP2B1). Cycling of glutamate across the placenta involving efflux via OAT4 and OATP2B1 and subsequent reuptake will drive placental uptake of organic anions from the fetal circulation.

  15. Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum.

    PubMed

    Unger, Tina; Bette, Stefanie; Zhang, Jiong; Theis, Martin; Engele, Jürgen

    2012-01-01

    The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex.

  16. Glutamate transport and xanthan gum production in the plant pathogen Xanthomonas axonopodis pv. citri.

    PubMed

    Rojas, Robert; Nishidomi, Sabrina; Nepomuceno, Roberto; Oshiro, Elisa; de Cassia Café Ferreira, Rita

    2013-11-01

    L-glutamate plays a central role in nitrogen metabolism in all living organisms. In the genus Xanthomonas, the nitrogen nutrition is an important factor involved in the xanthan gum production, an important exopolysaccharide with various industrial and biotechnological applications. In this report, we demonstrate that the use of L-glutamate by the phytopathogen Xanthomonas axonopodis pv. citri as a nitrogen source in defined medium significantly increases the production of xanthan gum. This increase is dependent on the L-glutamate concentration. In addition, we have also characterized a glutamate transport system that is dependent on a proton gradient and on ATP and is modulated by amino acids that are structurally related to glutamate. This is the first biochemical characterization of an energy substrate transport system observed in a bacterial phytopathogen with a broad economic and industrial impact due to xanthan gum production. PMID:23719672

  17. Glutamate Transporters and the Excitotoxic Path to Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis

    PubMed Central

    Foran, Emily

    2009-01-01

    Abstract Responsible for the majority of excitatory activity in the central nervous system (CNS), glutamate interacts with a range of specific receptor and transporter systems to establish a functional synapse. Excessive stimulation of glutamate receptors causes excitotoxicity, a phenomenon implicated in both acute and chronic neurodegenerative diseases [e.g., ischemia, Huntington's disease, and amyotrophic lateral sclerosis (ALS)]. In physiology, excitotoxicity is prevented by rapid binding and clearance of synaptic released glutamate by high-affinity, Na+-dependent glutamate transporters and amplified by defects to the glutamate transporter and receptor systems. ALS pathogenetic mechanisms are not completely understood and characterized, but excitotoxicity has been regarded as one firm mechanism implicated in the disease because of data obtained from ALS patients and animal and cellular models as well as inferred by the documented efficacy of riluzole, a generic antiglutamatergic drug, has in patients. In this article, we critically review the several lines of evidence supporting a role for glutamate-mediated excitotoxicity in the death of motor neurons occurring in ALS, putting a particular emphasis on the impairment of the glutamate-transport system. Antioxid. Redox Signal. 11, 1587–1602. PMID:19413484

  18. Neuroprotection Promoted by Guanosine Depends on Glutamine Synthetase and Glutamate Transporters Activity in Hippocampal Slices Subjected to Oxygen/Glucose Deprivation.

    PubMed

    Dal-Cim, Tharine; Martins, Wagner C; Thomaz, Daniel T; Coelho, Victor; Poluceno, Gabriela Godoy; Lanznaster, Débora; Vandresen-Filho, Samuel; Tasca, Carla I

    2016-05-01

    Guanosine (GUO) has been shown to act as a neuroprotective agent against glutamatergic excitotoxicity by increasing glutamate uptake and decreasing its release. In this study, a putative effect of GUO action on glutamate transporters activity modulation was assessed in hippocampal slices subjected to oxygen and glucose deprivation (OGD), an in vitro model of brain ischemia. Slices subjected to OGD showed increased excitatory amino acids release (measured by D-[(3)H]aspartate release) that was prevented in the presence of GUO (100 µM). The glutamate transporter blockers, DL-TBOA (10 µM), DHK (100 µM, selective inhibitor of GLT-1), and sulfasalazine (SAS, 250 µM, Xc(-) system inhibitor) decreased OGD-induced D-aspartate release. Interestingly, DHK or DL-TBOA blocked the decrease in glutamate release induced by GUO, whereas SAS did not modify the GUO effect. GUO protected hippocampal slices from cellular damage by modulation of glutamate transporters, however selective blockade of GLT-1 or Xc- system only did not affect this protective action of GUO. OGD decreased hippocampal glutamine synthetase (GS) activity and GUO recovered GS activity to control levels without altering the kinetic parameters of GS activity, thus suggesting GUO does not directly interact with GS. Additionally, the pharmacological inhibition of GS activity with methionine sulfoximine abolished the effect of GUO in reducing D-aspartate release and cellular damage evoked by OGD. Altogether, results in hippocampal slices subjected to OGD show that GUO counteracts the release of excitatory amino acids, stimulates the activity of GS, and decreases the cellular damage by modulation of glutamate transporters activity.

  19. Mitochondrial transporters involved in oleic acid utilization and glutamate metabolism in yeast.

    PubMed

    Trotter, Pamela J; Adamson, Amy L; Ghrist, Angela C; Rowe, Lindsay; Scott, Lori R; Sherman, Matthew P; Stites, Nicole C; Sun, Yue; Tawiah-Boateng, Mary Anne; Tibbetts, Anne S; Wadington, Megan C; West, Aaron C

    2005-10-01

    Utilization of fatty acids such as oleic acid as sole carbon source by the yeast Saccharomyces cerevisiae requires coordinated function of peroxisomes, where the fatty acids are degraded, and the mitochondria, where oxidation is completed. We identified two mitochondrial oxodicarboxylate transporters, Odc1p and Odc2p, as important in efficient utilization of oleic acid in yeast [Tibbetts et al., Arch. Biochem. Biophys. 406 (2002) 96-104]. Yet, the growth phenotype of odc1delta odc2delta strains indicated that additional transporter(s) were also involved. Here, we identify two putative transporter genes, YMC1 and YMC2, as able to suppress the odc1delta odc2delta growth phenotype. The mRNA levels for both are elevated in the presence of glycerol or oleic acid, as compared to glucose. Ymc1p and Ymc2p are localized to the mitochondria in oleic acid-grown cells. Deletion of all four transporters (quad mutant) prevents growth on oleic acid as sole carbon source, while growth on acetate is retained. It is known that the glutamate-sensitive retrograde signaling pathway is important for upregulation of peroxisomal function in response to oleic acid and the oxodicarboxylate alpha-ketoglutarate is transported out of the mitochondria for synthesis of glutamate. So, citric acid cycle function and glutamate synthesis were examined in transporter mutants. The quad mutant has significantly decreased citrate synthase activity and whole cell alpha-ketoglutarate levels, while isocitrate dehydrogenase activity is unaffected and glutamate dehydrogenase activity is increased 10-fold. Strains carrying only two or three transporter deletions exhibit intermediate affects. 13C NMR metabolic enrichment experiments confirm a defect in glutamate biosynthesis in the quad mutant and, in double and triple mutants, suggest increased cycling of the glutamate backbone in the mitochondria before export. Taken together these studies indicate that these four transporters have overlapping activity, and

  20. Chronic postnatal stress induces voluntary alcohol intake and modifies glutamate transporters in adolescent rats.

    PubMed

    Odeon, María Mercedes; Andreu, Marcela; Yamauchi, Laura; Grosman, Mauricio; Acosta, Gabriela Beatriz

    2015-01-01

    Postnatal stress alters stress responses for life, with serious consequences on the central nervous system (CNS), involving glutamatergic neurotransmission and development of voluntary alcohol intake. Several drugs of abuse, including alcohol and cocaine, alter glutamate transport (GluT). Here, we evaluated effects of chronic postnatal stress (CPS) on alcohol intake and brain glutamate uptake and transporters in male adolescent Wistar rats. For CPS from postnatal day (PD) 7, pups were separated from their mothers and exposed to cold stress (4 °C) for 1 h daily for 20 days; controls remained with their mothers. Then they were exposed to either voluntary ethanol (6%) or dextrose (1%) intake for 7 days (5-7 rats per group), then killed. CPS: (1) increased voluntary ethanol intake, (2) did not affect body weight gain or produce signs of toxicity with alcohol exposure, (3) increased glutamate uptake by hippocampal synaptosomes in vitro and (4) reduced protein levels (Western measurements) in hippocampus and frontal cortex of glial glutamate transporter-1 (GLT-1) and excitatory amino-acid transporter-3 (EAAT-3) but increased glutamate aspartate transporter (GLAST) levels. We propose that CPS-induced decrements in GLT-1 and EAAT-3 expression levels are opposed by activation of a compensatory mechanism to prevent excitotoxicity. A greater role for GLAST in total glutamate uptake to prevent enlarged extracellular glutamate levels is inferred. Although CPS strongly increased intake of ethanol, this had little impact on effects of CPS on brain glutamate uptake or transporters. However, the impact of early life adverse events on glutamatergic neurotransmission may underlie increased alcohol consumption in adulthood.

  1. Effects of strongly anisosmotic and NaCl deficient solutions on muscimol- and glutamate evoked whole-cell currents in freshly dissociated hippocampal neurons.

    PubMed

    Vreugdenhil, M; Somjen, G G; Wadman, W J

    1995-01-23

    Sudden exposure of dissociated hippocampal neurons to strongly hypo- or hyperosmotic solutions suppresses voltage gated Na+, K+ and Ca2+ currents. We investigated whether ligand gated ion currents were similarly shut down by exposure to anisosmotic solutions. The effect of hypo-osmotic, NaCl deficient (mannitol-substituted), or hyper-osmotic test solutions delivered from a flow pipette was tested on voltage gated Ca2+ currents and on currents and conductance changes evoked by brief administration of either the GABAA-agonist muscimol or glutamate. Hyper-osmotic solution caused cells to shrink, but cell membrane capacitance did not change. Muscimol-induced conductance increases were depressed by hypo-osmotic and by NaCl deficient solutions and often by hyper-osmotic solution. Voltage gated Ca2+ currents were depressed by anisosmotic, but not by NaCl deficient isosmotic solution. NMDA- and non-NMDA evoked conductance increases were depressed by hyperosmotic solution; hypo-osmotic and NaCl deficient solutions were not tested on glutamate induced currents. Ligand gated currents are suppressed by anisosmotic solutions more slowly than are voltage gated channels. The changes caused by anisosmotic and NaCl deficient solutions were much greater then expected from calculated electrochemical effects and are probably the result of change in receptor controlled channels. PMID:7536614

  2. Cell-specific abnormalities of glutamate transporters in schizophrenia: sick astrocytes and compensating relay neurons?

    PubMed

    McCullumsmith, R E; O'Donovan, S M; Drummond, J B; Benesh, F S; Simmons, M; Roberts, R; Lauriat, T; Haroutunian, V; Meador-Woodruff, J H

    2016-06-01

    Excitatory amino-acid transporters (EAATs) bind and transport glutamate, limiting spillover from synapses due to their dense perisynaptic expression primarily on astroglia. Converging evidence suggests that abnormalities in the astroglial glutamate transporter localization and function may underlie a disease mechanism with pathological glutamate spillover as well as alterations in the kinetics of perisynaptic glutamate buffering and uptake contributing to dysfunction of thalamo-cortical circuits in schizophrenia. We explored this hypothesis by performing cell- and region-level studies of EAAT1 and EAAT2 expression in the mediodorsal nucleus of the thalamus in an elderly cohort of subjects with schizophrenia. We found decreased protein expression for the typically astroglial-localized glutamate transporters in the mediodorsal and ventral tier nuclei. We next used laser-capture microdissection and quantitative polymerase chain reaction to assess cell-level expression of the transporters and their splice variants. In the mediodorsal nucleus, we found lower expression of transporter transcripts in a population of cells enriched for astrocytes, and higher expression of transporter transcripts in a population of cells enriched for relay neurons. We confirmed expression of transporter protein in neurons in schizophrenia using dual-label immunofluorescence. Finally, the pattern of transporter mRNA and protein expression in rodents treated for 9 months with antipsychotic medication suggests that our findings are not due to the effects of antipsychotic treatment. We found a compensatory increase in transporter expression in neurons that might be secondary to a loss of transporter expression in astrocytes. These changes suggest a profound abnormality in astrocyte functions that support, nourish and maintain neuronal fidelity and synaptic activity.

  3. Icilin evokes a dose- and time-dependent increase in glutamate within the dorsal striatum of rats.

    PubMed

    Werkheiser, J L; Rawls, S M; Cowan, A

    2006-05-01

    Icilin, the peripheral cold channel agonist, activates TRPM8 and TRPA1, localized on dorsal root ganglia and trigeminal neurons in rats. Icilin precipitates immediate wet-dog shakes in this species, which are antagonized by centrally acting mu and kappa opioid agonists, implicating the central nervous system in the behavioral response. We studied the effect icilin has on glutamate levels in the dorsal striatum, a brain region involved in movement. Icilin (0.25, 0.5 and 0.75 mg/kg, i.p.) elicited a dose- and time-dependent increase in glutamate within the striatum, indicative of icilin's neurochemical effect in rats.

  4. Glutamate receptors and transporters in genetic and acquired models of epilepsy.

    PubMed

    Meldrum, B S; Akbar, M T; Chapman, A G

    1999-09-01

    Glutamate, the principal excitatory neurotransmitter in the brain, acts on three families of ionotropic receptor--AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid), kainate and NMDA (N-methyl-D-aspartate) receptors and three families of metabotropic receptor (Group I: mGlu1 and mGlu5; Group II: mGlu2 and mGlu3; Group III: mGlu4, mGlu6, mGlu7 and mGlu8). Glutamate is removed from the synaptic cleft and the extracellular space by Na+-dependent transporters (GLAST/EAAT1, GLT/EAAT2, EAAC/EAAT3, EAAT4, EAAT5). In rodents, genetic manipulations relating to the expression or function of glutamate receptor proteins can induce epilepsy syndromes or raise seizure threshold. Decreased expression of glutamate transporters (EAAC knockdown, GLT knockout) can lead to seizures. In acquired epilepsy syndromes, a wide variety of changes in receptors and transporters have been described. Electrically-induced kindling in the rat is associated with functional potentiation of NMDA receptor-mediated responses at various limbic sites. Group I metabotropic responses are enhanced in the amygdala. To date, no genetic epilepsy in man has been identified in which the primary genetic defect involves glutamate receptors or transporters. Changes are found in some acquired syndromes, including enhanced NMDA receptor responses in dentate granule cells in patients with hippocampal sclerosis. PMID:10515165

  5. Effects of Ceftriaxone on Glial Glutamate Transporters in Wistar Rats Administered Sequential Ethanol and Methamphetamine

    PubMed Central

    Althobaiti, Yusuf S.; Alshehri, Fahad S.; Almalki, Atiah H.; Sari, Youssef

    2016-01-01

    Methamphetamine (METH) is one of the psychostimulants that is co-abused with ethanol. Repeated exposure to high dose of METH has been shown to cause increases in extracellular glutamate concentration. We have recently reported that ethanol exposure can also increase the extracellular glutamate concentration and downregulate the expression of glutamate transporter subtype 1 (GLT-1). GLT-1 is a glial transporter that regulates the majority of extracellular glutamate. A Wistar rat model of METH and ethanol co-abuse was used to examine the expression of GLT-1 as well as other glutamate transporters such as cystine/glutamate exchanger (xCT) and glutamate aspartate transporter (GLAST). We also examined the body temperature in rats administered METH, ethanol or both drugs. We further investigated the effects of ceftriaxone (CEF), a β-lactam antibiotic known to upregulate GLT-1, in this METH/ethanol co-abuse rat model. After 7 days of either ethanol (6 g/kg) or water oral gavage, Wistar rats received either saline or METH (10 mg/kg i.p. every 2 h × 4), followed by either saline or CEF (200 mg/kg) posttreatment. METH administered alone decreased GLT-1 expression in the nucleus accumbens (NAc) and prefrontal cortex (PFC) and increased body temperature, but did not reduce either xCT or GLAST expression in ethanol and water-pretreated rats. Interestingly, ethanol and METH were found to have an additive effect on the downregulation of GLT-1 expression in the NAc but not in the PFC. Moreover, ethanol alone caused GLT-1 downregulation in the NAc and elevated body temperature compared to control. Finally, CEF posttreatment significantly reversed METH-induced hyperthermia, restored GLT-1 expression, and increased xCT expression. These findings suggest the potential therapeutic role of CEF against METH- or ethanol/METH-induced hyperglutamatergic state and hyperthermia. PMID:27713684

  6. Differing effects of transport inhibitor on glutamate uptake by nerve terminals before and after exposure of rats to artificial gravity.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Krisanova, N.; Himmelreich, N.

    Glutamate is the major excitatory neurotransmitter in the brain. Subsequent to its release from glutamatergic neurons and activation of receptors, it is removed from extracellular space by high affinity Na^+-dependent glutamate transporters, which utilize the Na^+/K^+ electrochemical gradient as a driving force and located in nerve terminals and astrocytes. The glutamate transporters may modify the time course of synaptic events. Like glutamate itself, glutamate transporters are somehow involved in almost all aspects of normal and abnormal brain activity (e.g. cerebral ischemia, amyotrophic lateral sclerosis, Alzheimer's disease, traumatic brain injury, epilepsy and schizophrenia). The present study assessed transporter inhibitor for the ability to inhibit glutamate uptake by synaptosomes at the normal and hypergravity conditions (rats were rotated in a long-arm centrifuge at ten-G during one-hour period). DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a newly developed competitive inhibitor of the high-affinity, Na^+-dependent glutamate transporters. As a potent, non- transported inhibitor of glutamate transporters, DL-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms. We demonstrated that DL-TBOA inhibited glutamate uptake ( 100 μM glutamate, 30 sec incubation period) in dose-dependent manner as in control as in hypergravity. The effect of this transport inhibitor on glutamate uptake by control synaptosomes and synaptosomes prepared of animals exposed to hypergravity was different. IC50 values calculated on the basis of curves of non-linear regression kinetic analysis was 18±2 μM and 11±2 μM ((P≤0,05) before and after exposure to artificial gravity, respectively. Inhibition caused by 10 μM DL-TBOA was significantly increased from 38,0±3,8 % in control group to 51,0±4,1 % in animals, exposed to hypergravity (P≤0,05). Thus, DL-TBOA had complex effect on glutamate uptake process and perhaps, became more potent under

  7. TM4 of the glutamate transporter GLT-1 experiences substrate-induced motion during the transport cycle

    PubMed Central

    Rong, Xiuliang; Tan, Feng; Wu, Xiaojuan; Zhang, Xiuping; Lu, Lingli; Zou, Xiaoming; Qu, Shaogang

    2016-01-01

    Excitatory amino acid transporter 2 (EAAT2), also known as glial glutamate transporter type 1 (GLT-1), plays an important role in maintaining the extracellular glutamate concentrations below neurotoxic levels. The highly conserved TM2 transmembrane domain of GLT-1 maintains a stable position during the transport cycle; however, the effect of the transport cycle on the topology of TM4 in not well established. To further reveal the function of TM4, two cysteine pairs between TM2 and TM4 were introduced using site-directed mutagenesis. A significant decrease of transport activity was observed in the I93C/V241C and I97C/V241C mutants upon application of the oxidative cross-linking reagent, copper (II) (1,10-phenanthroline)3 (CuPh), which suggests that a conformational shift is essential for transporter activity. Furthermore, the decrease in activity by CuPh crosslinking was enhanced in external media with glutamate or potassium, which suggests that TM2 and TM4 assume closer proximity in the inward-facing conformation of the transporter. Our results suggest that the TM4 domain of GLT-1, and potentially other glutamate transporters, undergoes a complex conformational shift during substrate translocation, which involves an increase in the proximity of the TM2 and TM4 domains in the inward-facing conformation. PMID:27698371

  8. Astrocyte VAMP3 vesicles undergo Ca2+-independent cycling and modulate glutamate transporter trafficking

    PubMed Central

    Li, Dongdong; Hérault, Karine; Zylbersztejn, Kathleen; Lauterbach, Marcel A; Guillon, Marc; Oheim, Martin; Ropert, Nicole

    2015-01-01

    Key points Mouse cortical astrocytes express VAMP3 but not VAMP2. VAMP3 vesicles undergo Ca2+-independent exo- and endocytotic cycling at the plasma membrane. VAMP3 vesicle traffic regulates the recycling of plasma membrane glutamate transporters. cAMP modulates VAMP3 vesicle cycling and glutamate uptake. Abstract Previous studies suggest that small synaptic-like vesicles in astrocytes carry vesicle-associated vSNARE proteins, VAMP3 (cellubrevin) and VAMP2 (synaptobrevin 2), both contributing to the Ca2+-regulated exocytosis of gliotransmitters, thereby modulating brain information processing. Here, using cortical astrocytes taken from VAMP2 and VAMP3 knock-out mice, we find that astrocytes express only VAMP3. The morphology and function of VAMP3 vesicles were studied in cultured astrocytes at single vesicle level with stimulated emission depletion (STED) and total internal reflection fluorescence (TIRF) microscopies. We show that VAMP3 antibodies label small diameter (∼80 nm) vesicles and that VAMP3 vesicles undergo Ca2+-independent exo-endocytosis. We also show that this pathway modulates the surface expression of plasma membrane glutamate transporters and the glutamate uptake by astrocytes. Finally, using pharmacological and optogenetic tools, we provide evidence suggesting that the cytosolic cAMP level influences astrocytic VAMP3 vesicle trafficking and glutamate transport. Our results suggest a new role for VAMP3 vesicles in astrocytes. PMID:25864578

  9. Effects of Amoxicillin and Augmentin on Cystine-Glutamate Exchanger and Glutamate Transporter 1 Isoforms as well as Ethanol Intake in Alcohol-Preferring Rats

    PubMed Central

    Hakami, Alqassem Y.; Hammad, Alaa M.; Sari, Youssef

    2016-01-01

    Alcohol dependence is associated with alteration of glutamate transport and glutamate neurotransmission. Glutamate transporter 1 (GLT-1) is a major transporter that regulates the majority of extracellular glutamate concentration, which is also regulated by cystine-glutamate exchanger (xCT). Importantly, we recently reported that amoxicillin and Augmentin (amoxicillin/clavulanate) upreglulated GLT-1 expression in nucleus accumbens (NAc) and prefrontal cortex (PFC) as well as reduced ethanol consumption in male P rats. In this study, we examined the effects of amoxicillin and Augmentin on GLT-1 isoforms (GLT-1a and GLT-1b), xCT, and glutamate/aspartate transporter (GLAST) expression in NAc and PFC as well as ethanol intake in male P rats. We found that both compounds significantly reduced ethanol intake, and increased GLT-1a, GLT-1b, and xCT expression in NAc. However, only Augmentin increased GLT-1a, GLT-1b, and xCT expression in PFC. There were no effects of these compounds on GLAST expression in NAc and PFC. These findings demonstrated that Augmentin and amoxicillin have the potential to upregulate GLT-1 isoforms and xCT expression, and consequently attenuate ethanol dependence. PMID:27199635

  10. Gabapentin increases extracellular glutamatergic level in the locus coeruleus via astroglial glutamate transporter-dependent mechanisms.

    PubMed

    Suto, Takashi; Severino, Amie L; Eisenach, James C; Hayashida, Ken-ichiro

    2014-06-01

    Gabapentin has shown to be effective in animals and humans with acute postoperative and chronic pain. Yet the mechanisms by which gabapentin reduces pain have not been fully addressed. The current study performed in vivo microdialysis in the locus coeruleus (LC) in normal and spinal nerve ligated (SNL) rats to examine the effect of gabapentin on extracellular glutamate concentration and its mechanisms of action with focus on presynaptic GABA-B receptors, astroglial glutamate transporter-1 (GLT-1), and interactions with α2δ subunits of voltage-gated Ca(2+) channels and endogenous noradrenaline. Basal extracellular concentration and tissue content of glutamate in the LC were greater in SNL rats than normal ones. Intravenously administered and LC-perfused gabapentin increased extracellular glutamate concentration in the LC. The net amount of glutamate increased by gabapentin is larger in SNL rats compared with normal ones, although the percentage increases from the baseline did not differ. The gabapentin-related α2δ ligand pregabalin increased extracellular glutamate concentration in the LC, whereas another α2δ ligand, 3-exo-aminobicyclo [2.2.1] heptane-2-exo-carboxylic acid (ABHCA), did not. Selective blockade by the dihydrokainic acid or knock-down of GLT-1 by the small interfering RNA abolished the gabapentin-induced glutamate increase in the LC, whereas blockade of GABA-B receptors by the CGP-35348 and depletion of noradrenalin by the dopamine-β-hydroxylase antibody conjugated to saporin did not. These results suggest that gabapentin induces glutamate release from astrocytes in the LC via GLT-1-dependent mechanisms to stimulate descending inhibition. The present study also demonstrates that this target of gabapentin in astrocytes does not require interaction with α2δ subunits in neurons.

  11. Position of the Third Na+ Site in the Aspartate Transporter GltPh and the Human Glutamate Transporter, EAAT1

    PubMed Central

    Bastug, Turgut; Heinzelmann, Germano; Kuyucak, Serdar; Salim, Marietta; Vandenberg, Robert J.; Ryan, Renae M.

    2012-01-01

    Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na+ ions, one H+ and the counter-transport of one K+ ion. Transport by an archaeal homologue of the human glutamate transporters, GltPh, whose three dimensional structure is known is also coupled to three Na+ ions but only two Na+ ion binding sites have been observed in the crystal structure of GltPh. In order to fully utilize the GltPh structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of GltPh and accurately determine the number and location of Na+ ions coupled to transport. Several sites have been proposed for the binding of a third Na+ ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for GltPh and reveal a new site for the third Na+ ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in GltPh, and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na+ compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na+ ion in GltPh and EAAT1. PMID:22427946

  12. Identification and metabolic role of the mitochondrial aspartate-glutamate transporter in Saccharomyces cerevisiae.

    PubMed

    Cavero, S; Vozza, A; del Arco, A; Palmieri, L; Villa, A; Blanco, E; Runswick, M J; Walker, J E; Cerdán, S; Palmieri, F; Satrústegui, J

    2003-11-01

    The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspartate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identified Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomyces cerevisiae. On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast. PMID:14622413

  13. Effects of diabetes mellitus on astrocyte GFAP and glutamate transporters in the CNS.

    PubMed

    Coleman, Elaine; Judd, Robert; Hoe, Lori; Dennis, John; Posner, Philip

    2004-11-01

    Diabetes mellitus increases the risk of central nervous system (CNS) disorders such as stroke, seizures, dementia, and cognitive impairment. The cellular mechanisms responsible for the increased risk of these disorders are incompletely understood. Astrocytes are proving critical for normal CNS function, and alterations in their activity could contribute to diabetes-related disturbances in the brain. We examined the effects of streptozotocin (STZ)-induced diabetes in rats on the level of the astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP), number of astrocytes, and levels of the astrocyte glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate/aspartate transporter (GLAST), in the cerebral cortex, hippocampus, and cerebellum by Western blotting (WB) and immunohistochemistry (IH). Studies were carried out at 4 and 8 weeks of diabetes duration. Diabetes resulted in a significant decrease in GFAP protein levels (WB) in the hippocampus and cerebellum at 4 weeks and in the cerebral cortex, hippocampus and cerebellum by 8 weeks. Attenuated GFAP immunoreactivity (IH) was evident in the hippocampus, cerebellum and white matter regions such as the corpus callosum and external capsule at both 4 and 8 weeks of diabetes. Astrocyte cell counts of adjacent sections immunoreactive for S-100B were not different between control and diabetic animals. No significant differences were noted in astrocyte glutamate transporter levels in the cerebral cortex, hippocampus, or cerebellum at either time period (WB, IH). With the expanding list of astrocyte functions in the CNS, the role of astrocytes in diabetes-induced CNS disorders clearly warrants further investigation.

  14. Bovine neuronal vesicular glutamate transporter activity is inhibited by ergovaline and other ergopeptines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    L-Glutamate (Glu) is the major excitatory neurotransmitter responsible for neurotransmission in the vertebrate central nervous system, including the gastrointestinal tract (GIT) of cattle. Vesicular Glu transporters VGLUT1 and VGLUT2 concentrate (50 mM) Glu (Km = 1 to 4 mM) into synaptic vesicles (S...

  15. Lobelane inhibits methamphetamine-evoked dopamine release via inhibition of the vesicular monoamine transporter-2.

    PubMed

    Nickell, Justin R; Krishnamurthy, Sairam; Norrholm, Seth; Deaciuc, Gabriela; Siripurapu, Kiran B; Zheng, Guangrong; Crooks, Peter A; Dwoskin, Linda P

    2010-02-01

    Lobeline is currently being evaluated in clinical trials as a methamphetamine abuse treatment. Lobeline interacts with nicotinic receptor subtypes, dopamine transporters (DATs), and vesicular monoamine transporters (VMAT2s). Methamphetamine inhibits VMAT2 and promotes dopamine (DA) release from synaptic vesicles, resulting ultimately in increased extracellular DA. The present study generated structure-activity relationships by defunctionalizing the lobeline molecule and determining effects on [(3)H]dihydrotetrabenazine binding, inhibition of [(3)H]DA uptake into striatal synaptic vesicles and synaptosomes, the mechanism of VMAT2 inhibition, and inhibition of methamphetamine-evoked DA release. Compared with lobeline, the analogs exhibited greater potency inhibiting DA transporter (DAT) function. Saturated analogs, lobelane and nor-lobelane, exhibited high potency (K(i) = 45 nM) inhibiting vesicular [(3)H]DA uptake, and lobelane competitively inhibited VMAT2 function. Lobeline and lobelane exhibited 67- and 35-fold greater potency, respectively, in inhibiting VMAT2 function compared to DAT function. Lobelane potently decreased (IC(50) = 0.65 microM; I(max) = 73%) methamphetamine-evoked DA overflow, and with a greater maximal effect compared with lobeline (IC(50) = 0.42 microM, I(max) = 56.1%). These results provide support for VMAT2 as a target for inhibition of methamphetamine effects. Both trans-isomers and demethylated analogs of lobelane had reduced or unaltered potency inhibiting VMAT2 function and lower maximal inhibition of methamphetamine-evoked DA release compared with lobelane. Thus, defunctionalization, cis-stereochemistry of the side chains, and presence of the piperidino N-methyl are structural features that afford greatest inhibition of methamphetamine-evoked DA release and enhancement of selectivity for VMAT2. The current results reveal that lobelane, a selective VMAT2 inhibitor, inhibits methamphetamine-evoked DA release and is a promising lead for

  16. Lobelane Inhibits Methamphetamine-Evoked Dopamine Release via Inhibition of the Vesicular Monoamine Transporter-2S⃞

    PubMed Central

    Nickell, Justin R.; Krishnamurthy, Sairam; Norrholm, Seth; Deaciuc, Gabriela; Siripurapu, Kiran B.; Zheng, Guangrong; Crooks, Peter A.

    2010-01-01

    Lobeline is currently being evaluated in clinical trials as a methamphetamine abuse treatment. Lobeline interacts with nicotinic receptor subtypes, dopamine transporters (DATs), and vesicular monoamine transporters (VMAT2s). Methamphetamine inhibits VMAT2 and promotes dopamine (DA) release from synaptic vesicles, resulting ultimately in increased extracellular DA. The present study generated structure-activity relationships by defunctionalizing the lobeline molecule and determining effects on [3H]dihydrotetrabenazine binding, inhibition of [3H]DA uptake into striatal synaptic vesicles and synaptosomes, the mechanism of VMAT2 inhibition, and inhibition of methamphetamine-evoked DA release. Compared with lobeline, the analogs exhibited greater potency inhibiting DA transporter (DAT) function. Saturated analogs, lobelane and nor-lobelane, exhibited high potency (Ki = 45 nM) inhibiting vesicular [3H]DA uptake, and lobelane competitively inhibited VMAT2 function. Lobeline and lobelane exhibited 67- and 35-fold greater potency, respectively, in inhibiting VMAT2 function compared to DAT function. Lobelane potently decreased (IC50 = 0.65 μM; Imax = 73%) methamphetamine-evoked DA overflow, and with a greater maximal effect compared with lobeline (IC50 = 0.42 μM, Imax = 56.1%). These results provide support for VMAT2 as a target for inhibition of methamphetamine effects. Both trans-isomers and demethylated analogs of lobelane had reduced or unaltered potency inhibiting VMAT2 function and lower maximal inhibition of methamphetamine-evoked DA release compared with lobelane. Thus, defunctionalization, cis-stereochemistry of the side chains, and presence of the piperidino N-methyl are structural features that afford greatest inhibition of methamphetamine-evoked DA release and enhancement of selectivity for VMAT2. The current results reveal that lobelane, a selective VMAT2 inhibitor, inhibits methamphetamine-evoked DA release and is a promising lead for the development of a

  17. Axonal Segregation and Role of the Vesicular Glutamate Transporter VGLUT3 in Serotonin Neurons

    PubMed Central

    Voisin, Aurore N.; Mnie-Filali, Ouissame; Giguère, Nicolas; Fortin, Guillaume M.; Vigneault, Erika; El Mestikawy, Salah; Descarries, Laurent; Trudeau, Louis-Éric

    2016-01-01

    A subset of monoamine neurons releases glutamate as a cotransmitter due to presence of the vesicular glutamate transporters VGLUT2 or VGLUT3. In addition to mediating vesicular loading of glutamate, it has been proposed that VGLUT3 enhances serotonin (5-HT) vesicular loading by the vesicular monoamine transporter (VMAT2) in 5-HT neurons. In dopamine (DA) neurons, glutamate appears to be released from specialized subsets of terminals and it may play a developmental role, promoting neuronal growth and survival. The hypothesis of a similar developmental role and axonal localization of glutamate co-release in 5-HT neurons has not been directly examined. Using postnatal mouse raphe neurons in culture, we first observed that in contrast to 5-HT itself, other phenotypic markers of 5-HT axon terminals such as the 5-HT reuptake transporter (SERT) show a more restricted localization in the axonal arborization. Interestingly, only a subset of SERT- and 5-HT-positive axonal varicosities expressed VGLUT3, with SERT and VGLUT3 being mostly segregated. Using VGLUT3 knockout mice, we found that deletion of this transporter leads to reduced survival of 5-HT neurons in vitro and also decreased the density of 5-HT-immunoreactivity in terminals in the dorsal striatum and dorsal part of the hippocampus in the intact brain. Our results demonstrate that raphe 5-HT neurons express SERT and VGLUT3 mainly in segregated axon terminals and that VGLUT3 regulates the vulnerability of these neurons and the neurochemical identity of their axonal domain, offering new perspectives on the functional connectivity of a cell population involved in anxiety disorders and depression. PMID:27147980

  18. Engineering the glutamate transporter homologue GltPh using protein semisynthesis.

    PubMed

    Focke, Paul J; Annen, Alvin W; Valiyaveetil, Francis I

    2015-03-01

    Glutamate transporters catalyze the concentrative uptake of glutamate from synapses and are essential for normal synaptic function. Despite extensive investigations of glutamate transporters, the mechanisms underlying substrate recognition, ion selectivity, and the coupling of substrate and ion transport are not well-understood. Deciphering these mechanisms requires the ability to precisely engineer the transporter. In this study, we describe the semisynthesis of GltPh, an archaeal homologue of glutamate transporters. Semisynthesis allows the precise engineering of GltPh through the incorporation of unnatural amino acids and peptide backbone modifications. In the semisynthesis, the GltPh polypeptide is initially assembled from a recombinantly expressed thioester peptide and a chemically synthesized peptide using the native chemical ligation reaction followed by in vitro folding to the native state. We have developed a robust procedure for the in vitro folding of GltPh. Biochemical characterization of the semisynthetic GltPh indicates that it is similar to the native transporter. We used semisynthesis to substitute Arg397, a highly conserved residue in the substrate binding site, with the unnatural analogue, citrulline. Our studies demonstrate that Arg397 is required for high-affinity substrate binding, and on the basis of our results, we propose that Arg397 is involved in a Na+-dependent remodeling of the substrate binding site required for high-affinity Asp binding. We anticipate that the semisynthetic approach developed in this study will be extremely useful in investigating functional mechanisms in GltPh. Further, the approach developed in this study should also be applicable to other membrane transport proteins.

  19. The Density of EAAC1 (EAAT3) Glutamate Transporters Expressed by Neurons in the Mammalian CNS

    PubMed Central

    Holmseth, Silvia; Dehnes, Yvette; Huang, Yanhua H.; Follin-Arbelet, Virginie V.; Grutle, Nina J.; Mylonakou, Maria N.; Plachez, Celine; Zhou, Yun; Furness, David N.; Bergles, Dwight E.

    2012-01-01

    The extracellular levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was ∼0.013 mg/g tissue (∼130 molecules μm−3), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-d-aspartate (4-methoxy-7-nitroindolinyl-d-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and d-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses. PMID:22539860

  20. Different functional roles of arginine residues 39 and 61 and tyrosine residue 98 in transport and channel mode of the glutamate transporter EAAC1.

    PubMed

    Zhu, Yani; Vasilets, Larisa A; Fei, Jian; Guo, Lihe; Schwarz, Wolfgang

    2004-10-11

    The excitatory amino acid transporter EAAC1 is an electrogenic Na+ - and K+ -gradient-driven transporter. In addition, the transporter mediates in the presence of Na+ and glutamate an anion conductance uncoupled from the transport of the glutamate. The first two N-terminal domains, important for forming the conductance mode, are extracellularly bordered by positively charged arginine residues, R39 and R61, being completely conserved throughout the transporter family. Also the conserved tyrosine residue Y98 could be important for Cl- conductance. We have investigated, by measurements of glutamate uptake and glutamate-induced currents, the effects of mutation of the arginines and the tyrosine to alanine. The mutation R39A hardly affects transport and channel mode. The mutation R61A, on the other hand, reduces the activity of transport but stimulates the channel conductance. In addition, the apparent Km values for glutamate uptake and for the glutamate-activated current are reduced. Glutamate stimulation of current seems to be associated with a voltage-dependent step, and the apparent valence of charge moved during binding is reduced in the R61A mutant. The mutation Y98A leads to reduced function with reduced apparent Km value for glutamate, and with strong reduction of the selectivity ration between NO3- and Cl- of the conductance mode.

  1. In vivo and in vitro studies of glycine- and glutamate-evoked acetylcholinesterase release from spinal motor neurones: implications for amyotrophic lateral sclerosis/motor neurone disease pathogenesis.

    PubMed

    Rodríguez-Ithurralde, D; Olivera, S; Vincent, O; Maruri, A

    1997-10-01

    To investigate the spinal cellular structures and molecular mechanisms involved in acetylcholinesterase (AChE) release evoked by both glycine (GLY) and glutamate (GLU)--responses that might play a role in chronic neurotoxicity--we analysed AChE histochemistry and histology upon systemic administration of aspartate (ASP), and conducted in vitro experiments in synaptosomes and slices prepared from mouse spinal ventral horns. Upon superfusion and incubation exposure of these preparations to GLY- and GLU-receptor agonists, we assayed both tissue content and release of AChE, butyrylcholinesterase and lactic dehydrogenase. Histochemical reduction of motor neurone (MN) AChE, calcium dependency, decreases in intracellular AChE and the ratio amongst molecular forms released, suggest that both synaptosomal GLY-evoked AChE release (GLY-EAR) and GLU-receptor-elicited AChE release (GEAR) have release sites located at MN presynaptic terminals. These responses exhibited remarkable postnatal regulation. GEAR seems to be mediated through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptors after the fourth postnatal week and through both NMDA and non-NMDA receptors at earlier stages. Sustained rises of extracellular AChE might link acute excitotoxic injury with several long-lasting pathways leading to chronic neurotoxicity, since AChE molecular properties include: (1) the ability to block cholinergic mechanisms that protect MN against overactivity; (2) activation of ATP-dependent potassium channels; (3) promotion of neurite and axon outgrowth; and possibly (4) stimulation of brain macrophage migration and activation.

  2. Conditional deletion of the glutamate transporter GLT-1 reveals that astrocytic GLT-1 protects against fatal epilepsy while neuronal GLT-1 contributes significantly to glutamate uptake into synaptosomes.

    PubMed

    Petr, Geraldine T; Sun, Yan; Frederick, Natalie M; Zhou, Yun; Dhamne, Sameer C; Hameed, Mustafa Q; Miranda, Clive; Bedoya, Edward A; Fischer, Kathryn D; Armsen, Wencke; Wang, Jianlin; Danbolt, Niels C; Rotenberg, Alexander; Aoki, Chiye J; Rosenberg, Paul A

    2015-04-01

    GLT-1 (EAAT2; slc1a2) is the major glutamate transporter in the brain, and is predominantly expressed in astrocytes, but at lower levels also in excitatory terminals. We generated a conditional GLT-1 knock-out mouse to uncover cell-type-specific functional roles of GLT-1. Inactivation of the GLT-1 gene was achieved in either neurons or astrocytes by expression of synapsin-Cre or inducible human GFAP-CreERT2. Elimination of GLT-1 from astrocytes resulted in loss of ∼80% of GLT-1 protein and of glutamate uptake activity that could be solubilized and reconstituted in liposomes. This loss was accompanied by excess mortality, lower body weight, and seizures suggesting that astrocytic GLT-1 is of major importance. However, there was only a small (15%) reduction that did not reach significance of glutamate uptake into crude forebrain synaptosomes. In contrast, when GLT-1 was deleted in neurons, both the GLT-1 protein and glutamate uptake activity that could be solubilized and reconstituted in liposomes were virtually unaffected. These mice showed normal survival, weight gain, and no seizures. However, the synaptosomal glutamate uptake capacity (Vmax) was reduced significantly (40%). In conclusion, astrocytic GLT-1 performs critical functions required for normal weight gain, resistance to epilepsy, and survival. However, the contribution of astrocytic GLT-1 to glutamate uptake into synaptosomes is less than expected, and the contribution of neuronal GLT-1 to synaptosomal glutamate uptake is greater than expected based on their relative protein expression. These results have important implications for the interpretation of the many previous studies assessing glutamate uptake capacity by measuring synaptosomal uptake. PMID:25834045

  3. Conditional Deletion of the Glutamate Transporter GLT-1 Reveals That Astrocytic GLT-1 Protects against Fatal Epilepsy While Neuronal GLT-1 Contributes Significantly to Glutamate Uptake into Synaptosomes

    PubMed Central

    Petr, Geraldine T.; Sun, Yan; Frederick, Natalie M.; Zhou, Yun; Dhamne, Sameer C.; Hameed, Mustafa Q.; Miranda, Clive; Bedoya, Edward A.; Fischer, Kathryn D.; Armsen, Wencke; Wang, Jianlin; Danbolt, Niels C.; Rotenberg, Alexander; Aoki, Chiye J.

    2015-01-01

    GLT-1 (EAAT2; slc1a2) is the major glutamate transporter in the brain, and is predominantly expressed in astrocytes, but at lower levels also in excitatory terminals. We generated a conditional GLT-1 knock-out mouse to uncover cell-type-specific functional roles of GLT-1. Inactivation of the GLT-1 gene was achieved in either neurons or astrocytes by expression of synapsin-Cre or inducible human GFAP-CreERT2. Elimination of GLT-1 from astrocytes resulted in loss of ∼80% of GLT-1 protein and of glutamate uptake activity that could be solubilized and reconstituted in liposomes. This loss was accompanied by excess mortality, lower body weight, and seizures suggesting that astrocytic GLT-1 is of major importance. However, there was only a small (15%) reduction that did not reach significance of glutamate uptake into crude forebrain synaptosomes. In contrast, when GLT-1 was deleted in neurons, both the GLT-1 protein and glutamate uptake activity that could be solubilized and reconstituted in liposomes were virtually unaffected. These mice showed normal survival, weight gain, and no seizures. However, the synaptosomal glutamate uptake capacity (Vmax) was reduced significantly (40%). In conclusion, astrocytic GLT-1 performs critical functions required for normal weight gain, resistance to epilepsy, and survival. However, the contribution of astrocytic GLT-1 to glutamate uptake into synaptosomes is less than expected, and the contribution of neuronal GLT-1 to synaptosomal glutamate uptake is greater than expected based on their relative protein expression. These results have important implications for the interpretation of the many previous studies assessing glutamate uptake capacity by measuring synaptosomal uptake. PMID:25834045

  4. Resveratrol Prevents Retinal Dysfunction by Regulating Glutamate Transporters, Glutamine Synthetase Expression and Activity in Diabetic Retina.

    PubMed

    Zeng, Kaihong; Yang, Na; Wang, Duozi; Li, Suping; Ming, Jian; Wang, Jing; Yu, Xuemei; Song, Yi; Zhou, Xue; Yang, Yongtao

    2016-05-01

    This study investigated the effects of resveratrol (RSV) on retinal functions, glutamate transporters (GLAST) and glutamine synthetase (GS) expression in diabetic rats retina, and on glutamate uptake, GS activity, GLAST and GS expression in high glucose-cultured Müller cells. The electroretinogram was used to evaluate retinal functions. Müller cells cultures were prepared from 5- to 7-day-old Sprague-Dawley rats. The expression of GLAST and GS was examined by qRT-PCR, ELISA and western-blotting. Glutamate uptake was measured as (3)H-glutamate contents of the lysates. GS activity was assessed by a spectrophotometric assay. 1- to 7-month RSV administrations (5 and 10 mg/kg/day) significantly alleviated hyperglycemia and weight loss in diabetic rats. RSV administrations also significantly attenuated diabetes-induced decreases in amplitude of a-wave in rod response, decreases in amplitude of a-, and b-wave in cone and rod response and decreases in amplitude of OP2 in oscillatory potentials. 1- to 7-month RSV treatments also significantly inhibited diabetes-induced delay in OP2 implicit times in scotopic 3.0 OPS test. The down-regulated mRNA and protein expression of GLAST and GS in diabetic rats retina was prevented by RSV administrations. In high glucose-treated cultures, Müller cells' glutamate uptake, GS activity, GLAST and GS expression were decreased significantly compared with normal control cultures. RSV (10, 20, and 30 mmol/l) significantly inhibited the HG-induced decreases in glutamate uptake, GS activity, GLAST and GS expression (at least P < 0.05). These beneficial results suggest that RSV may be considered as a therapeutic option to prevent from diabetic retinopathy.

  5. Elevated venous glutamate levels in (pre)catabolic conditions result at least partly from a decreased glutamate transport activity.

    PubMed

    Hack, V; Stütz, O; Kinscherf, R; Schykowski, M; Kellerer, M; Holm, E; Dröge, W

    1996-06-01

    Abnormally high postabsorptive venous plasma glutamate levels have been reported for several diseases that are associated with a loss of body cell mass including cancer, human/simian immunodeficiency virus infection, and amyotrophic lateral sclerosis. Studies on exchange rates in well-nourished cancer patients now show that high venous plasma glutamate levels may serve as a bona fide indicator for a decreased uptake of glutamate by the peripheral muscle tissue in the postabsorptive period and may be indicative for a precachectic state. High glutamate levels are also moderately correlated with a decreased uptake of glucose and ketone bodies. Relatively high venous glutamate levels have also been found in non-insulin-dependent diabetes mellitus and to some extent also in the cubital vein of normal elderly subjects, i.e., in conditions commonly associated with a decreased glucose tolerance and progressive loss of body cell mass.

  6. Developmental and target-dependent regulation of vesicular glutamate transporter expression by dopamine neurons.

    PubMed

    Mendez, Jose Alfredo; Bourque, Marie-Josée; Dal Bo, Gregory; Bourdeau, Mathieu L; Danik, Marc; Williams, Sylvain; Lacaille, Jean-Claude; Trudeau, Louis-Eric

    2008-06-18

    Mesencephalic dopamine (DA) neurons have been suggested to use glutamate as a cotransmitter. Here, we suggest a mechanism for this form of cotransmission by showing that a subset of DA neurons both in vitro and in vivo expresses vesicular glutamate transporter 2 (VGluT2). Expression of VGluT2 decreases with age. Moreover, when DA neurons are grown in isolation using a microculture system, there is a marked upregulation of VGluT2 expression. We provide evidence that expression of this transporter is normally repressed through a contact-dependent interaction with GABA and other DA neurons, thus providing a partial explanation for the highly restricted expression of VGluT2 in DA neurons in vivo. Our results demonstrate that the neurotransmitter phenotype of DA neurons is both developmentally and dynamically regulated. These findings may have implications for a better understanding of the fast synaptic action of DA neurons as well as basal ganglia circuitry. PMID:18562601

  7. Altered hippocampal expression of glutamate receptors and transporters in GRM2 and GRM3 knockout mice.

    PubMed

    Lyon, Louisa; Kew, James N C; Corti, Corrado; Harrison, Paul J; Burnet, Philip W J

    2008-11-01

    Group II metabotropic glutamate receptors (mGluR2 and mGluR3, also called mGlu2 and mGlu3, encoded by GRM2 and GRM3, respectively) are therapeutic targets for several psychiatric disorders. GRM3 may also be a schizophrenia susceptibility gene. mGluR2-/- and mGluR3-/- mice provide the only unequivocal means to differentiate between these receptors, yet interpretation of in vivo findings may be complicated by secondary effects on expression of other genes. To address this issue, we examined the expression of NMDA receptor subunits (NR1, NR2A, NR2B) and glutamate transporters (EAAT1-3), as well as the remaining group II mGluR, in the hippocampus of mGluR2-/- and mGluR3-/- mice, compared with wild-type controls. mGluR2 mRNA was increased in mGluR3-/- mice, and vice versa. NR2A mRNA was increased in both knockout mice. EAAT1 (GLAST) mRNA and protein, and EAAT2 (GLT-1) protein, were reduced in mGluR3-/- mice, whereas EAAT3 (EAAC1) mRNA was decreased in mGluR2-/- mice. Transcripts for NR1 and NR2B were unchanged. The findings show a compensatory upregulation of the remaining group II metabotropic glutamate receptor in the knockout mice. Upregulation of NR2A expression suggests modified NMDA receptor signaling in mGluR2-/- and mGluR3-/- mice, and downregulation of glutamate transporter expression suggests a response to altered synaptic glutamate levels. The results show a mutual interplay between mGluR2 and mGluR3, and also provide a context in which to interpret behavioral and electrophysiological results in these mice. PMID:18720515

  8. Transplantation of Glial Progenitors That Overexpress Glutamate Transporter GLT1 Preserves Diaphragm Function Following Cervical SCI

    PubMed Central

    Li, Ke; Javed, Elham; Hala, Tamara J; Sannie, Daniel; Regan, Kathleen A; Maragakis, Nicholas J; Wright, Megan C; Poulsen, David J; Lepore, Angelo C

    2015-01-01

    Approximately half of traumatic spinal cord injury (SCI) cases affect cervical regions, resulting in chronic respiratory compromise. The majority of these injuries affect midcervical levels, the location of phrenic motor neurons (PMNs) that innervate the diaphragm. A valuable opportunity exists following SCI for preventing PMN loss that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxicity due to dysregulation of extracellular glutamate homeostasis. Astrocytes express glutamate transporter 1 (GLT1), which is responsible for the majority of CNS glutamate clearance. Given our observations of GLT1 dysfunction post-SCI, we evaluated intraspinal transplantation of Glial-Restricted Precursors (GRPs)—a class of lineage-restricted astrocyte progenitors—into ventral horn following cervical hemicontusion as a novel strategy for reconstituting GLT1 function, preventing excitotoxicity and protecting PMNs in the acutely injured spinal cord. We find that unmodified transplants express low levels of GLT1 in the injured spinal cord. To enhance their therapeutic properties, we engineered GRPs with AAV8 to overexpress GLT1 only in astrocytes using the GFA2 promoter, resulting in significantly increased GLT1 protein expression and functional glutamate uptake following astrocyte differentiation in vitro and after transplantation into C4 hemicontusion. Compared to medium-only control and unmodified GRPs, GLT1-overexpressing transplants reduced lesion size, diaphragm denervation and diaphragm dysfunction. Our findings demonstrate transplantation-based replacement of astrocyte GLT1 is a promising approach for SCI. PMID:25492561

  9. A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility.

    PubMed

    Siksou, Léa; Silm, Kätlin; Biesemann, Christoph; Nehring, Ralf B; Wojcik, Sonja M; Triller, Antoine; El Mestikawy, Salah; Marty, Serge; Herzog, Etienne

    2013-05-01

    Synaptic vesicles (SVs) from excitatory synapses carry vesicular glutamate transporters (VGLUTs) that fill the vesicles with neurotransmitter. Although the essential function of VGLUTs as glutamate transporters has been well established, the evidence for additional cell-biological functions is more controversial. Both VGLUT1 and VGLUT2 disruptions in mice result in a reduced number of SVs away from release sites, flattening of SVs, and the appearance of tubular structures. Therefore, we analysed the morphology, biochemical composition and trafficking of SVs at synapses of VGLUT1(-/-) mice in order to test for a function of VGLUTs in the formation or clustering of SVs. Analyses with high-pressure freezing immobilisation and electron tomography pointed to a role of VGLUT1 transport function in the tonicity of excitatory SVs, explaining the aldehyde-induced flattening of SVs observed in VGLUT1(-/-) synapses. We confirmed the steep reduction in the number of SVs previously observed in VGLUT1(-/-) presynaptic terminals, but did not observe accumulation of endocytotic intermediates. Furthermore, SV proteins of adult VGLUT1(-/-) mouse brain tissue were expressed at normal levels in all subcellular fractions, suggesting that they were not displaced to another organelle. We thus assessed the mobility of the recently documented superpool of SVs. Synaptobrevin2-enhanced green fluorescent protein time lapse experiments revealed an oversized superpool of SVs in VGLUT1(-/-) neurons. Our results support the idea that, beyond glutamate loading, VGLUT1 enhances the tonicity of excitatory SVs and stabilises SVs at presynaptic terminals. PMID:23581566

  10. Efficient production of gamma-aminobutyric acid using Escherichia coli by co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter.

    PubMed

    Dung Pham, Van; Somasundaram, Sivachandiran; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2016-01-01

    Gamma-aminobutyric acid (GABA) is an important bio-product, which is used in pharmaceutical formulations, nutritional supplements, and biopolymer monomer. The traditional GABA process involves the decarboxylation of glutamate. However, the direct production of GABA from glucose is a more efficient process. To construct the recombinant strains of Escherichia coli, a novel synthetic scaffold was introduced. By carrying out the co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter, we redirected the TCA cycle flux to GABA pathway. The genetically engineered E. coli strain produced 1.08 g/L of GABA from 10 g/L of initial glucose. Thus, with the introduction of a synthetic scaffold, we increased GABA production by 2.2-fold. The final GABA concentration was increased by 21.8% by inactivating competing pathways.

  11. Distribution of Vesicular Glutamate Transporter 2 and Ionotropic Glutamate Receptors in the Auditory Ganglion and Cochlear Nuclei of Pigeons (Columba livia).

    PubMed

    Karim, M R; Atoji, Y

    2016-02-01

    Glutamate is a principal excitatory neurotransmitter in the auditory system. Our previous studies revealed localization of glutamate receptor mRNAs in the pigeon cochlear nuclei, suggesting the existence of glutamatergic input from the auditory nerve to the brainstem. This study demonstrated localization of mRNAs for vesicular glutamate transporter 2 (vGluT2) and ionotropic glutamate receptors (AMPA, kainate and NMDA) in the auditory ganglion (AG) and cochlear nuclei (magnocellular, angular and laminar nuclei). VGluT2 mRNA was intensely expressed in AG and intensely or moderately in the cochlear nuclei. The AG and cochlear nuclei showed intense-to-moderate mRNA signals for GluA2, GluA3, GluA4, GluK4 and GluN1. These results suggest that the pigeon AG neurons receives glutamatergic input from hair cells and in turn projects to the magnocellular and angular nuclei. Glutamate may play a pivotal role in the excitatory synapse transmission in the peripheral auditory pathway of birds.

  12. Abnormal Expression of Glutamate Transporter and Transporter Interacting Molecules in Prefrontal Cortex in Elderly Patients with Schizophrenia

    PubMed Central

    Bauer, Deborah; Gupta, Daya; Harotunian, Vahram; Meador-Woodruff, James H.; McCullumsmith, Robert E.

    2008-01-01

    Glutamate cycling is critically important for neurotransmission, and may be altered in schizophrenia. The excitatory amino acid transporters (EAATs) facilitate the reuptake of glutamate from the synaptic cleft and have a key role in glutamate cycling. We hypothesized that expression of the EAATs and the EAAT regulating proteins ARHGEF11, JWA, G protein suppressor pathway 1 (GPS1), and KIAA0302 are altered in the brain in schizophrenia. To test this, we measured expression of EAAT1, EAAT2, EAAT3, and EAAT interacting proteins in postmortem tissue from the dorsolateral prefrontal and anterior cingulate cortex of patients with schizophrenia and a comparison group using in situ hybridization and Western blot analysis. We found increased EAAT1 transcripts and decreased protein expression, increased EAAT3 transcripts and protein, and elevated protein expression of both GPS1 and KIAA0302 protein. We did not find any changes in expression of EAAT2. These data indicate that proteins involved in glutamate reuptake and cycling are altered in the cortex in schizophrenia, and may provide potential targets for future treatment strategies. PMID:18678470

  13. Pharmacological evaluation of glutamate transporter 1 (GLT-1) mediated neuroprotection following cerebral ischemia/reperfusion injury.

    PubMed

    Verma, Rajkumar; Mishra, Vikas; Sasmal, Dinakar; Raghubir, Ram

    2010-07-25

    Recently glutamate transporters have emerged as a potential therapeutic target in a wide range of acute and chronic neurological disorders, owing to their novel mode of action. The modulation of GLT-1, a major glutamate transporter has been shown to exert neuroprotection in various models of ischemic injury and motoneuron degeneration. Therefore, an attempt was made to explore its neuroprotective potential in cerebral ischemia/reperfusion injury using ceftriaxone, a GLT-1 modulator. Pre-treatment with ceftriaxone (100mg/kg. i.v) for five days resulted in a significant reduction (P<0.01) in neurological deficit as well as cerebral infarct volume after 1h of ischemia followed by 24h of reperfusion injury. It also caused a significant (P<0.05) upregulation of GLT-1 mRNA, protein and glutamine synthetase (GS) activity. Furthermore, inhibition of ceftriaxone-mediated increased glutamine synthetase activity by dihydrokainate (DHK), a GLT-1 specific inhibitor, confirms the specific effect of ceftriaxone on GLT-1 activity. In addition, ceftriaxone also induced a significant (P<0.01) increase in [(3)H]-glutamate uptake, mediated by GLT-1 in glial enriched preparation, as evidenced by use of DHK and DL-threo-beta-benzyloxyaspartate (DL-TBOA). Thus, the present study provides overwhelming evidence that modulation of GLT-1 protein expression and activity confers neuroprotection in cerebral ischemia/reperfusion injury.

  14. Transcriptional profiles for glutamate transporters reveal differences between organophosphates but similarities with unrelated neurotoxicants.

    PubMed

    Slotkin, Theodore A; Lobner, Doug; Seidler, Frederic J

    2010-08-30

    The developmental neurotoxicity of organophosphates involves mechanisms other than their shared property as cholinesterase inhibitors, among which are excitotoxicity and oxidative stress. We used PC12 cells as a neurodevelopmental model to compare the effects of chlorpyrifos and diazinon on the expression of genes encoding glutamate transporters. Chlorpyrifos had a greater effect in cells undergoing nerve growth factor-induced neurodifferentiation as compared to undifferentiated PC12 cells, with peak sensitivity at the initiation of differentiation, reflecting a global upregulation of all the glutamate transporter genes expressed in this cell line. In differentiating cells, chlorpyrifos had a significantly greater effect than did diazinon and concordance analysis indicated no resemblance in their expression patterns. At the same time, the smaller effects of diazinon were highly concordant with those of an organochlorine pesticide (dieldrin) and a metal (divalent nickel). We also performed similar evaluations for the cystine/glutamate exchanger, which provides protection against oxidative stress by moving cystine into the cell; again, chlorpyrifos had the greatest effect, in this case reducing expression in undifferentiated and differentiating cells. Our results point to excitotoxicity and oxidative stress as major contributors to the noncholinesterase mechanisms that distinguish the neurodevelopmental outcomes between different organophosphates while providing a means whereby apparently unrelated neurotoxicants may produce similar outcomes. PMID:20600679

  15. Loss-of-function mutations in the glutamate transporter SLC1A1 cause human dicarboxylic aminoaciduria

    PubMed Central

    Bailey, Charles G.; Ryan, Renae M.; Thoeng, Annora D.; Ng, Cynthia; King, Kara; Vanslambrouck, Jessica M.; Auray-Blais, Christiane; Vandenberg, Robert J.; Bröer, Stefan; Rasko, John E.J.

    2010-01-01

    Solute carrier family 1, member 1 (SLC1A1; also known as EAAT3 and EAAC1) is the major epithelial transporter of glutamate and aspartate in the kidneys and intestines of rodents. Within the brain, SLC1A1 serves as the predominant neuronal glutamate transporter and buffers the synaptic release of the excitatory neurotransmitter glutamate within the interneuronal synaptic cleft. Recent studies have also revealed that polymorphisms in SLC1A1 are associated with obsessive-compulsive disorder (OCD) in early-onset patient cohorts. Here we report that SLC1A1 mutations leading to substitution of arginine to tryptophan at position 445 (R445W) and deletion of isoleucine at position 395 (I395del) cause human dicarboxylic aminoaciduria, an autosomal recessive disorder of urinary glutamate and aspartate transport that can be associated with mental retardation. These mutations of conserved residues impeded or abrogated glutamate and cysteine transport by SLC1A1 and led to near-absent surface expression in a canine kidney cell line. These findings provide evidence that SLC1A1 is the major renal transporter of glutamate and aspartate in humans and implicate SLC1A1 in the pathogenesis of some neurological disorders. PMID:21123949

  16. Protonation of a Glutamate Residue Modulates the Dynamics of the Drug Transporter EmrE

    PubMed Central

    Gayen, Anindita; Leninger, Maureen; Traaseth, Nathaniel J.

    2015-01-01

    Secondary active transport proteins play a central role in conferring bacterial multidrug resistance. In this work, we investigated the proton-coupled transport mechanism for the Escherichia coli drug efflux pump EmrE using nuclear magnetic resonance (NMR) spectroscopy. Our results show that the global conformational motions necessary for transport are modulated in an allosteric fashion by the protonation state of a membrane-embedded glutamate residue. These observations directly correlate with the resistance phenotype for EmrE and the E14D mutant as a function of pH. Furthermore, our results support a model in which the pH gradient across the inner membrane of E. coli may be used on a mechanistic level to shift the equilibrium of the transporter in favor of an inward-open resting conformation poised for drug binding. PMID:26751516

  17. Astrocytes and glutamate homoeostasis in Alzheimer's disease: a decrease in glutamine synthetase, but not in glutamate transporter-1, in the prefrontal cortex.

    PubMed

    Kulijewicz-Nawrot, Magdalena; Syková, Eva; Chvátal, Alexander; Verkhratsky, Alexei; Rodríguez, José J

    2013-10-07

    Astrocytes control tissue equilibrium and hence define the homoeostasis and function of the CNS (central nervous system). Being principal homoeostatic cells, astroglia are fundamental for various forms of neuropathology, including AD (Alzheimer's disease). AD is a progressive neurodegenerative disorder characterized by the loss of cognitive functions due to specific lesions in mnesic-associated regions, including the mPFC (medial prefrontal cortex). Here, we analyzed the expression of GS (glutamine synthetase) and GLT-1 (glutamate transporter-1) in astrocytes in the mPFC during the progression of AD in a triple-transgenic mouse model (3xTg-AD). GS is an astrocyte-specific enzyme, responsible for the intracellular conversion of glutamate into glutamine, whereas the removal of glutamate from the extracellular space is accomplished mainly by astroglia-specific GLT-1. We found a significant decrease in the numerical density (Nv, cells/mm3) of GS-positive astrocytes from early to middle ages (1-9 months; at the age of 1 month by 17%, 6 months by 27% and 9 months by 27% when compared with control animals) in parallel with a reduced expression of GS (determined by Western blots), which started at the age of 6 months and was sustained up to 12 months of age. We did not, however, find any changes in the expression of GLT-1, which implies an intact glutamate uptake mechanism. Our results indicate that the decrease in GS expression may underlie a gradual decline in the vital astrocyte-dependent glutamate-glutamine conversion pathway, which in turn may compromise glutamate homoeostasis, leading towards failures in synaptic connectivity with deficient cognition and memory.

  18. Inducible expression and pharmacology of the human excitatory amino acid transporter 2 subtype of L-glutamate transporter.

    PubMed

    Dunlop, J; Lou, Z; Zhang, Y; McIlvain, H B

    1999-12-01

    1. In this study we have examined the use of the ecdysone-inducible mammalian expression system (Invitrogen) for the regulation of expression of the predominant L-glutamate transporter EAAT2 (Excitatory Amino Acid Transporter) in HEK 293 cells. 2. HEK 293 cells which were stably transformed with the regulatory vector pVgRXR (EcR 293 cells) were used for transfection of the human EAAT2 cDNA using the inducible vector pIND and a clone designated HEK/EAAT2 was selected for further characterization. 3. Na+-dependent L-glutamate uptake activity (3.2 pmol min-1 mg-1) was observed in EcR 293 cells and this was increased approximately 2 fold in the uninduced HEK/EAAT2 cells, indicating a low level of basal EAAT2 activity in the absence of exogenous inducing agent. Exposure of HEK/EAAT2 cells to the ecdysone analogue Ponasterone A (10 microM for 24 h) resulted in a > or = 10 fold increase in the Na+-dependent activity. 4. L-glutamate uptake into induced HEK/EAAT2 cells followed first-order Michaelis-Menten kinetics and Eadie-Hofstee transformation of the saturable uptake data produced estimates of kinetic parameters as follows; Km 52.7+/-7.5 microM, Vmax 3.8+/-0.9 nmol min-1 mg-1 protein. 5. The pharmacological profile of the EAAT2 subtype was characterized using a series of L-glutamate transport inhibitors and the rank order of inhibitory potency was similar to that described previously for the rat homologue GLT-1 and in synaptosomal preparations from rat cortex. 6. Addition of the EAAT2 modulator arachidonic acid resulted in an enhancement (155+/-5% control in the presence of 30 microM) of the L-glutamate transport capacity in the induced HEK/EAAT2 cells. 7. This study demonstrates that the expression of EAAT2 can be regulated in a mammalian cell line using the ecdysone-inducible mammalian expression system.

  19. Gabapentin inhibits the activity of the rat excitatory glutamate transporter 3 expressed in Xenopus oocytes.

    PubMed

    Gil, Yang Sook; Kim, Jong Hak; Kim, Chi Hyo; Han, Jong In; Zuo, Zhiyi; Baik, Hee Jung

    2015-09-01

    Gabapentin, a derivative of γ-aminobutyric acid (GABA), is used to treat epilepsy and neuropathic pain. The pharmacological mechanisms for gabapentin effects are not completely elucidated. We investigated the effect of gabapentin on the activity of excitatory amino acid transporter 3 (EAAT3) that can regulate extracellular glutamate concentrations. EAAT3 was expressed in Xenopus oocytes. Membrane currents were recorded after application of l-glutamate in the presence or absence of different concentrations of gabapentin (1-300μM) by using a two-electrode voltage clamp. To determine the effect of gabapentin on Vmax and Km of EAAT3 for l-glutamate, l-glutamate at 3-300μM was used. To study the effects of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) on gabapentin-induced changes in EAAT3 activity, oocytes were incubated with the PKC activator (Phorbol 12-myristate 13-acetate, PMA), the PKC inhibitors (chelerythrine or staurosporine), and the PI3K inhibitor wortmannin. Gabapentin decreased EAAT3 activity in a concentration-dependent manner and EAAT3 activity was significantly inhibited by 10-300μM gabapentin. Gabapentin significantly decreased Vmax without affecting Km. PMA increased EAAT3 activity; however, gabapentin attenuated the PMA-induced increase in EAAT3 activity. Pre-incubation of oocytes with chelerythrine, staurosporine, or wortmannin decreased basal EAAT3 activity, which was further reduced by gabapentin. We conclude that gabapentin decreases EAAT3 activity at clinically relevant and higher concentrations, in which PKC and PI3K may not be involved. The results suggest that EAAT3 might not be a target for the anticonvulsant action of gabapentin.

  20. 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes.

    PubMed

    Keller, J N; Mark, R J; Bruce, A J; Blanc, E; Rothstein, J D; Uchida, K; Waeg, G; Mattson, M P

    1997-10-01

    Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimer's, Parkinson's and Huntington's diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4

  1. Inhibition of vesicular glutamate transporters contributes to attenuate methamphetamine-induced conditioned place preference in rats.

    PubMed

    He, Zongsheng; Chen, Yuan; Dong, Huajin; Su, Ruibin; Gong, Zehui; Yan, Lingdi

    2014-07-01

    Accumulating evidence suggests that glutamatergic system plays a crucial role in methamphetamine (METH) addiction. In the glutamatergic transmission, vesicular glutamate transporters (VGLUTs) are responsible for transporting glutamate into synaptic vesicles and affect the glutamate concentrations in the synaptic cleft. It is well documented that VGLUTs play an essential role in pathophysiology of several psychiatric and neurological diseases, however, whether VGLUTs also have a role in addiction caused by psychostimulant drugs is still unknown. The present study was underwent to investigate the effect of inhibition of VGLUTs on METH-induced induce conditioned place preference in rats. Rats were induced to conditioned place preference with METH (0.5, 1.0 and 2.0mg/kg) by intraperitoneal injection. Intracerebroventricular administration of 1.0 or 5.0μg Chicago sky blue 6B (CSB6B), a VGLUTs inhibitor, and 2.5h prior to METH was to observe its effect on METH-induced conditioned place preference in rats. The rats receiving METH showed stronger place preference at the dose of 1.0mg/kg than that of other doses. The intracerebroventricular administration of CSB6B (1.0, 5.0μg) 2.5h prior to the exposure to METH attenuated the acquisition of METH-induced conditioned place preference, while CSB6B itself had no effect on place preference. These results indicate that VGLUTs are involved in the effect of METH-induced conditioned place preference and may be a new target against METH addiction.

  2. Inhibition of vesicular glutamate transporters contributes to attenuate methamphetamine-induced conditioned place preference in rats.

    PubMed

    He, Zongsheng; Chen, Yuan; Dong, Huajin; Su, Ruibin; Gong, Zehui; Yan, Lingdi

    2014-07-01

    Accumulating evidence suggests that glutamatergic system plays a crucial role in methamphetamine (METH) addiction. In the glutamatergic transmission, vesicular glutamate transporters (VGLUTs) are responsible for transporting glutamate into synaptic vesicles and affect the glutamate concentrations in the synaptic cleft. It is well documented that VGLUTs play an essential role in pathophysiology of several psychiatric and neurological diseases, however, whether VGLUTs also have a role in addiction caused by psychostimulant drugs is still unknown. The present study was underwent to investigate the effect of inhibition of VGLUTs on METH-induced induce conditioned place preference in rats. Rats were induced to conditioned place preference with METH (0.5, 1.0 and 2.0mg/kg) by intraperitoneal injection. Intracerebroventricular administration of 1.0 or 5.0μg Chicago sky blue 6B (CSB6B), a VGLUTs inhibitor, and 2.5h prior to METH was to observe its effect on METH-induced conditioned place preference in rats. The rats receiving METH showed stronger place preference at the dose of 1.0mg/kg than that of other doses. The intracerebroventricular administration of CSB6B (1.0, 5.0μg) 2.5h prior to the exposure to METH attenuated the acquisition of METH-induced conditioned place preference, while CSB6B itself had no effect on place preference. These results indicate that VGLUTs are involved in the effect of METH-induced conditioned place preference and may be a new target against METH addiction. PMID:24613241

  3. Neuronal activity mediated regulation of glutamate transporter GLT‐1 surface diffusion in rat astrocytes in dissociated and slice cultures

    PubMed Central

    Al Awabdh, Sana; Gupta‐Agarwal, Swati; Sheehan, David F.; Muir, James; Norkett, Rosalind; Twelvetrees, Alison E.; Griffin, Lewis D.

    2016-01-01

    The astrocytic GLT‐1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live‐cell imaging to study the mechanisms regulating GLT‐1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP‐time lapse imaging, we show that GLT‐1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity‐dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT‐1 is more stable than diffuse GLT‐1 and that glutamate increases GLT‐1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT‐1 isoforms expressed in the brain, GLT‐1a and GLT‐1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT‐1b more so. GLT‐1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT‐1 isoforms. Altogether, these data reveal that astrocytic GLT‐1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252–1264 PMID:27189737

  4. Kinetic properties of a phosphate-bond-driven glutamate-glutamine transport system in Streptococcus lactis and Streptococcus cremoris.

    PubMed

    Poolman, B; Smid, E J; Konings, W N

    1987-06-01

    In Streptococcus lactis ML3 and Streptococcus cremoris Wg2 the uptake of glutamate and glutamine is mediated by the same transport system, which has a 30-fold higher affinity for glutamine than for glutamate at pH 6.0. The apparent affinity constant for transport (KT) of glutamine is 2.5 +/- 0.3 microM, independent of the extracellular pH. The KTS for glutamate uptake are 3.5, 11.2, 77, and 1200 microM at pH 4.0, 5.1, 6.0, and 7.0, respectively. Recalculation of the affinity constants based on the concentration of glutamic acid in the solution yield KTS of 1.8 +/- 0.5 microM independent of the external pH, indicating that the protonated form of glutamate, i.e., glutamic acid, and glutamine are the transported species. The maximal rates of glutamate and glutamine uptake are independent of the extracellular pH as long as the intracellular pH is kept constant, despite large differences in the magnitude and composition of the components of the proton motive force. Uptake of glutamate and glutamine requires the synthesis of ATP either from glycolysis or from arginine metabolism and appears to be essentially unidirectional. Cells are able to maintain glutamate concentration gradients exceeding 4 X 10(3) for several hours even in the absence of metabolic energy. The t1/2s of glutamate efflux are 2, 12, and greater than 30 h at pH 5.0, 6.0, and 7.0, respectively. After the addition of lactose as energy source, the rate of glutamine uptake and the level of ATP are both very sensitive to arsenate. When the intracellular pH is kept constant, both parameters decrease approximately in parallel (between 0.2 and 1.0 mM ATP) with increasing concentrations of the inhibitor. These results suggest that the accumulation of glutamate and glutamine is energized by ATP or an equivalent energy-rich phosphorylated intermediate and not by the the proton motive force.

  5. Neural Stem Cell Transplantation Induces Stroke Recovery by Upregulating Glutamate Transporter GLT-1 in Astrocytes

    PubMed Central

    Russo, Gianluca Luigi; Peruzzotti-Jametti, Luca; Rossi, Silvia; Sandrone, Stefano; Butti, Erica; De Ceglia, Roberta; Bergamaschi, Andrea; Motta, Caterina; Gallizioli, Mattia; Studer, Valeria; Colombo, Emanuela; Farina, Cinthia; Comi, Giancarlo; Politi, Letterio Salvatore; Muzio, Luca; Villani, Claudia; Invernizzi, Roberto William; Hermann, Dirk Matthias; Centonze, Diego

    2016-01-01

    Ischemic stroke is the leading cause of disability, but effective therapies are currently widely lacking. Recovery from stroke is very much dependent on the possibility to develop treatments able to both halt the neurodegenerative process as well as to foster adaptive tissue plasticity. Here we show that ischemic mice treated with neural precursor cell (NPC) transplantation had on neurophysiological analysis, early after treatment, reduced presynaptic release of glutamate within the ipsilesional corticospinal tract (CST), and an enhanced NMDA-mediated excitatory transmission in the contralesional CST. Concurrently, NPC-treated mice displayed a reduced CST degeneration, increased axonal rewiring, and augmented dendritic arborization, resulting in long-term functional amelioration persisting up to 60 d after ischemia. The enhanced functional and structural plasticity relied on the capacity of transplanted NPCs to localize in the peri-ischemic and ischemic area, to promote the upregulation of the glial glutamate transporter 1 (GLT-1) on astrocytes and to reduce peri-ischemic extracellular glutamate. The upregulation of GLT-1 induced by transplanted NPCs was found to rely on the secretion of VEGF by NPCs. Blocking VEGF during the first week after stroke reduced GLT-1 upregulation as well as long-term behavioral recovery in NPC-treated mice. Our results show that NPC transplantation, by modulating the excitatory–inhibitory balance and stroke microenvironment, is a promising therapy to ameliorate disability, to promote tissue recovery and plasticity processes after stroke. SIGNIFICANCE STATEMENT Tissue damage and loss of function occurring after stroke can be constrained by fostering plasticity processes of the brain. Over the past years, stem cell transplantation for repair of the CNS has received increasing interest, although underlying mechanism remain elusive. We here show that neural stem/precursor cell transplantation after ischemic stroke is able to foster

  6. Centrifuge-induced hypergravity: [ 3H]GABA and L-[ 14C]glutamate uptake, exocytosis and efflux mediated by high-affinity, sodium-dependent transporters

    NASA Astrophysics Data System (ADS)

    Borisova, T. A.; Himmelreich, N. H.

    The effects of centrifuge-induced hypergravity on the presynaptic events have been investigated in order to provide further insight into regulation of glutamate and GABA neurotransmission and correlation between excitatory and inhibitory responses under artificial gravity conditions. Exposure of animals to hypergravity (centrifugation of rats at 10 G for 1 h) has been found to cause changes in the synaptic processes of brain, in particular neurotransmitter release and uptake in rat brain synaptosomes. Hypergravity loading resulted in more than two-fold enhancement of GABA transporter activity ( Vmax increased from 1.4 ± 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for the animals exposed to hypergravity ( P ⩽ 0.05)). The maximal velocity of L-[ 14C]glutamate uptake decreased from 12.5 ± 3.2 to 5.6 ± 0.9 nmol/min/mg of protein under artificial gravity conditions. Depolarization-evoked exocytotic release of the neurotransmitters has also changed in response to hypergravity. It increased for GABA (7.2 ± 0.54% and 11.74 ± 1.2% of total accumulated label for control and hypergravity, respectively ( P ⩽ 0.05)), but reduced for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%, for control and hypergravity, respectively). Thus, comparative analysis of the neurotransmitter uptake and release has demonstrated that short-term centrifuge-induced 10 G hypergravity loading intensified inhibitory and attenuated excitatory processes in nerve terminals. The activation or reduction of neurotransmitter uptake appeared to be coupled with similarly directed alterations of the neurotransmitter release.

  7. Vesicular glutamate transporter 2 is required for the respiratory and parasympathetic activation produced by optogenetic stimulation of catecholaminergic neurons in the rostral ventrolateral medulla of mice in vivo.

    PubMed

    Abbott, Stephen B G; Holloway, Benjamin B; Viar, Kenneth E; Guyenet, Patrice G

    2014-01-01

    Catecholaminergic neurons of the rostral ventrolateral medulla (RVLM-CA neurons; C1 neurons) contribute to the sympathetic, parasympathetic and neuroendocrine responses elicited by physical stressors such as hypotension, hypoxia, hypoglycemia, and infection. Most RVLM-CA neurons express vesicular glutamate transporter (VGLUT)2, and may use glutamate as a ionotropic transmitter, but the importance of this mode of transmission in vivo is uncertain. To address this question, we genetically deleted VGLUT2 from dopamine-β-hydroxylase-expressing neurons in mice [DβH(Cre/0) ;VGLUT2(flox/flox) mice (cKO mice)]. We compared the in vivo effects of selectively stimulating RVLM-CA neurons in cKO vs. control mice (DβH(Cre/0) ), using channelrhodopsin-2 (ChR2-mCherry) optogenetics. ChR2-mCherry was expressed by similar numbers of rostral ventrolateral medulla (RVLM) neurons in each strain (~400 neurons), with identical selectivity for catecholaminergic neurons (90-99% colocalisation with tyrosine hydroxylase). RVLM-CA neurons had similar morphology and axonal projections in DβH(Cre/0) and cKO mice. Under urethane anesthesia, photostimulation produced a similar pattern of activation of presumptive ChR2-positive RVLM-CA neurons in DβH(Cre/0) and cKO mice. Photostimulation in conscious mice produced frequency-dependent respiratory activation in DβH(Cre/0) mice but no effect in cKO mice. Similarly, photostimulation under urethane anesthesia strongly activated efferent vagal nerve activity in DβH(Cre/0) mice only. Vagal responses were unaffected by α1 -adrenoreceptor blockade. In conclusion, two responses evoked by RVLM-CA neuron stimulation in vivo require the expression of VGLUT2 by these neurons, suggesting that the acute autonomic responses driven by RVLM-CA neurons are mediated by glutamate.

  8. Cystathionine is a novel substrate of cystine/glutamate transporter: implications for immune function.

    PubMed

    Kobayashi, Sho; Sato, Mami; Kasakoshi, Takayuki; Tsutsui, Takumi; Sugimoto, Masahiro; Osaki, Mitsuhiko; Okada, Futoshi; Igarashi, Kiharu; Hiratake, Jun; Homma, Takujiro; Conrad, Marcus; Fujii, Junichi; Soga, Tomoyoshi; Bannai, Shiro; Sato, Hideyo

    2015-04-01

    The cystine/glutamate transporter, designated as system xc(-), is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system xc(-), xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues, such as the thymus and spleen. Although cystine and glutamate are the well established substrates of system xc(-) and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analyzed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged as being absent specifically in the thymus and spleen of xCT-deficient mice. No expression of either cystathionine β-synthase or cystathionine γ-lyase was observed in the thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knock-out cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation. We thus conclude that cystathionine is a novel physiological substrate of system xc(-) and that the accumulation of cystathionine in immune tissues is exclusively mediated by system xc(-).

  9. Sumoylation of the astroglial glutamate transporter EAAT2 governs its intracellular compartmentalization

    PubMed Central

    Foran, E.; Rosenblum, L.; Bogush, A.; Pasinelli, P.; Trotti, D.

    2014-01-01

    EAAT2 is a predominantly astroglial glutamate transporter responsible for the majority of synaptic glutamate clearance in the mammalian CNS. Its dysfunction has been linked to many neurological disorders, including amyotrophic lateral sclerosis (ALS). Decreases in EAAT2 expression and function have been implicated in causing motor neuron excitotoxic death in ALS. Nevertheless, increasing EAAT2 expression does not significantly improve ALS phenotype in mouse models or in clinical trials. In the SOD1-G93A mouse model of inherited ALS, the cytosolic carboxy-terminal domain is cleaved from EAAT2, conjugated to SUMO1, and accumulated in astrocytes where it triggers astrocyte-mediated neurotoxic effects as disease progresses. However, it is not known whether this fragment is sumoylated after cleavage or if full-length EAAT2 is already sumoylated prior to cleavage as part of physiological regulation. In this study, we show that a fraction of full-length EAAT2 is constitutively sumoylated in primary cultures of astrocytes in vitro and in the CNS in vivo. Furthermore, the extent of sumoylation of EAAT2 does not change during the course of ALS in the SOD1-G93A mouse and is not affected by the expression of ALS-causative mutant SOD1 proteins in astrocytes in vitro, indicating that EAAT2 sumoylation is not driven by pathogenic mechanisms. Most interestingly, sumoylated EAAT2 localizes to intracellular compartments, while non-sumoylated EAAT2 resides on the plasma membrane. In agreement, promoting desumoylation in primary astrocytes causes increased EAAT2–mediated glutamate uptake. These findings could have implications for optimizing therapeutic approaches aimed at increasing EAAT2 activity in the dysfunctional or diseased CNS. PMID:24753081

  10. Analogies between respiration and a light-driven proton pump as sources of energy for active glutamate transport in Halobacterium halobium

    NASA Technical Reports Server (NTRS)

    Belliveau, J. W.; Lanyi, J. K.

    1977-01-01

    Halobacterium halobium is known to contain sheets of bacteriorhodopsin, a pigment which upon exposure to light undergoes cyclic protonation and deprotonation, resulting in net H(+) translocation. In this paper, experiments were conducted to test H. halobium cell envelope vesicles for respiration-induced glutamate uptake. It is shown that glutamate transport in H. halobium cell envelope vesicles can occur as a result of respiration, as well as light acting on bacteriorhodopsin. Glutamate transport can be energized by the oxidation of dimethyl phenylenediamine, and the properties of the transport system are entirely analogous to those observed with illumination as the source of energy. In the case of respiration-dependent glutamate transport, the transportation is also driven by a Na(+) gradient, thereby confirming the existence of a single glutamate transport system independent of the source of energy. The analogy observed is indirect evidence that the cytochrome oxidase of H. halobium functions as a H(+) pump.

  11. Transport rates of a glutamate transporter homologue are influenced by the lipid bilayer.

    PubMed

    McIlwain, Benjamin C; Vandenberg, Robert J; Ryan, Renae M

    2015-04-10

    The aspartate transporter from Pyrococcus horikoshii (GltPh) is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of GltPh provide insight into mechanisms of ion coupling and substrate transport; however, structures have been solved in the absence of a lipid bilayer so they provide limited information regarding interactions that occur between the protein and lipids of the membrane. Here, we investigated the effect of the lipid environment on aspartate transport by reconstituting GltPh into liposomes of defined lipid composition where the primary lipid is phosphatidylethanolamine (PE) or its methyl derivatives. We showed that the rate of aspartate transport and the transmembrane orientation of GltPh were influenced by the primary lipid in the liposomes. In PE liposomes, we observed the highest transport rate and showed that 85% of the transporters were orientated right-side out, whereas in trimethyl PE liposomes, 50% of transporters were right-side out, and we observed a 4-fold reduction in transport rate. Differences in orientation can only partially explain the lipid composition effect on transport rate. Crystal structures of GltPh revealed a tyrosine residue (Tyr-33) that we propose interacts with lipid headgroups during the transport cycle. Based on site-directed mutagenesis, we propose that a cation-π interaction between Tyr-33 and the lipid headgroups can influence conformational flexibility of the trimerization domain and thus the rate of transport. These results provide a specific example of how interactions between membrane lipids and membrane-bound proteins can influence function and highlight the importance of the role of the membrane in transporter function.

  12. Conformationally-restricted amino acid analogues bearing a distal sulfonic acid show selective inhibition of system x(c)(-) over the vesicular glutamate transporter.

    PubMed

    Etoga, Jean-Louis G; Ahmed, S Kaleem; Patel, Sarjubhai; Bridges, Richard J; Thompson, Charles M

    2010-04-15

    A panel of amino acid analogs and conformationally-restricted amino acids bearing a sulfonic acid were synthesized and tested for their ability to preferentially inhibit the obligate cysteine-glutamate transporter system x(c)(-) versus the vesicular glutamate transporter (VGLUT). Several promising candidate molecules were identified: R/S-4-[4'-carboxyphenyl]-phenylglycine, a biphenyl substituted analog of 4-carboxyphenylglycine and 2-thiopheneglycine-5-sulfonic acid both of which reduced glutamate uptake at system x(c)(-) by 70-75% while having modest to no effect on glutamate uptake at VGLUT.

  13. Effect of environmental enrichment on dopamine and serotonin transporters and glutamate neurotransmission in medial prefrontal and orbitofrontal cortex.

    PubMed

    Darna, Mahesh; Beckmann, Joshua S; Gipson, Cassandra D; Bardo, Michael T; Dwoskin, Linda P

    2015-03-01

    Recent studies have reported that rats raised in an enriched condition (EC) have decreased dopamine transporter (DAT) function and expression in medial prefrontal cortex (mPFC), as well as increased d-amphetamine-induced glutamate release in nucleus accumbens compared to rats raised in an isolated condition (IC). In these previous studies, DAT function and expression were evaluated using mPFC pooled from four rats for each condition to obtain kinetic parameters due to sparse DAT expression in mPFC. In contrast, accumbal glutamate release was determined using individual rats. The current study extends the previous work and reports on the optimization of DAT and serotonin transporter (SERT) functional assays, as well as cell surface expression assays using both mPFC and orbitofrontal cortex (OFC) from individual EC or IC rats. In addition, the effect of d-amphetamine on glutamate release in mPFC and OFC of EC and IC rats was determined using in vivo microdialysis. Results show that environmental enrichment decreased maximal transport velocity (Vmax) for [(3)H]dopamine uptake in mPFC, but increased Vmax for [(3)H]dopamine uptake in OFC. Corresponding changes in DAT cell surface expression were not found. In contrast, Vmax for [(3)H]serotonin uptake and cellular localization of SERT in mPFC and OFC were not different between EC and IC rats. Further, acute d-amphetamine (2mg/kg, s.c.) increased extracellular glutamate concentrations in mPFC of EC rats only and in OFC of IC rats only. Overall, these results suggest that enrichment produces long-lasting alterations in mPFC and OFC DAT function via a trafficking-independent mechanism, as well as differential glutamate release in mPFC and OFC. Rearing-induced modulation of DAT function and glutamate release in prefrontal cortical subregions may contribute to the known protective effects of enrichment on drug abuse vulnerability.

  14. Diabetes Impairs Wnt3 Protein-induced Neurogenesis in Olfactory Bulbs via Glutamate Transporter 1 Inhibition.

    PubMed

    Wakabayashi, Tamami; Hidaka, Ryo; Fujimaki, Shin; Asashima, Makoto; Kuwabara, Tomoko

    2016-07-15

    Diabetes is associated with impaired cognitive function. Streptozotocin (STZ)-induced diabetic rats exhibit a loss of neurogenesis and deficits in behavioral tasks involving spatial learning and memory; thus, impaired adult hippocampal neurogenesis may contribute to diabetes-associated cognitive deficits. Recent studies have demonstrated that adult neurogenesis generally occurs in the dentate gyrus of the hippocampus, the subventricular zone, and the olfactory bulbs (OB) and is defective in patients with diabetes. We hypothesized that OB neurogenesis and associated behaviors would be affected in diabetes. In this study, we show that inhibition of Wnt3-induced neurogenesis in the OB causes several behavioral deficits in STZ-induced diabetic rats, including impaired odor discrimination, cognitive dysfunction, and increased anxiety. Notably, the sodium- and chloride-dependent GABA transporters and excitatory amino acid transporters that localize to GABAergic and glutamatergic terminals decreased in the OB of diabetic rats. Moreover, GAT1 inhibitor administration also hindered Wnt3-induced neurogenesis in vitro Collectively, these data suggest that STZ-induced diabetes adversely affects OB neurogenesis via GABA and glutamate transporter systems, leading to functional impairments in olfactory performance. PMID:27226528

  15. Role of transcription factor yin yang 1 in manganese-induced reduction of astrocytic glutamate transporters: Putative mechanism for manganese-induced neurotoxicity.

    PubMed

    Karki, Pratap; Smith, Keisha; Johnson, James; Aschner, Michael; Lee, Eunsook

    2015-09-01

    Astrocytes are the most abundant non-neuronal glial cells in the brain. Once relegated to a mere supportive role for neurons, contemporary dogmas ascribe multiple active roles for these cells in central nervous system (CNS) function, including maintenance of optimal glutamate levels in synapses. Regulation of glutamate levels in the synaptic cleft is crucial for preventing excitotoxic neuronal injury. Glutamate levels are regulated predominantly by two astrocytic glutamate transporters, glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST). Indeed, the dysregulation of these transporters has been linked to several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and Parkinson's disease (PD), as well as manganism, which is caused by overexposure to the trace metal, manganese (Mn). Although Mn is an essential trace element, its excessive accumulation in the brain as a result of chronic occupational or environmental exposures induces a neurological disorder referred to as manganism, which shares common pathological features with Parkinsonism. Mn decreases the expression and function of both GLAST and GLT-1. Astrocytes are commonly targeted by Mn, and thus reduction in astrocytic glutamate transporter function represents a critical mechanism of Mn-induced neurotoxicity. In this review, we will discuss the role of astrocytic glutamate transporters in neurodegenerative diseases and Mn-induced neurotoxicity.

  16. Role of transcription factor yin yang 1 in manganese-induced reduction of astrocytic glutamate transporters: putative mechanism for manganese-induced neurotoxicity

    PubMed Central

    Karki, Pratap; Smith, Keisha; Johnson, James; Aschner, Michael; Lee, Eunsook

    2014-01-01

    Astrocytes are the most abundant non-neuronal glial cells in the brain. Once relegated to a mere supportive role for neurons, contemporary dogmas ascribe multiple active roles for these cells in central nervous system (CNS) function, including maintenance of optimal glutamate levels in synapses. Regulation of glutamate levels in the synaptic cleft is crucial for preventing excitotoxic neuronal injury. Glutamate levels are regulated predominantly by two astrocytic glutamate transporters, glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST). Indeed, the dysregulation of these transporters has been linked to several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and Parkinson's disease (PD), as well as manganism, which is caused by overexposure to the trace metal, manganese (Mn). Although Mn is an essential trace element, its excessive accumulation in the brain as a result of chronic occupational or environmental exposures induces a neurological disorder referred to as manganism, which shares common pathological features with Parkinsonism. Mn decreases the expression and function of both GLAST and GLT-1.Astrocytes are commonly targeted by Mn, and thus reduction in astrocytic glutamate transporter function represents a critical mechanism of Mn-induced neurotoxicity. In this review, we will discuss the role of astrocytic glutamate transporters in neurodegenerative diseases and Mn-induced neurotoxicity. PMID:25128239

  17. Rapid Microelectrode Measurements and the Origin and Regulation of Extracellular Glutamate in Rat Prefrontal Cortex

    PubMed Central

    Hascup, E.R.; Hascup, K.N.; Stephens, M.; Pomerleau, F.; Huettl, P.; Gratton, A.; Gerhardt, G.A.

    2010-01-01

    Glutamate in the prefrontal cortex (PFC) plays a significant role in several mental illnesses, including schizophrenia, addiction and anxiety. Previous studies on PFC glutamate-mediated function have used techniques that raise questions on the neuronal vs. astrocytic origin of glutamate. The present studies used enzyme-based microelectrode arrays (MEAs) to monitor second-by-second resting glutamate levels in the PFC of awake rats. Locally-applied drugs were employed in an attempt to discriminate between the neuronal or glial components of the resting glutamate signal. Local application of tetrodotoxin (TTX; sodium channel blocker), produced a significant (~40%) decline in resting glutamate levels. In addition significant reductions in extracellular glutamate were seen with locally-applied ω-conotoxin (MVIIC; ~50%; calcium channel blocker), and the mGluR⅔ agonist, LY379268 (~20%), and a significant increase with the mGluR⅔ antagonist LY341495 (~40%), effects all consistent with a large neuronal contribution to the resting glutamate levels. Local administration of D,L-threo-β-benzyloxyaspartate (TBOA; glutamate transporter inhibitor) produced an ~120% increase in extracellular glutamate levels, supporting that excitatory amino acid transporters, which are largely located on glia, modulate clearance of extracellular glutamate. Interestingly, local application of (S)-4-carboxyphenylglycine (CPG; cystine/glutamate antiporter inhibitor), produced small, non-significant bi-phasic changes in extracellular glutamate versus vehicle control. Finally, pre-administration of TTX completely blocked the glutamate response to tail pinch stress. Taken together, these results support that PFC resting glutamate levels in rats as measured by the MEA technology are at least 40-50% derived from neurons. Furthermore, these data support that the impulse flow-dependent glutamate release from a physiologically-evoked event is entirely neuronally derived. PMID:20969570

  18. Effect of MS-153, a glutamate transporter activator, on the conditioned rewarding effects of morphine, methamphetamine and cocaine in mice.

    PubMed

    Nakagawa, Takayuki; Fujio, Mayumi; Ozawa, Tohru; Minami, Masabumi; Satoh, Masamichi

    2005-01-30

    There is a body of evidence implying the involvement of the glutamatergic system in the conditioned rewarding effects of drugs of abuse. It is recognized that the release of extracellular glutamate from nerve terminals is counterbalanced by the functions of neuronal and glial glutamate transporters. In the present study, we investigated the effects of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), a glutamate transporter activator, on the induction of the conditioned place preference to morphine, methamphetamine and cocaine in mice. In the conditioned place preference paradigm, mice were conditioned with repeated subcutaneous injections of morphine (5 mg/kg), methamphetamine (2 mg/kg) or cocaine (8 mg/kg) in combination with or without MS-153 (3 and 10 mg/kg). Co-administration of MS-153 at a dose of 10 mg/kg, but not 3 mg/kg, significantly attenuated the induction of conditioned place preference to morphine, methamphetamine and cocaine. However, MS-153 itself produced neither conditioned place preference nor aversion. On the other hand, co-administration of MS-153 (10 mg/kg) did not alter the acute locomotor activation elicited by a single injection of morphine, methamphetamine and cocaine. These results suggest that MS-153, a glutamate transporter activator, has an inhibitory effect on the conditioned rewarding effects of morphine, methamphetamine and cocaine without affecting their acute locomotor responses.

  19. Interaction of Peptide Transporter 1 With D-Glucose and L-Glutamic Acid; Possible Involvement of Taste Receptors.

    PubMed

    Arakawa, Hiroshi; Ohmachi, Taichi; Ichiba, Kiko; Kamioka, Hiroki; Tomono, Takumi; Kanagawa, Masahiko; Idota, Yoko; Hatano, Yasuko; Yano, Kentaro; Morimoto, Kaori; Ogihara, Takuo

    2016-01-01

    We investigated the influence of sweet and umami (savory) tastants on the intestinal absorption of cephalexin (CEX), a substrate of peptide transporter 1 (PEPT1, SLC15A1) in rats. After oral administration of glucose or mannitol to rats, CEX was administered together with a second dose of glucose or mannitol. Western blot analysis indicated that expression of PEPT1 in rat jejunum membrane was decreased by glucose, compared to mannitol. Furthermore, the maximum plasma concentration (Cmax) of orally administered CEX was reduced by glucose compared to mannitol. The effect of glucose was diminished by nifedipine, a L-type Ca(2+) channel blocker. We also found that Cmax of orally administered CEX was reduced by treatment with L-glutamic acid, compared to D-glutamic acid. Thus, excessive intake of glucose and L-glutamic acid may impair oral absorption of PEPT1 substrates. PMID:26852864

  20. Cysteine mutagenesis reveals alternate proximity between transmembrane domain 2 and hairpin loop 1 of the glutamate transporter EAAT1.

    PubMed

    Zhang, Yunlong; Zhang, Xiuping; Qu, Shaogang

    2014-07-01

    Excitatory amino acid transporter 1 (EAAT1) plays an important role in restricting the neurotoxicity of glutamate. Previous structure-function studies have provided evidence that reentrant helical hairpin loop (HP) 1 has predominant function during the transport cycle. The proposed internal gate HP1 is packed against transmembrane domain (TM) 2 and TM5 in its closed state, and two residues located in TM2 and HP2 of EAAT1 are in close proximity. However, the spatial relationship between TM2 and HP1 during the transport cycle remains unknown. In this study, we used chemical cross-linking of introduced cysteine pair (V96C and S366C) in a cysteine-less version of EAAT1 to assess the proximity of TM2 and HP1. Here, we show that inhibition of transport by copper(II)(1,10-phenanthroline)3 (CuPh) and cadmium ion (Cd(2+)) were observed in the V96C/S366C mutant. Glutamate or potassium significantly protected against the inhibition of transport activity of V96C/S366C by CuPh, while TBOA potentiated the inhibition of transport activity of V96C/S366C by CuPh. We also checked the kinetic parameters of V96C/S366C treated with or without CuPh in the presence of NaCl, NaCl + L-glutamate, NaCl + TBOA, and KCl, respectively. The sensitivity of V96C and S366C to membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] was attenuated by glutamate or potassium. TBOA had no effect on the sensitivity of V96C and S366C to MTSET. These data suggest that the spatial relationship between Val-96 of TM2 and Ser-366 of HP1 is altered in the transport cycle.

  1. Distribution of vesicular glutamate transporter 2 and glutamate receptor 1 mRNA in the central nervous system of the pigeon (Columba livia).

    PubMed

    Islam, Mohammad Rafiqul; Atoji, Yasuro

    2008-12-10

    Glutamate acts as the excitatory neurotransmitter in the central nervous system (CNS) and is mediated largely by the vesicular glutamate transporters (VGLUT1-3) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) types of glutamate receptors (GluR1-4) in mammals. In the present study, we determined the cDNA sequences of pigeon VGLUT2 and GluR1 and mapped the distribution of their mRNA in the pigeon CNS. The predicted amino acids of pigeon VGLUT2 and GluR1 showed a 93% identity to human VGLUT2 and GluR1 both. In situ hybridization autoradiograms showed VGLUT2 mRNA expression exclusively in the pallium of the telencephalon, and no expression in the subpallium. Within the diencephalon, VGLUT2 mRNA was more abundant in the thalamus than in the hypothalamus. Rich VGLUT2 mRNA expression was found in the optic tectum, nucleus mesencephalicus lateralis, pars dorsalis, nucleus isthmi, pars parvocellularis, isthmo-optic nucleus, pontine nuclei, and granular layer of the cerebellum. Moderate expression was noted in the cerebellar nuclei, vestibular nuclei, cochlear nuclei, inferior olivary nucleus, and gray matter of the spinal cord. GluR1 mRNA was expressed abundantly in the pallium and subpallium of the telencephalon, but it was poor in the diencephalon, midbrain, medulla, cerebellar cortex, and gray matter of the spinal cord. These results suggest that the cDNA sequences of VGLUT2 and GluR1 in the pigeon are comparable to those of VGLUT2 and GluR1 in mammals, respectively. The distribution of pigeon GluR1 mRNA resembles that of mammals, but the distribution of VGLUT2 mRNA resembles that of both VGLUT1 and VGLUT2 in mammals.

  2. Oral administration of MSG increases expression of glutamate receptors and transporters in the gastrointestinal tract of young piglets.

    PubMed

    Zhang, Jun; Yin, Yulong; Shu, Xu Gang; Li, Tiejun; Li, Fengna; Tan, Bie; Wu, Zhenlong; Wu, Guoyao

    2013-11-01

    Glutamate receptors and transporters, including T1R1 and T1R3 (taste receptor 1, subtypes 1 and 3), mGluRs (metabotropic glutamate receptors), EAAC-1 (excitatory amino acid carrier-1), GLAST-1 (glutamate-aspartate transporter-1), and GLT-1 (glutamate transporter-1), are expressed in the gastrointestinal tract. This study determined effects of oral administration of monosodium glutamate [MSG; 0, 0.06, 0.5, or 1 g/kg body weight (BW)/day] for 21 days on expression of glutamate receptors and transporters in the stomach and jejunum of sow-reared piglets. Both mRNA and protein levels for gastric T1R1, T1R3, mGluR1, mGluR4, EAAT1, EAAT2, EAAT3, and EAAT4 and mRNA levels for jejunal T1R1, T1R3, EAAT1, EAAT2, EAAT3 and EAAT4 were increased (P < 0.05) by MSG supplementation. Among all groups, mRNA levels for gastric EAAT1, EAAT2, EAAT3, and EAAT4 were highest (P < 0.05) in piglets receiving 1 g MSG/kg BW/day. EAAT1 and EAAT2 mRNA levels in the stomach and jejunum of piglets receiving 0.5 g MSG/kg BW/day, as well as jejunal EAAT3 and EAAT4 mRNA levels in piglets receiving 1 g MSG/kg BW/day, were higher (P < 0.05) than those in the control and in piglets receiving 0.06 g MSG/kg BW/day. Furthermore, protein levels for jejunal T1R1 and EAAT3 were higher (P < 0.05) in piglets receiving 1 g MSG/kg BW/day than those in the control and in piglets receiving 0.06 g MSG/kg BW/day. Collectively, these findings indicate that dietary MSG may beneficially stimulate glutamate signaling and sensing in the stomach and jejunum of young pigs, as well as their gastrointestinal function.

  3. Proteome Analysis and Conditional Deletion of the EAAT2 Glutamate Transporter Provide Evidence against a Role of EAAT2 in Pancreatic Insulin Secretion in Mice*

    PubMed Central

    Zhou, Yun; Waanders, Leonie F.; Holmseth, Silvia; Guo, Caiying; Berger, Urs V.; Li, Yuchuan; Lehre, Anne-Catherine; Lehre, Knut P.; Danbolt, Niels C.

    2014-01-01

    Islet function is incompletely understood in part because key steps in glutamate handling remain undetermined. The glutamate (excitatory amino acid) transporter 2 (EAAT2; Slc1a2) has been hypothesized to (a) provide islet cells with glutamate, (b) protect islet cells against high extracellular glutamate concentrations, (c) mediate glutamate release, or (d) control the pH inside insulin secretory granules. Here we floxed the EAAT2 gene to produce the first conditional EAAT2 knock-out mice. Crossing with Nestin-cyclization recombinase (Cre) eliminated EAAT2 from the brain, resulting in epilepsy and premature death, confirming the importance of EAAT2 for brain function and validating the genetic construction. Crossing with insulin-Cre lines (RIP-Cre and IPF1-Cre) to obtain pancreas-selective deletion did not appear to affect survival, growth, glucose tolerance, or β-cell number. We found (using TaqMan RT-PCR, immunoblotting, immunocytochemistry, and proteome analysis) that the EAAT2 levels were too low to support any of the four hypothesized functions. The proteome analysis detected more than 7,000 islet proteins of which more than 100 were transporters. Although mitochondrial glutamate transporters and transporters for neutral amino acids were present at high levels, all other transporters with known ability to transport glutamate were strikingly absent. Glutamate-metabolizing enzymes were abundant. The level of glutamine synthetase was 2 orders of magnitude higher than that of glutaminase. Taken together this suggests that the uptake of glutamate by islets from the extracellular fluid is insignificant and that glutamate is intracellularly produced. Glutamine synthetase may be more important for islets than assumed previously. PMID:24280215

  4. Effect of the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon on the glutamate release from rat brain nerve terminals under altered gravity conditions.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Krisanova, N.

    L-glutamate acts within the mammalian central nervous system as the predominant excitatory neurotransmitter and as a potent neurotoxin The balance between these physiological and pathological actions of glutamate is thought to be kept in check by the rapid removal of the neurotransmitter from the synaptic cleft The majority of uptake is mediated by the high-affinity Na -dependent glutamate transporters Depolarization leads to stimulation of glutamate efflux mediated by reversal of the high-affinity glutamate transporters The effects of the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon FCCP on the glutamate release from isolated nerve terminals rat brain synaptosomes were investigated in control and after centrifuge-induced hypergravity rats were rotated in a long-arm centrifuge at ten-G during one-hour period The treatment of synaptosomes with 1 mu M FCCP during 11 min resulted in the increase in L- 14 C glutamate release by 23 0 pm 2 3 of total accumulated synaptosomal label in control animals and 24 0 pm 2 3 animals subjected to hypergravity FCCP evoked release of L- 14 C glutamate from synaptosomes was not altered in animals exposed to hypergravity as compared to control Glutamate transport is of electrogenic nature and thus depends on the membrane potential The high-KCl stimulated L- 14 C glutamate release in Ca 2 -free media occurred due to reversal of the glutamate transporters Carrier --mediated release of L- 14 C glutamate 6 min slightly increased as a result of

  5. Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

    NASA Astrophysics Data System (ADS)

    Brew, Helen; Attwell, David

    1987-06-01

    Glutamate is taken up avidly by glial cells in the central nervous system1. Glutamate uptake may terminate the transmitter action of glutamate released from neurons1, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique2. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.

  6. Astroglial Glutamate Transporter Deficiency Increases Synaptic Excitability and Leads to Pathological Repetitive Behaviors in Mice

    PubMed Central

    Aida, Tomomi; Yoshida, Junichi; Nomura, Masatoshi; Tanimura, Asami; Iino, Yusuke; Soma, Miho; Bai, Ning; Ito, Yukiko; Cui, Wanpeng; Aizawa, Hidenori; Yanagisawa, Michiko; Nagai, Terumi; Takata, Norio; Tanaka, Kenji F; Takayanagi, Ryoichi; Kano, Masanobu; Götz, Magdalena; Hirase, Hajime; Tanaka, Kohichi

    2015-01-01

    An increase in the ratio of cellular excitation to inhibition (E/I ratio) has been proposed to underlie the pathogenesis of neuropsychiatric disorders, such as autism spectrum disorders (ASD), obsessive-compulsive disorder (OCD), and Tourette's syndrome (TS). A proper E/I ratio is achieved via factors expressed in neuron and glia. In astrocytes, the glutamate transporter GLT1 is critical for regulating an E/I ratio. However, the role of GLT1 dysfunction in the pathogenesis of neuropsychiatric disorders remains unknown because mice with a complete deficiency of GLT1 exhibited seizures and premature death. Here, we show that astrocyte-specific GLT1 inducible knockout (GLASTCreERT2/+/GLT1flox/flox, iKO) mice exhibit pathological repetitive behaviors including excessive and injurious levels of self-grooming and tic-like head shakes. Electrophysiological studies reveal that excitatory transmission at corticostriatal synapse is normal in a basal state but is increased after repetitive stimulation. Furthermore, treatment with an N-methyl-D-aspartate (NMDA) receptor antagonist memantine ameliorated the pathological repetitive behaviors in iKO mice. These results suggest that astroglial GLT1 has a critical role in controlling the synaptic efficacy at corticostriatal synapses and its dysfunction causes pathological repetitive behaviors. PMID:25662838

  7. Astroglial glutamate transporter deficiency increases synaptic excitability and leads to pathological repetitive behaviors in mice.

    PubMed

    Aida, Tomomi; Yoshida, Junichi; Nomura, Masatoshi; Tanimura, Asami; Iino, Yusuke; Soma, Miho; Bai, Ning; Ito, Yukiko; Cui, Wanpeng; Aizawa, Hidenori; Yanagisawa, Michiko; Nagai, Terumi; Takata, Norio; Tanaka, Kenji F; Takayanagi, Ryoichi; Kano, Masanobu; Götz, Magdalena; Hirase, Hajime; Tanaka, Kohichi

    2015-06-01

    An increase in the ratio of cellular excitation to inhibition (E/I ratio) has been proposed to underlie the pathogenesis of neuropsychiatric disorders, such as autism spectrum disorders (ASD), obsessive-compulsive disorder (OCD), and Tourette's syndrome (TS). A proper E/I ratio is achieved via factors expressed in neuron and glia. In astrocytes, the glutamate transporter GLT1 is critical for regulating an E/I ratio. However, the role of GLT1 dysfunction in the pathogenesis of neuropsychiatric disorders remains unknown because mice with a complete deficiency of GLT1 exhibited seizures and premature death. Here, we show that astrocyte-specific GLT1 inducible knockout (GLAST(CreERT2/+)/GLT1(flox/flox), iKO) mice exhibit pathological repetitive behaviors including excessive and injurious levels of self-grooming and tic-like head shakes. Electrophysiological studies reveal that excitatory transmission at corticostriatal synapse is normal in a basal state but is increased after repetitive stimulation. Furthermore, treatment with an N-methyl-D-aspartate (NMDA) receptor antagonist memantine ameliorated the pathological repetitive behaviors in iKO mice. These results suggest that astroglial GLT1 has a critical role in controlling the synaptic efficacy at corticostriatal synapses and its dysfunction causes pathological repetitive behaviors. PMID:25662838

  8. Restored glial glutamate transporter EAAT2 function as a potential therapeutic approach for Alzheimer's disease.

    PubMed

    Takahashi, Kou; Kong, Qiongman; Lin, Yuchen; Stouffer, Nathan; Schulte, Delanie A; Lai, Liching; Liu, Qibing; Chang, Ling-Chu; Dominguez, Sky; Xing, Xuechao; Cuny, Gregory D; Hodgetts, Kevin J; Glicksman, Marcie A; Lin, Chien-Liang Glenn

    2015-03-01

    Glutamatergic systems play a critical role in cognitive functions and are known to be defective in Alzheimer's disease (AD) patients. Previous literature has indicated that glial glutamate transporter EAAT2 plays an essential role in cognitive functions and that loss of EAAT2 protein is a common phenomenon observed in AD patients and animal models. In the current study, we investigated whether restored EAAT2 protein and function could benefit cognitive functions and pathology in APPSw,Ind mice, an animal model of AD. A transgenic mouse approach via crossing EAAT2 transgenic mice with APPSw,Ind. mice and a pharmacological approach using a novel EAAT2 translational activator, LDN/OSU-0212320, were conducted. Findings from both approaches demonstrated that restored EAAT2 protein function significantly improved cognitive functions, restored synaptic integrity, and reduced amyloid plaques. Importantly, the observed benefits were sustained one month after compound treatment cessation, suggesting that EAAT2 is a potential disease modifier with therapeutic potential for AD. PMID:25711212

  9. Astroglial glutamate transporter deficiency increases synaptic excitability and leads to pathological repetitive behaviors in mice.

    PubMed

    Aida, Tomomi; Yoshida, Junichi; Nomura, Masatoshi; Tanimura, Asami; Iino, Yusuke; Soma, Miho; Bai, Ning; Ito, Yukiko; Cui, Wanpeng; Aizawa, Hidenori; Yanagisawa, Michiko; Nagai, Terumi; Takata, Norio; Tanaka, Kenji F; Takayanagi, Ryoichi; Kano, Masanobu; Götz, Magdalena; Hirase, Hajime; Tanaka, Kohichi

    2015-06-01

    An increase in the ratio of cellular excitation to inhibition (E/I ratio) has been proposed to underlie the pathogenesis of neuropsychiatric disorders, such as autism spectrum disorders (ASD), obsessive-compulsive disorder (OCD), and Tourette's syndrome (TS). A proper E/I ratio is achieved via factors expressed in neuron and glia. In astrocytes, the glutamate transporter GLT1 is critical for regulating an E/I ratio. However, the role of GLT1 dysfunction in the pathogenesis of neuropsychiatric disorders remains unknown because mice with a complete deficiency of GLT1 exhibited seizures and premature death. Here, we show that astrocyte-specific GLT1 inducible knockout (GLAST(CreERT2/+)/GLT1(flox/flox), iKO) mice exhibit pathological repetitive behaviors including excessive and injurious levels of self-grooming and tic-like head shakes. Electrophysiological studies reveal that excitatory transmission at corticostriatal synapse is normal in a basal state but is increased after repetitive stimulation. Furthermore, treatment with an N-methyl-D-aspartate (NMDA) receptor antagonist memantine ameliorated the pathological repetitive behaviors in iKO mice. These results suggest that astroglial GLT1 has a critical role in controlling the synaptic efficacy at corticostriatal synapses and its dysfunction causes pathological repetitive behaviors.

  10. Physical and Functional Interaction of NCX1 and EAAC1 Transporters Leading to Glutamate-Enhanced ATP Production in Brain Mitochondria

    PubMed Central

    Arcangeli, Sara; Nasti, Annamaria Assunta; Giordano, Antonio; Amoroso, Salvatore

    2012-01-01

    Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production. PMID:22479505

  11. Neurological effects of inorganic arsenic exposure: altered cysteine/glutamate transport, NMDA expression and spatial memory impairment

    PubMed Central

    Ramos-Chávez, Lucio A.; Rendón-López, Christian R. R.; Zepeda, Angélica; Silva-Adaya, Daniela; Del Razo, Luz M.; Gonsebatt, María E.

    2015-01-01

    Inorganic arsenic (iAs) is an important natural pollutant. Millions of individuals worldwide drink water with high levels of iAs. Chronic exposure to iAs has been associated with lower IQ and learning disabilities as well as memory impairment. iAs is methylated in tissues such as the brain generating mono and dimethylated species. iAs methylation requires cellular glutathione (GSH), which is the main antioxidant in the central nervous system (CNS). In humans, As species cross the placenta and are found in cord blood. A CD1 mouse model was used to investigate effects of gestational iAs exposure which can lead to oxidative damage, disrupted cysteine/glutamate transport and its putative impact in learning and memory. On postnatal days (PNDs) 1, 15 and 90, the expression of membrane transporters related to GSH synthesis and glutamate transport and toxicity, such as xCT, EAAC1, GLAST and GLT1, as well as LAT1, were analyzed. Also, the expression of the glutamate receptor N-methyl-D-aspartate (NMDAR) subunits NR2A and B as well as the presence of As species in cortex and hippocampus were investigated. On PND 90, an object location task was performed to associate exposure with memory impairment. Gestational exposure to iAs affected the expression of cysteine/glutamate transporters in cortex and hippocampus and induced a negative modulation of NMDAR NR2B subunit in the hippocampus. Behavioral tasks showed significant spatial memory impairment in males while the effect was marginal in females. PMID:25709567

  12. Expression of Vesicular Glutamate Transporters VGLUT1 and VGLUT2 in the Rat Dental Pulp and Trigeminal Ganglion following Inflammation

    PubMed Central

    Hong, Jae Hyun; Kim, Yun Sook; Choi, So Young; Kim, Tae Heon; Cho, Yi Sul; Bae, Yong Chul

    2014-01-01

    Background There is increasing evidence that peripheral glutamate signaling mechanism is involved in the nociceptive transmission during pathological conditions. However, little is known about the glutamate signaling mechanism and related specific type of vesicular glutamate transporter (VGLUT) in the dental pulp following inflammation. To address this issue, we investigated expression and protein levels of VGLUT1 and VGLUT2 in the dental pulp and trigeminal ganglion (TG) following complete Freund’s adjuvant (CFA) application to the rat dental pulp by light microscopic immunohistochemistry and Western blot analysis. Results The density of VGLUT2− immunopositive (+) axons in the dental pulp and the number of VGLUT2+ soma in the TG increased significantly in the CFA-treated group, compared to control group. The protein levels of VGLUT2 in the dental pulp and TG were also significantly higher in the CFA-treated group than control group by Western blot analysis. The density of VGLUT1+ axons in the dental pulp and soma in the TG remained unchanged in the CFA-treated group. Conclusions These findings suggest that glutamate signaling that is mediated by VGLUT2 in the pulpal axons may be enhanced in the inflamed dental pulp, which may contribute to pulpal axon sensitization leading to hyperalgesia following inflammation. PMID:25290694

  13. Glutamate transporters alterations in the reorganizing dentate gyrus are associated with progressive seizure activity in chronic epileptic rats.

    PubMed

    Gorter, Jan A; Van Vliet, Erwin A; Proper, Evelien A; De Graan, Pierre N E; Ghijsen, Wim E J M; Lopes Da Silva, Fernando H; Aronica, Eleonora

    2002-01-21

    The expression of glial and neuronal glutamate transporter proteins was investigated in the hippocampal region at different time points after electrically induced status epilepticus (SE) in the rat. This experimental rat model for mesial temporal lobe epilepsy is characterized by cell loss, gliosis, synaptic reorganization, and chronic seizures after a latent period. Despite extensive gliosis, immunocytochemistry revealed only an up-regulation of both glial transporters localized at the outer aspect of the inner molecular layer (iml) in chronic epileptic rats. The neuronal EAAC1 transporter was increased in many somata of individual CA1-3 neurons and granule cells that had survived after SE; this up-regulation was still present in the chronic epileptic phase. In contrast, a permanent decrease of EAAC1 immunoreactivity was observed in the iml of the dentate gyrus. This permanent decrease in EAAC1 expression, which was only observed in rats that experienced progressive spontaneous seizure activity, could lead to abnormal glutamate levels in the iml once new abnormal glutamatergic synaptic contacts are formed by means of sprouted mossy fibers. Considering the steady growth of reorganizing mossy fibers in the iml, the absence of a glutamate reuptake mechanism in this region could contribute to progression of spontaneous seizure activity, which occurs with a similar time course.

  14. Regulation of the mouse Na+-dependent glutamate/aspartate transporter GLAST: putative role of an AP-1 DNA binding site.

    PubMed

    Ramírez-Sotelo, Guadalupe; López-Bayghen, Esther; Hernández-Kelly, L Clara R; Arias-Montaño, J Antonio; Bernabé, Alfonso; Ortega, Arturo

    2007-01-01

    Appropriate removal of L: -glutamate from the synaptic cleft is important for prevention of the excitotoxic effects of this neurotransmitter. The Na+-dependent glutamate/aspartate transporter GLAST is regulated in the short term, by a transporter-dependent decrease in uptake activity while in the long term, a receptor's-dependent decrease in GLAST protein levels leads to a severe reduction in glutamate uptake. The promoter region of the mouse glast gene harbors an Activator Protein-1 site (AP-1). To gain insight into the molecular mechanisms triggered by Glu-receptors activation involved in GLAST regulation, we took advantage of the neonatal mouse cerebellar prisms model. We characterized the glutamate uptake activity; the glutamate-dependent effect on GLAST protein levels and over the interaction of nuclear proteins with a mouse glast promoter AP-1 probe. A time and dose dependent decrease in transporter activity matching with a decrease in GLAST levels was recorded upon glutamate treatment. Moreover, a significant increase in glast AP-1 DNA binding was found. Pharmacological experiments established that both effects are mediated through alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors, favoring the notion of the critical involvement of glutamate in the regulation of its binding partners: receptors and transporters.

  15. A critical role of glutamate transporter type 3 in the learning and memory of mice.

    PubMed

    Wang, Zhi; Park, Sang-Hon; Zhao, Huijuan; Peng, Shuling; Zuo, Zhiyi

    2014-10-01

    Hippocampus-dependent learning and memory are associated with trafficking of excitatory amino acid transporter type 3 (EAAT3) to the plasma membrane. To assess whether this trafficking is an intrinsic component of the biochemical responses underlying learning and memory, 7- to 9-week old male EAAT3 knockout mice and CD-1 wild-type mice were subjected to fear conditioning. Their hippocampal CA1 regions, amygdalae and entorhinal cortices were harvested before, or 30 min or 3 h after the fear conditioning stimulation. We found that EAAT3 knockout mice had worse contextual and tone-related learning and memory than did the wild-type mice. The expression of EAAT3, glutamate receptor (GluR)1 and GluR2 in the plasma membrane and of phospho-GluR1 (at Ser 831) and phospho-CaMKII in the hippocampus of the wild-type mice was increased at 30 min after the fear conditioning stimulation. Similar biochemical changes occurred in the amygdala. Fear conditioning also increased the expression of c-Fos and activity-regulated cytoskeleton-associated protein (Arc) in the CA1 regions and of Arc in the entorhinal cortices of the wild-type mice. These biochemical responses were attenuated in the EAAT3 knockout mice. These results suggest that EAAT3 plays a critical role in learning and memory. Our results also provide initial evidence that EAAT3 may have receptor-like functions to participate in the biochemical reactions underlying learning and memory.

  16. Vesicular Glutamate Transporter 2 Expression in the Rat Pineal Gland: Detailed Analysis of Expression Pattern and Regulatory Mechanism

    NASA Astrophysics Data System (ADS)

    Yoshida, Sachine; Hisano, Setsuji

    Melatonin, a hormone secreted by the pineal gland, is closely related physiologically to circadian rhythm, sleep and reproduction, and also psychiatrically to mood disorders in humans. Under circadian control, melatonin secretion is modulated via nocturnal autonomic (adrenergic) stimulation to the gland, which expresses vesicular glutamate transporter (VGLUT) 1, VGLUT2 and a VGLUT1 splice variant (VGLUT1v), glutamatergic markers. Expression of VGLUT2 gene and protein in the intact gland has been reported to exhibit a rhythmic change during a day. To study VGLUT2 expression is under adrenergic control, we here performed an in vitro experiment using dispersed pineal cells of rats. Stimulation of either β-adrenergic receptor or cAMP production to the pineal cells was shown to increase mRNA level of VGLUT2, but not VGLUT1 and VGLUT1v. Because an ability of glutamate to inhibit melatonin production was previously reported in the cultured gland, it is likely that pineal VGLUT2 transports glutamate engaged in the inhibition of melatonin production.

  17. Spinal astrocyte gap junction and glutamate transporter expression contributes to a rat model of bortezomib-induced peripheral neuropathy

    PubMed Central

    Robinson, Caleb R.; Dougherty, Patrick M.

    2014-01-01

    There is increasing evidence implicating astrocytes in multiple forms of chronic pain, as well as in the specific context of chemotherapy-induced peripheral neuropathy (CIPN). However, it is still unclear what the exact role of astrocytes may be in the context of CIPN. Findings in oxaliplatin and paclitaxel models have displayed altered expression of astrocytic gap junctions and glutamate transporters as means by which astrocytes may contribute to observed behavioral changes. The current study investigated whether these changes were also generalizable to the bortezomib CIPN. Changes in mechanical sensitivity were verified in bortezomib-treated animals, and these changes were prevented by co-treatment with a glial activation inhibitor (minocycline), a gap junction decoupler (carbenoxolone), and by a glutamate transporter upregulator (ceftriaxone). Immunohistochemistry data at day 30 in bortezomib-treated animals showed increases in expression of GFAP and connexin 43 but decrease in GLAST expression. These changes were prevented by co-treatment with minocycline. Follow-up Western blotting data showed a shift in connexin 43 from a non-phosphorylated state to a phosphorylated state, indicating increased trafficking of expressed connexin 43 to the cell membrane. These data suggest that increases in behavioral sensitivity to cutaneous stimuli may be tied to persistent synaptic glutamate resulting from increased calcium flow between spinal astrocytes. PMID:25446343

  18. The peroxisome proliferator activated receptor gamma agonist pioglitazone increases functional expression of the glutamate transporter excitatory amino acid transporter 2 (EAAT2) in human glioblastoma cells

    PubMed Central

    Ching, Jared; Amiridis, Stephanie; Stylli, Stanley S.; Bjorksten, Andrew R.; Kountouri, Nicole; Zheng, Thomas; Paradiso, Lucy; Luwor, Rodney B.; Morokoff, Andrew P.; O'Brien, Terence J.; Kaye, Andrew H.

    2015-01-01

    Glioma cells release glutamate through expression of system xc−, which exchanges intracellular glutamate for extracellular cysteine. Lack of the excitatory amino acid transporter 2 (EAAT2) expression maintains high extracellular glutamate levels in the glioma microenvironment, causing excitotoxicity to surrounding parenchyma. Not only does this contribute to the survival and proliferation of glioma cells, but is involved in the pathophysiology of tumour-associated epilepsy (TAE). We investigated the role of the peroxisome proliferator activated receptor gamma (PPARγ) agonist pioglitazone in modulating EAAT2 expression in glioma cells. We found that EAAT2 expression was increased in a dose dependent manner in both U87MG and U251MG glioma cells. Extracellular glutamate levels were reduced with the addition of pioglitazone, where statistical significance was reached in both U87MG and U251MG cells at a concentration of ≥ 30 μM pioglitazone (p < 0.05). The PPARγ antagonist GW9662 inhibited the effect of pioglitazone on extracellular glutamate levels, indicating PPARγ dependence. In addition, pioglitazone significantly reduced cell viability of U87MG and U251MG cells at ≥ 30 μM and 100 μM (p < 0.05) respectively. GW9662 also significantly reduced viability of U87MG and U251MG cells with 10 μM and 30 μM (p < 0.05) respectively. The effect on viability was partially dependent on PPARγ activation in U87MG cells but not U251MG cells, whereby PPARγ blockade with GW9662 had a synergistic effect. We conclude that PPARγ agonists may be therapeutically beneficial in the treatment of gliomas and furthermore suggest a novel role for these agents in the treatment of tumour associated seizures through the reduction in extracellular glutamate. PMID:26046374

  19. Regulation of astrocyte glutamate transporter-1 (GLT1) and aquaporin-4 (AQP4) expression in a model of epilepsy.

    PubMed

    Hubbard, Jacqueline A; Szu, Jenny I; Yonan, Jennifer M; Binder, Devin K

    2016-09-01

    Astrocytes regulate extracellular glutamate and water homeostasis through the astrocyte-specific membrane proteins glutamate transporter-1 (GLT1) and aquaporin-4 (AQP4), respectively. The role of astrocytes and the regulation of GLT1 and AQP4 in epilepsy are not fully understood. In this study, we investigated the expression of GLT1 and AQP4 in the intrahippocampal kainic acid (IHKA) model of temporal lobe epilepsy (TLE). We used real-time polymerase chain reaction (RT-PCR), Western blot, and immunohistochemical analysis at 1, 4, 7, and 30days after kainic acid-induced status epilepticus (SE) to determine hippocampal glial fibrillary acidic protein (GFAP, a marker for reactive astrocytes), GLT1, and AQP4 expression changes during the development of epilepsy (epileptogenesis). Following IHKA, all mice had SE and progressive increases in GFAP immunoreactivity and GFAP protein expression out to 30days post-SE. A significant initial increase in dorsal hippocampal GLT1 immunoreactivity and protein levels were observed 1day post SE and followed by a marked downregulation at 4 and 7days post SE with a return to near control levels by 30days post SE. AQP4 dorsal hippocampal protein expression was significantly downregulated at 1day post SE and was followed by a gradual return to baseline levels with a significant increase in ipsilateral protein levels by 30days post SE. Transient increases in GFAP and AQP4 mRNA were also observed. Our findings suggest that specific molecular changes in astrocyte glutamate transporters and water channels occur during epileptogenesis in this model, and suggest the novel therapeutic strategy of restoring glutamate and water homeostasis. PMID:27155358

  20. Hypersensitive Glutamate Signaling Correlates with the Development of Late-Onset Behavioral Morbidity in Diffuse Brain-Injured Circuitry

    PubMed Central

    Thomas, Theresa Currier; Hinzman, Jason M.; Gerhardt, Greg A.

    2012-01-01

    Abstract In diffuse brain-injured rats, robust sensory sensitivity to manual whisker stimulation develops over 1 month post-injury, comparable to agitation expressed by brain-injured individuals with overstimulation. In the rat, whisker somatosensation relies on thalamocortical glutamatergic relays between the ventral posterior medial (VPM) thalamus and barrel fields of somatosensory cortex (S1BF). Using novel glutamate-selective microelectrode arrays coupled to amperometry, we test the hypothesis that disrupted glutamatergic neurotransmission underlies the whisker sensory sensitivity associated with diffuse brain injury. We report hypersensitive glutamate neurotransmission that parallels and correlates with the development of post-traumatic sensory sensitivity. Hypersensitivity is demonstrated by significant 110% increases in VPM extracellular glutamate levels, and 100% increase in potassium-evoked glutamate release in the VPM and S1BF, with no change in glutamate clearance. Further, evoked glutamate release showed 50% greater sensitivity to a calcium channel antagonist in brain-injured over uninjured VPM. In conjunction with no changes in glutamate transporter gene expression and exogenous glutamate clearance efficiency, these data support a presynaptic origin for enduring post-traumatic circuit alterations. In the anatomically-distinct whisker circuit, the injury-induced functional alterations correlate with the development of late-onset behavioral morbidity. Effective therapies to modulate presynaptic glutamate function in diffuse-injured circuits may translate into improvements in essential brain function and behavioral performance in other brain-injured circuits in rodents and in humans. PMID:21939393

  1. Inhibition of mitochondrial pyruvate transport by zaprinast causes massive accumulation of aspartate at the expense of glutamate in the retina.

    PubMed

    Du, Jianhai; Cleghorn, Whitney M; Contreras, Laura; Lindsay, Ken; Rountree, Austin M; Chertov, Andrei O; Turner, Sally J; Sahaboglu, Ayse; Linton, Jonathan; Sadilek, Martin; Satrústegui, Jorgina; Sweet, Ian R; Paquet-Durand, François; Hurley, James B

    2013-12-13

    Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing (13)C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia.

  2. Inhibition of Mitochondrial Pyruvate Transport by Zaprinast Causes Massive Accumulation of Aspartate at the Expense of Glutamate in the Retina*

    PubMed Central

    Du, Jianhai; Cleghorn, Whitney M.; Contreras, Laura; Lindsay, Ken; Rountree, Austin M.; Chertov, Andrei O.; Turner, Sally J.; Sahaboglu, Ayse; Linton, Jonathan; Sadilek, Martin; Satrústegui, Jorgina; Sweet, Ian R.; Paquet-Durand, François; Hurley, James B.

    2013-01-01

    Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing 13C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia. PMID:24187136

  3. Distribution of glutamate transporter 1 mRNA in the central nervous system of the pigeon (Columba livia).

    PubMed

    Atoji, Yasuro; Islam, Mohammad Rafiqul

    2009-07-01

    Glutamate transporter 1 (GLT1) in glial cells removes glutamate that diffuses from the synaptic cleft into the extracellular space. Previously, we have shown the distribution of glutamatergic neurons in the central nervous system (CNS) of the pigeon. In the present study, we identified cDNA sequence of the pigeon GLT1, and mapped the distribution of the mRNA-expressing cells in CNS to examine whether GLT1 is associated with glutamatergic terminal areas. The cDNA sequence of the pigeon GLT1 consisted of 1889bp nucleotides and the amino acids showed 97% and 87% identity to the chicken and human GLT1, respectively. In situ hybridization autoradiograms revealed GLT1 mRNA expression in glial cells and produced regional differences of GLT1 mRNA distribution in CNS. GLT1 mRNA was expressed preferentially in the pallium than the subpallium. Moderate expression was seen in the hyperpallium, Field L, mesopallium, and hippocampal formation. In the thalamus, moderate expression was found in the ovoidal nucleus, rotundal nucleus, triangular nucleus, and lateral spiriform nucleus, while the dorsal thalamic nuclei were weak. In the brainstem, the isthmic nuclei, optic tectum, vestibular nuclei, and cochlear nuclei expressed moderately, but the cerebellar cortex showed strong expression. Bergmann glial cells expressed GLT1 mRNA very strongly. The results indicate that cDNA sequence of the pigeon GLT1 is comparable with that of the mammalian GLT1, and a large number of GLT1 mRNA-expressing areas correspond with areas where AMPA-type glutamate receptors are located. Avian GLT1 in glial cells probably maintain microenvironment of glutamate concentration around synapses as in mammalian GLT1.

  4. Immunocytochemical localization of the high-affinity glutamate transporter, EAAC1, in the retina of representative vertebrate species.

    PubMed

    Schultz, K; Stell, W K

    1996-06-28

    The glutamate transporter, EAAC1, was localized immunocytochemically in goldfish, salamander, turtle, chicken, and rat retinas, using affinity-purified oligopeptide antibodies. Immunoreactive (IR) EAAC1 was present in the inner plexiform layer of all species, and in cell bodies of bipolar, amacrine, and ganglion cells of most species, but absent from photoreceptors and Müller's glial cells. Western blots revealed an IR-EAAC1 band at 70 kDa. Staining was abolished by preabsorption with EAAC1 peptide. PMID:8817573

  5. Individual synaptic vesicles from the electroplaque of Torpedo californica, a classic cholinergic synapse, also contain transporters for glutamate and ATP.

    PubMed

    Li, Huinan; Harlow, Mark L

    2014-01-01

    The type of neurotransmitter secreted by a neuron is a product of the vesicular transporters present on its synaptic vesicle membranes and the available transmitters in the local cytosolic environment where the synaptic vesicles reside. Synaptic vesicles isolated from electroplaques of the marine ray, Torpedo californica, have served as model vesicles for cholinergic neurotransmission. Many lines of evidence support the idea that in addition to acetylcholine, additional neurotransmitters and/or neuromodulators are also released from cholinergic synapses. We identified the types of vesicular neurotransmitter transporters present at the electroplaque using immunoblot and immunofluoresence techniques with antibodies against the vesicle acetylcholine transporter (VAChT), the vesicular glutamate transporters (VGLUT1, 2, and 3), and the vesicular nucleotide transporter (VNUT). We found that VAChT, VNUT, VGLUT 1 and 2, but not 3 were present by immunoblot, and confirmed that the antibodies were specific to proteins of the axons and terminals of the electroplaque. We used a single-vesicle imaging technique to determine whether these neurotransmitter transporters were present on the same or different populations of synaptic vesicles. We found that greater than 85% of vesicles that labeled for VAChT colabeled with VGLUT1 or VGLUT2, and approximately 70% colabeled with VNUT. Based upon confidence intervals, at least 52% of cholinergic vesicles isolated are likely to contain all four transporters. The presence of multiple types of neurotransmitter transporters - and potentially neurotransmitters - in individual synaptic vesicles raises fundamental questions about the role of cotransmitter release and neurotransmitter synergy at cholinergic synapses.

  6. Co-release of glutamate and GABA from single vesicles in GABAergic neurons exogenously expressing VGLUT3

    PubMed Central

    Zimmermann, Johannes; Herman, Melissa A.; Rosenmund, Christian

    2015-01-01

    The identity of the vesicle neurotransmitter transporter expressed by a neuron largely corresponds with the primary neurotransmitter that cell releases. However, the vesicular glutamate transporter subtype 3 (VGLUT3) is mainly expressed in non-glutamatergic neurons, including cholinergic, serotonergic, or GABAergic neurons. Though a functional role for glutamate release from these non-glutamatergic neurons has been demonstrated, the interplay between VGLUT3 and the neuron’s characteristic neurotransmitter transporter, particularly in the case of GABAergic neurons, at the synaptic and vesicular level is less clear. In this study, we explore how exogenous expression of VGLUT3 in striatal GABAergic neurons affects the packaging and release of glutamate and GABA in synaptic vesicles (SVs). We found that VGLUT3 expression in isolated, autaptic GABAergic neurons leads to action potential evoked release of glutamate. Under these conditions, glutamate and GABA could be packaged together in single vesicles release either spontaneously or asynchronously. However, the presence of glutamate in GABAergic vesicles did not affect uptake of GABA itself, suggesting a lack of synergy in vesicle filling for these transmitters. Finally, we found postsynaptic detection of glutamate released from GABAergic terminals difficult when bona fide glutamatergic synapses were present, suggesting that co-released glutamate cannot induce postsynaptic glutamate receptor clustering. PMID:26441632

  7. No evidence for genetic association between glutamate transporter EAAT2 and Devic's neuromyelitis optica in caucasians and afro-caribbeans.

    PubMed

    Hanoux, Vincent; Coulbault, Laurent; Derache, Nathalie; Cabre, Philippe; De Seze, Jérôme; Marignier, Romain; Rudolf, Gabrielle; Emmanuelle Dugué, Audrey; Allouche, Stéphane; Defer, Gilles

    2014-01-01

    Devic's neuromyelitis optica (NMO) is a severe inflammatory and autoimmune disease producing demyelinating lesions. Recent data suggest that a complex genetic component could be involved. While impairment of glutamate homeostasis has emerged as a contributing etiological factor in NMO, a genetic alteration of Excitatory Amino Acid Transporter 2 (EAAT2/SLC1A2), the major glutamate transporter in the Central Nervous System (CNS), could contribute to glutamate excitotoxicity and then must be considered. We evaluated whether mutations and/or single nucleotide polymorphisms (SNPs) in EAAT2 gene, are associated with susceptibility to NMO. We studied a cohort of NMO sporadic cases including afro-caribbean patients (n=81; French cohort of Devic's neuromyelitis optica-NOMADMUS cohort) and compared to control subjects (n=56). We sequenced the whole coding region of EAAT2 gene and splicing consensus sequences flanking each exon. The results obtained from all NMO samples did not show any novel mutations and/or SNPs both in the coding region and splicing sites of EAAT2 gene compared to controls subjects. We reported three synonymous SNPs (rs752949, rs1042113 and rs7102949) but only rs7102949 was found in afro-caribbean. Genotype frequencies did not differ between patients and controls for the three SNPs in caucasians and afro-caribbeans (rs752949: p=0.71 and p=0.37, respectively; rs1042113: p=0.73 and p=0.35, respectively; rs7102949: p=0.08 in afro-caribbeans). Our data showed no evidence for a genetic association between EAAT2 gene and Devic's neuromyelitis optica. PMID:25877978

  8. Nippostrongylus brasiliensis infection evokes neuronal abnormalities and alterations in neurally regulated electrolyte transport in rat jejunum.

    PubMed

    Masson, S D; McKay, D M; Stead, R H; Agro, A; Stanisz, A; Perdue, M H

    1996-08-01

    Neuronal abnormalities have been described in the intestine of helminth-infected rats. However, the physiological ramifications of these changes have not been determined. Here, we examined epithelial ion secretion, indicated by increases in short-circuit current (Isc), evoked by electrical transmural stimulation (TS) of enteric nerves in Ussing-chambered jejunal tissues from Nippostrongylus brasiliensis-infected rats. Rats were examined at 10 and 35 days post-infection (p.i.); non-infected rats served as controls. TS resulted in significantly reduced ion secretion in jejunum from 10 day p.i. rats compared to controls or jejunum from 35 day p.i. rats. The TS response in tissue from infected rats had, unlike controls, no cholinergic component. Tissues from both non-infected and infected rats were equally responsive to the muscarinic agonist bethanechol, suggesting that the cholinergic defect was neuronal and not an inability of the epithelium to respond to cholinergic stimulation. However, increases in Isc evoked by exogenous substance P (SP) in tissue from rats 10 day p.i. were reduced in magnitude to approximately 25% of control values. Concomitant with these physiological changes, tissue from infected rats contained increased amounts of substance P immunoreactivity and intestinal sections displayed increased numbers of substance P-immunoreactive nerve fibre profiles at both 10 and 35 days p.i. Thus, following N. brasiliensis infection there is a shift in the enteric nervous system away from cholinergic to non-cholinergic regulation, associated with increased amounts of the pro-inflammatory neuropeptide, substance P. We speculate that changes in neuronal structure and function are intimately involved in the co-ordinated multicellular response to intestinal parasitic infection and subsequent gut recovery.

  9. Clavulanic acid enhances glutamate transporter subtype I (GLT-1) expression and decreases reinforcing efficacy of cocaine in mice.

    PubMed

    Kim, Jae; John, Joel; Langford, Dianne; Walker, Ellen; Ward, Sara; Rawls, Scott M

    2016-03-01

    The β-lactam antibiotic ceftriaxone (CTX) reduces cocaine reinforcement and relapse in preclinical assays through a mechanism involving activation of glutamate transporter subtype 1 (GLT-1). However, its poor brain penetrability and intravenous administration route may limit its therapeutic utility for indications related to CNS diseases. An alternative is clavulanic acid (CA), a structural analog of CTX that retains the β-lactam core required for GLT-1 activity but displays enhanced brain penetrability and oral activity relative to CTX. Here, we tested the hypothesis that CA (1, 10 mg/kg ip) would enhance GLT-1 expression and decrease cocaine self-administration (SA) in mice, but at lower doses than CTX. Experiments revealed that GLT-1 transporter expression in the nucleus accumbens of mice treated with repeated CA (1, 10 mg/kg) was enhanced relative to saline-treated mice. Repeated CA treatment (1 mg/kg) reduced the reinforcing efficacy of cocaine (0.56 mg/kg/inf) in mice maintained on a progressive-ratio (PR) schedule of reinforcement but did not affect acquisition of cocaine SA under fixed-ratio responding or acquisition or retention of learning. These findings suggest that the β-lactamase inhibitor CA can activate the cellular glutamate reuptake system in the brain reward circuit and reduce cocaine's reinforcing efficacy at 100-fold lower doses than CTX.

  10. Clavulanic acid enhances glutamate transporter subtype I (GLT-1) expression and decreases reinforcing efficacy of cocaine in mice.

    PubMed

    Kim, Jae; John, Joel; Langford, Dianne; Walker, Ellen; Ward, Sara; Rawls, Scott M

    2016-03-01

    The β-lactam antibiotic ceftriaxone (CTX) reduces cocaine reinforcement and relapse in preclinical assays through a mechanism involving activation of glutamate transporter subtype 1 (GLT-1). However, its poor brain penetrability and intravenous administration route may limit its therapeutic utility for indications related to CNS diseases. An alternative is clavulanic acid (CA), a structural analog of CTX that retains the β-lactam core required for GLT-1 activity but displays enhanced brain penetrability and oral activity relative to CTX. Here, we tested the hypothesis that CA (1, 10 mg/kg ip) would enhance GLT-1 expression and decrease cocaine self-administration (SA) in mice, but at lower doses than CTX. Experiments revealed that GLT-1 transporter expression in the nucleus accumbens of mice treated with repeated CA (1, 10 mg/kg) was enhanced relative to saline-treated mice. Repeated CA treatment (1 mg/kg) reduced the reinforcing efficacy of cocaine (0.56 mg/kg/inf) in mice maintained on a progressive-ratio (PR) schedule of reinforcement but did not affect acquisition of cocaine SA under fixed-ratio responding or acquisition or retention of learning. These findings suggest that the β-lactamase inhibitor CA can activate the cellular glutamate reuptake system in the brain reward circuit and reduce cocaine's reinforcing efficacy at 100-fold lower doses than CTX. PMID:26543027

  11. Clinical aspects of urea cycle dysfunction and altered brain energy metabolism on modulation of glutamate receptors and transporters in acute and chronic hyperammonemia.

    PubMed

    Natesan, Vijayakumar; Mani, Renuka; Arumugam, Ramakrishnan

    2016-07-01

    In living organisms, nitrogen arise primarily as ammonia (NH3) and ammonium (NH4(+)), which is a main component of the nucleic acid pool and proteins. Although nitrogen is essential for growth and maintenance in animals, but when the nitrogenous compounds exceeds the normal range which can quickly lead to toxicity and death. Urea cycle is the common pathway for the disposal of excess nitrogen through urea biosynthesis. Hyperammonemia is a consistent finding in many neurological disorders including congenital urea cycle disorders, reye's syndrome and acute liver failure leads to deleterious effects. Hyperammonemia and liver failure results in glutamatergic neurotransmission which contributes to the alteration in the function of the glutamate-nitric oxide-cGMP pathway, modulates the important cerebral process. Even though ammonia is essential for normal functioning of the central nervous system (CNS), in particular high concentrations of ammonia exposure to the brain leads to the alterations of glutamate transport by the transporters. Several glutamate transporters have been recognized in the central nervous system and each has a unique physiological property and distribution. The loss of glutamate transporter activity in brain during acute liver failure and hyperammonemia is allied with increased extracellular brain glutamate concentrations which may be conscientious for the cerebral edema and ultimately cell death. PMID:27261594

  12. Effects of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline on glutamate transporter 1 and cysteine/glutamate exchanger as well as ethanol drinking behavior in male, alcohol-preferring rats.

    PubMed

    Aal-Aaboda, Munaf; Alhaddad, Hasan; Osowik, Francis; Nauli, Surya M; Sari, Youssef

    2015-06-01

    Alcohol consumption is largely associated with alterations in the extracellular glutamate concentrations in several brain reward regions. We recently showed that glutamate transporter 1 (GLT-1) is downregulated following chronic exposure to ethanol for 5 weeks in alcohol-preferring (P) rats and that upregulation of the GLT-1 levels in nucleus accumbens and prefrontal cortex results, in part, in attenuating ethanol consumption. Cystine glutamate antiporter (xCT) is also downregulated after chronic ethanol exposure in P rats, and its upregulation could be valuable in attenuating ethanol drinking. This study examines the effect of a synthetic compound, (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), on ethanol drinking and expressions of GLT-1 and xCT in the amygdala and the hippocampus of P rats. P rats were exposed to continuous free-choice access to water, 15% and 30% ethanol, and food for 5 weeks, after which they received treatments of MS-153 or vehicle for 5 days. The results show that MS-153 treatment significantly reduces ethanol consumption. It was revealed that GLT-1 and xCT expressions were downregulated in both the amygdala and the hippocampus of ethanol-vehicle-treated rats (ethanol-vehicle group) compared with water-control animals. MS-153 treatment upregulated GLT-1 and xCT expressions in these brain regions. These findings demonstrate an important role for MS-153 in these glutamate transporters for the attenuation of ethanol-drinking behavior.

  13. Partial mitochondrial complex I inhibition induces oxidative damage and perturbs glutamate transport in primary retinal cultures. Relevance to Leber Hereditary Optic Neuropathy (LHON).

    PubMed

    Beretta, Simone; Wood, John P M; Derham, Barry; Sala, Gessica; Tremolizzo, Lucio; Ferrarese, Carlo; Osborne, Neville N

    2006-11-01

    Leber Hereditary Optic Neuropathy (LHON) is a maternally inherited form of visual loss, due to selective degeneration of retinal ganglion cells. Despite the established aetiological association between LHON and mitochondrial DNA mutations affecting complex I of the electron transport chain, the pathophysiology of this disorder remains obscure. Primary rat retinal cultures were exposed to increasing concentrations of rotenone to titrate complex I inhibition. Neural cells were more sensitive than Müller glial cells to rotenone toxicity. Rotenone induced an increase in mitochondrial-derived free radicals and lipid peroxidation. Sodium-dependent glutamate uptake, which is mostly mediated by the glutamate transporter GLAST expressed by Müller glial cells, was reduced dose-dependently by rotenone with no changes in GLAST expression. Our findings suggest that complex I-derived free radicals and disruption of glutamate transport might represent key elements for explaining the selective retinal ganglion cell death in LHON.

  14. Determination of glutamate uptake by high performance liquid chromatography (HPLC) in preparations of retinal tissue.

    PubMed

    Moraes, Edinaldo Rogério da Silva; Grisolia, Alan Barroso Araújo; Oliveira, Karen Renata Matos; Picanço-Diniz, Domingos Luiz Wanderley; Crespo-López, Maria Elena; Maximino, Caio; Batista, Evander de Jesus Oliveira; Herculano, Anderson Manoel

    2012-10-15

    The present study describes a simple and efficient method utilizing high performance liquid chromatography (HPLC) coupled to fluorescence detection for the determination of kinetic parameters of glutamate uptake in nervous tissue. Retinal tissue obtained from 7-day-old chicks was incubated with known concentrations of glutamate (50-2000 μM) for 10 min, and the levels of the o-phtaldehyde (OPA)-derivatized neurotransmitter in the incubation medium were measured. By assessing the difference between initial and final concentrations of glutamate in the medium, a saturable uptake mechanism was characterized (K(m)=8.2 and V(max)=9.8 nmol/mg protein/min). This measure was largely sodium- and temperature-dependent, strongly supporting that the mechanism for concentration decrements is indeed uptake by high-affinity transporters. Added to this, our results also demonstrated that zinc chloride (an inhibitor of glutamate/aspartate transporters) evoked a concentration-dependent decrease in glutamate uptake, demonstrating the specificity of our methodology. Overall, the present work characterizes an alternative methodology to evaluate glutamate uptake in nervous tissue using HPLC. This approach could be an important tool for studies associated to the characterization of minute alterations in glutamate transport related with central nervous system injury.

  15. Ferulic Acid Suppresses Glutamate Release Through Inhibition of Voltage-Dependent Calcium Entry in Rat Cerebrocortical Nerve Terminals

    PubMed Central

    Lin, Tzu Yu; Lu, Cheng Wei; Huang, Shu-Kuei

    2013-01-01

    Abstract This study investigated the effects and possible mechanism of ferulic acid, a naturally occurring phenolic compound, on endogenous glutamate release in the nerve terminals of the cerebral cortex in rats. Results show that ferulic acid inhibited the release of glutamate evoked by the K+ channel blocker 4-aminopyridine (4-AP). The effect of ferulic acid on the evoked glutamate release was prevented by chelating the extracellular Ca2+ ions, but was insensitive to the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate. Ferulic acid suppressed the depolarization-induced increase in a cytosolic-free Ca2+ concentration, but did not alter 4-AP–mediated depolarization. Furthermore, the effect of ferulic acid on evoked glutamate release was abolished by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but not by blocking ryanodine receptors or mitochondrial Na+/Ca2+ exchange. These results show that ferulic acid inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca2+ entry. PMID:23342970

  16. Assignment of the gene coding for the human high-affinity glutamate transporter EAAC1 to 9p24: Potential role in dicarboxylic aminoaciduria and neurodegenerative disorders

    SciTech Connect

    Smith, C.P.; Kanai, Y.; Stelzner, M.; Hediger, M.A.; Weremowicz, S.; Morton, C.C. )

    1994-03-15

    Functional defects of high-affinity glutamate transporters have been implicated in the pathophysiology of neurodegenerative diseases such as amyotrophic lateral sclerosis. In small intestine and kidney, in which the high-affinity glutamate transporter mediates net absorption of glutamate and aspartate across epithelial cells, an inborn error of glutamate transport is thought to cause dicarboxylic aminoaciduria. This disorder is characterized by increased urinary excretion of glutamate and aspartate and is, in general, associated with neurologic and developmental abnormalities. Recently, the authors isolated a cDNA encoding a high-affinity glutamate transporter (EAAC1) that also transports aspartate but not other amino acids. EAAC1 is ubiquitously expressed throughout the body, particularly in brain (neurons), intestine, and kidney. Here, the authors present mapping of the chromosome location of EAAC1 using Southern analysis of a panel of human/rodent somatic cell hybrids and fluorescence in situ hybridization (FISH). Southern analysis of EcoRI-digested DNA gave bands at 6.5, 5.6, 5.1, and 1.2 kb for human genomic DNA; 7.5 kb for mouse genomic DNA; and 7.3, 3.2, and 1 kb for hamster genomic DNA. All four human EAAC1-specific bands were observed in the lane corresponding to the human/Chinese hamster hybrid containing chromosome 9 but not in lanes corresponding to any other hybrid. Because the human/Chinese hamster hybrid is the only one retaining chromosome 9, this result unambiguously assigns human EAAC1 to chromosome 9. For precise chromosome assignment of the human EAAC1 gene, they employed FISH. Map position of the EAAC1 probe was assigned by visual inspection of the fluorescent signal on the DAPI-stained metaphase chromosomes. The human EAAC1 gene was assigned to 9p24.

  17. Meningococcal internalization into human endothelial and epithelial cells is triggered by the influx of extracellular L-glutamate via GltT L-glutamate ABC transporter in Neisseria meningitidis.

    PubMed

    Takahashi, Hideyuki; Kim, Kwang Sik; Watanabe, Haruo

    2011-01-01

    Meningococcal internalization into human cells is likely to be a consequence of meningococcal adhesion to human epithelial and endothelial cells. Here, we identified three transposon mutants of Neisseria meningitidis that were primarily defective in the internalization of human brain microvascular endothelial cells (HBMEC), with insertions occurring in the gltT (a sodium-independent L-glutamate transporter) gene or its neighboring gene, NMB1964 (unknown function). NMB1964 was tentatively named gltM in this study because of the presence of a mammalian cell entry (MCE)-related domain in the deduced amino acid sequences. The null ΔgltT-ΔgltM N. meningitidis mutant was also defective in the internalization into human umbilical vein endothelial cells and the human lung carcinoma epithelial cell line A549, and the defect was suppressed by transcomplementation of the mutants with gltT(+)-gltM(+) genes. The intracellular survival of the ΔgltT-ΔgltM mutant in HBMEC was not largely different from that of the wild-type strain under our experimental conditions. Introduction of a1-bp deletion and amber or ochre mutations in gltT-gltM genes resulted in the loss of efficient internalization into HBMEC. The defect in meningococcal internalization into HBMEC and L-glutamate uptake in the ΔgltT-ΔgltM mutant were suppressed only in strains expressing both GltT and GltM proteins. The efficiency of meningococcal invasion to HBMEC decreased under L-glutamate-depleted conditions. Furthermore, ezrin, a key membrane-cytoskeleton linker, accumulated beneath colonies of the gltT(+)-gltM(+) N. meningitidis strain but not of the ΔgltT-ΔgltM mutant. These findings suggest that l-glutamate influx via the GltT-GltM L-glutamate ABC transporter serves as a cue for N. meningitidis internalization into host cells. PMID:20956569

  18. Localization of vesicular glutamate transporter 2 mRNA in the dorsal root ganglion of the pigeon (Columba livia).

    PubMed

    Atoji, Y; Islam, M R

    2009-11-01

    Our previous study showed localization of glutamate receptor 1 (GluR1) mRNA in neurons of the pigeon spinal cord, suggesting glutamatergic input from intrinsic and extrinsic origins. The present study examined localization of vesicular glutamate transporter 2 (VGLUT2) mRNA to confirm an extrinsic origin of glutamatergic neurons in the dorsal root ganglion (DRG). GluR1 and GluR2 mRNAs were examined in DRG and spinal cord to seek projection regions from VGLUT2 mRNA-expressing neurons. VGLUT2 mRNA was expressed in most DRG neurons and labelling intensity varied from weakly to intensely. Intense VGLUT2 mRNA expression was mainly seen in medium to large neurons. GluR1 and GluR2 mRNAs were expressed in the dorsal horn and GluR2 mRNA signal was also seen in the marginal nucleus. The results suggest that the pigeon DRG has an extrinsic glutamatergic origin that project to the dorsal horn and marginal nucleus of the spinal cord. PMID:19821818

  19. Expression of vesicular glutamate transporters in transient receptor potential melastatin 8 (TRPM8)-positive dental afferents in the mouse.

    PubMed

    Kim, Y S; Kim, T H; McKemy, D D; Bae, Y C

    2015-09-10

    Transient receptor potential melastatin 8 (TRPM8) is activated by innocuous cool and noxious cold and plays a crucial role in cold-induced acute pain and pain hypersensitivity. To help understand the mechanism of TRPM8-mediated cold perception under normal and pathologic conditions, we used light microscopic immunohistochemistry and Western blot analysis in mice expressing a genetically encoded axonal tracer in TRPM8-positive (+) neurons. We investigated the coexpression of TRPM8 and vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 in the trigeminal ganglion (TG) and the dental pulp before and after inducing pulpal inflammation. Many TRPM8+ neurons in the TG and axons in the dental pulp expressed VGLUT2, while none expressed VGLUT1. TRPM8+ axons were dense in the pulp horn and peripheral pulp and also frequently observed in the dentinal tubules. Following pulpal inflammation, the proportion of VGLUT2+ and of VGLUT2+/TRPM8+ neurons increased significantly, whereas that of TRPM8+ neurons remained unchanged. Our findings suggest the existence of VGLUT2 (but not VGLUT1)-mediated glutamate signaling in TRPM8+ neurons possibly underlying the cold-induced acute pain and hypersensitivity to cold following pulpal inflammation. PMID:26166724

  20. Inhibition of glutamate uptake by a polypeptide toxin (phoneutriatoxin 3-4) from the spider Phoneutria nigriventer.

    PubMed

    Reis, H J; Prado, M A; Kalapothakis, E; Cordeiro, M N; Diniz, C R; De Marco, L A; Gomez, M V; Romano-Silva, M A

    1999-10-15

    Glutamate concentration increases significantly in the extracellular compartment during brain ischaemia and anoxia. This increase has an important Ca(2+)-independent component, which is due in part to the reversal of glutamate transporters of the plasma membrane of neurons and glia. The toxin phoneutriatoxin 3-4 (Tx3-4) from the spider Phoneutria nigriventer has been reported to decrease the evoked glutamate release from synaptosomes by inhibiting Ca(2+) entry via voltage-dependent Ca(2+) channels. However, we report here that Tx3-4 is also able to inhibit the uptake of glutamate by synaptosomes in a time-dependent manner and that this inhibition in turn leads to a decrease in the Ca(2+)-independent release of glutamate. No other polypeptide toxin so far described has this effect. Our results suggest that Tx3-4 can be a valuable tool in the investigation of function and dysfunction of glutamatergic neurotransmission in diseases such as ischaemia.

  1. Hispidulin inhibits the release of glutamate in rat cerebrocortical nerve terminals

    SciTech Connect

    Lin, Tzu-Yu; Lu, Cheng-Wei; Wang, Chia-Chuan; Lu, Jyh-Feng; Wang, Su-Jane

    2012-09-01

    Hispidulin, a naturally occurring flavone, has been reported to have an antiepileptic profile. An excessive release of glutamate is considered to be related to neuropathology of epilepsy. We investigated whether hispidulin affected endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes) and explored the possible mechanism. Hispidulin inhibited the release of glutamate evoked by the K{sup +} channel blocker 4-aminopyridine (4-AP). The effects of hispidulin on the evoked glutamate release were prevented by the chelation of extracellular Ca{sup 2+} ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate did not have any effect on hispidulin action. Hispidulin reduced the depolarization-induced increase in cytosolic free Ca{sup 2+} concentration ([Ca{sup 2+}]{sub C}), but did not alter 4-AP-mediated depolarization. Furthermore, the effect of hispidulin on evoked glutamate release was abolished by blocking the Ca{sub v}2.2 (N-type) and Ca{sub v}2.1 (P/Q-type) channels, but not by blocking ryanodine receptors or mitochondrial Na{sup +}/Ca{sup 2+} exchange. Mitogen-activated protein kinase kinase (MEK) inhibition also prevented the inhibitory effect of hispidulin on evoked glutamate release. Western blot analyses showed that hispidulin decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, a major presynaptic substrate for ERK; this decrease was also blocked by the MEK inhibitor. Moreover, the inhibition of glutamate release by hispidulin was strongly attenuated in mice without synapsin I. These results show that hispidulin inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca{sup 2+} entry and ERK/synapsin I signaling pathway. -- Highlights: ► Hispidulin inhibited glutamate release from rat

  2. Co-localization of glutamic acid decarboxylase and vesicular GABA transporter in cytochrome oxidase patches of macaque striate cortex.

    PubMed

    Adams, Daniel L; Economides, John R; Horton, Jonathan C

    2015-01-01

    The patches in primary visual cortex constitute hot spots of metabolic activity, manifested by enhanced levels of cytochrome oxidase (CO) activity. They are also labeled preferentially by immunostaining for glutamic acid decarboxylase (GAD), γ-aminobutyric acid (GABA), and parvalbumin. However, calbindin shows stronger immunoreactivity outside patches. In light of this discrepancy, the distribution of the vesicular GABA transporter (VGAT) was examined in striate cortex of two normal macaques. VGAT immunoreactivity was strongest in layers 4B, 4Cα, and 5. In tangential sections, the distribution of CO, GAD, and VGAT was compared in layer 2/3. There was a close match between all three labels. This finding indicates that GABA synthesis is enriched in patches, and that inhibitory synapses are more active in patches than interpatches. PMID:26579566

  3. Arundic acid attenuates retinal ganglion cell death by increasing glutamate/aspartate transporter expression in a model of normal tension glaucoma

    PubMed Central

    Yanagisawa, M; Aida, T; Takeda, T; Namekata, K; Harada, T; Shinagawa, R; Tanaka, K

    2015-01-01

    Glaucoma is the second leading cause of blindness worldwide and is characterized by gradual visual impairment owing to progressive loss of retinal ganglion cells (RGCs) and their axons. Glutamate excitotoxicity has been implicated as a mechanism of RGC death in glaucoma. Consistent with this claim, we previously reported that glutamate/aspartate transporter (GLAST)-deficient mice show optic nerve degeneration that is similar to that observed in glaucoma. Therefore, drugs that upregulate GLAST may be useful for neuroprotection in glaucoma. Although many compounds are known to increase the expression of another glial glutamate transporter, EAAT2/GLT1, few compounds are shown to increase GLAST expression. Arundic acid is a glial modulating agent that ameliorates delayed ischemic brain damage by attenuating increases in extracellular glutamate. We hypothesized that arundic acid neuroprotection involves upregulation of GLAST. To test this hypothesis, we examined the effect of arundic acid on GLAST expression and glutamate uptake. We found that arundic acid induces GLAST expression in vitro and in vivo. In addition, arundic acid treatment prevented RGC death by upregulating GLAST in heterozygous (GLAST+/−) mice. Furthermore, arundic acid stimulates the human GLAST ortholog, EAAT1, expression in human neuroglioblastoma cells. Thus, discovering compounds that can enhance EAAT1 expression and activity may be a novel strategy for therapeutic treatment of glaucoma. PMID:25789968

  4. Brief Report: Glutamate Transporter Gene ("SLC1A1") Single Nucleotide Polymorphism (rs301430) and Repetitive Behaviors and Anxiety in Children with Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Gadow, Kenneth D.; Roohi, Jasmin; DeVincent, Carla J.; Kirsch, Sarah; Hatchwell, Eli

    2010-01-01

    Investigated association of single nucleotide polymorphism (SNP) rs301430 in glutamate transporter gene ("SLC1A1") with severity of repetitive behaviors (obsessive-compulsive behaviors, tics) and anxiety in children with autism spectrum disorder (ASD). Mothers and/or teachers completed a validated DSM-IV-referenced rating scale for 67 children…

  5. Effects of glutamate decarboxylase and gamma-aminobutyric acid (GABA) transporter on the bioconversion of GABA in engineered Escherichia coli.

    PubMed

    Le Vo, Tam Dinh; Kim, Tae Wan; Hong, Soon Ho

    2012-05-01

    Gamma-aminobutyric acid (GABA) is a non-essential amino acid and a precursor of pyrrolidone, a monomer of nylon 4. GABA can be biosynthesized through the decarboxylation of L: -glutamate by glutamate decarboxylase. In this study, the effects of glutamate decarboxylase (gadA, gadB), glutamate/GABA antiporter (gadC) and GABA aminotransferase (gabT) on GABA production were investigated in Escherichia coli. Glutamate decarboxylase was overexpressed alone or with the glutamate/GABA antiporter to enhance GABA synthesis. GABA aminotransferase, which redirects GABA into the TCA cycle, was knock-out mutated. When gadB and gadC were co-overexpressed in the gabT mutant strain, a final GABA concentration of 5.46 g/l was obtained from 10 g/l of monosodium glutamate (MSG), which corresponded to a GABA yield of 89.5%.

  6. Glutamate and Neurodegenerative Disease

    NASA Astrophysics Data System (ADS)

    Schaeffer, Eric; Duplantier, Allen

    As the main excitatory neurotransmitter in the mammalian central nervous system, glutamate is critically involved in most aspects of CNS function. Given this critical role, it is not surprising that glutamatergic dysfunction is associated with many CNS disorders. In this chapter, we review the literature that links aberrant glutamate neurotransmission with CNS pathology, with a focus on neurodegenerative diseases. The biology and pharmacology of the various glutamate receptor families are discussed, along with data which links these receptors with neurodegenerative conditions. In addition, we review progress that has been made in developing small molecule modulators of glutamate receptors and transporters, and describe how these compounds have helped us understand the complex pharmacology of glutamate in normal CNS function, as well as their potential for the treatment of neurodegenerative diseases.

  7. Variation of the genes encoding the human glutamate EAAT2, serotonin and dopamine transporters and Susceptibility to idiopathic generalized epilepsy.

    PubMed

    Sander, T; Berlin, W; Ostapowicz, A; Samochowiec, J; Gscheidel, N; Hoehe, M R

    2000-08-01

    Several interacting genetic factors are likely to be involved in the epileptogenesis of idiopathic generalized epilepsies (IGE). Neurotransmitter transporters play a central role in the fine tuning of neurotransmission by removal of released neurotransmitters from the synaptic cleft. The present association study tested the hypotheses that variation of the genes encoding neurotransmitter transporters confers susceptibility to IGE. The genotypes of 133 German IGE subjects and 223 ethnically matched controls were assessed for DNA polymorphisms of genes encoding the glutamate (EAAT2), the serotonin (SERT), and dopamine (DAT) transporters. To increase genetic homogeneity, a subgroup of 76 patients with idiopathic absence epilepsy (IAE) was analyzed separately. We found no evidence for an allelic association of either the silent G603A substitution polymorphism in exon 5 of the EAAT2 gene or the regulatory promoter polymorphism of the SERT gene with either IGE or IAE. The frequency of the nine-copy allele of the 40 base pair repeat polymorphism in the 3' un pop popd region of the DAT gene was significantly increased in the IGE patients (chi2 = 4.11, degrees of freedom (d.f.) = 1, P = 0.043) and, in particular, in the IAE patients (chi2 = 7.81, d.f. = 1, P = 0.005) compared with the controls. The present findings strengthen previous evidence that genetic variation of the DAT gene modulates neuronal network excitability and contributes to the epileptogenesis of IAE.

  8. Expression of vesicular glutamate transporter 3 mRNA in the brain and retina of the pigeon.

    PubMed

    Atoji, Yasuro; Karim, Mohammad Rabiul

    2014-11-01

    Vesicular glutamate transporters (vGluTs), which accumulate glutamate into synaptic vesicles, are classified into three subtypes in mammalian brains: vGluT1, vGluT2, and vGluT3. VGluT3 is localized in non-glutamatergic neurons of the brain and retinal amacrine cells. In birds, the vGluT3 genome is found, but its distribution in the brain or retina is unknown. The present study was conducted to analyze vGluT3 cDNA sequence and elucidate its distribution in the pigeon brain and retina. The vGluT3 cDNA comprises 1761bp and showed 95% and 88% identity to the chicken and zebra finch vGluT3 cDNAs, respectively, and 74% identity to human vGluT3 cDNA. In situ hybridization revealed that the vGluT3 mRNA was expressed in neurons of the caudal linear nucleus (LC) of the brain and in amacrine cells of the inner nuclear layer of the retina. A combination of in situ hybridization and serotonin immunohistochemistry revealed three types of stained cells in LC and retina: vGluT3(+)/serotonin(+), vGluT3(+)/serotonin(-), and vGluT3(-)/serotonin(+). The vGluT3(+)/serotonin(+) cells were approximately 22% in LC and 16% in the retina. The present results suggest that the pigeon vGluT3 mRNA is comparable with the mammalian type.

  9. Functional Characterization of a Vesicular Glutamate Transporter in an Interneuron That Makes Excitatory and Inhibitory Synaptic Connections in a Molluscan Neural Circuit.

    PubMed

    Jing, Jian; Alexeeva, Vera; Chen, Song-An; Yu, Ke; Due, Michael R; Tan, Li-Nuo; Chen, Ting-Ting; Liu, Dan-Dan; Cropper, Elizabeth C; Vilim, Ferdinand S; Weiss, Klaudiusz R

    2015-06-17

    Understanding circuit function requires the characterization of component neurons and their neurotransmitters. Previous work on radula protraction in the Aplysia feeding circuit demonstrated that critical neurons initiate feeding via cholinergic excitation. In contrast, it is less clear how retraction is mediated at the interneuronal level. In particular, glutamate involvement was suggested, but was not directly confirmed. Here we study a suspected glutamatergic retraction interneuron, B64. We used the representational difference analysis (RDA) method to successfully clone an Aplysia vesicular glutamate transporter (ApVGLUT) from B64 and from a glutamatergic motor neuron B38. Previously, RDA was used to characterize novel neuropeptides. Here we demonstrate its utility for characterizing other types of molecules. Bioinformatics suggests that ApVGLUT is more closely related to mammalian VGLUTs than to Drosophila and Caenorhabditis elegans VGLUTs. We expressed ApVGLUT in a cell line, and demonstrated that it indeed transports glutamate in an ATP and proton gradient-dependent manner. We mapped the ApVGLUT distribution in the CNS using in situ hybridization and immunocytochemistry. Further, we demonstrated that B64 is ApVGLUT positive, supporting the idea that it is glutamatergic. Although glutamate is primarily an excitatory transmitter in the mammalian CNS, B64 elicits inhibitory PSPs in protraction neurons to terminate protraction and excitatory PSPs in retraction neurons to maintain retraction. Pharmacological data indicated that both types of PSPs are mediated by glutamate. Thus, glutamate mediates the dual function of B64 in Aplysia. More generally, our systematic approaches based on RDA may facilitate analyses of transmitter actions in small circuits with identifiable neurons.

  10. The effects of combined application of inorganic Martian dust simulant and carbon dots on glutamate transport rat brain nerve terminals

    NASA Astrophysics Data System (ADS)

    Borisova, Tatiana; Krisanova, Natalia; Nazarova, Anastasiya; Borysov, Arseniy; Pastukhov, Artem; Pozdnyakova, Natalia; Dudarenko, Marina

    2016-07-01

    During inhalation, nano-/microsized particles are efficiently deposited in nasal, tracheobronchial, and alveolar regions and can be transported to the central nervous system (Oberdorster et al., 2004). Recently, the research team of this study found the minor fractions of nanoparticles with the size ~ 50 -60 nm in Lunar and Martian dust stimulants (JSC-1a and JSC, ORBITEC Orbital Technologies Corporation, Madison, Wisconsin), whereas the average size of the simulants was 1 mm and 4mm, respectively (Krisanova et al., 2013). Also, the research team of this study discovered new phenomenon - the neuromodulating and neurotoxic effect of carbon nano-sized particles - Carbon dots (C-dots), originated from ash of burned carbon-containing product (Borisova et al, 2015). The aims of this study was to analyse acute effects of upgraded stimulant of inorganic Martian dust derived from volcanic ash (JSC-1a/JSC, ORBITEC Orbital Technologies Corporation, Madison, Wisconsin) by the addition of carbon components, that is, carbon dots, on the key characteristic of synaptic neurotransmission. Acute administration of carbon-containing Martian dust analogue resulted in a significant decrease in transporter-mediated uptake of L-[14C]glutamate (the major excitatory neurotransmitter) by isolated rat brain nerve terminals. The ambient level of the neurotransmitter in the preparation of nerve terminals increased in the presence of carbon dot-contained Martian dust analogue. These effects were associated with action of carbon component of the upgraded Martian dust stimulant but not with its inorganic constituent.

  11. Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons.

    PubMed

    Widera, Darius; Klenke, Christin; Nair, Deepak; Heidbreder, Meike; Malkusch, Sebastian; Sibarita, Jean-Baptiste; Choquet, Daniel; Kaltschmidt, Barbara; Heilemann, Mike; Kaltschmidt, Christian

    2016-10-01

    Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The caliber of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. We quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient ([Formula: see text]) in neurites from [Formula: see text] to [Formula: see text] after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from [Formula: see text] to [Formula: see text], whereas a velocity increase from [Formula: see text] to [Formula: see text] was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons. PMID:27226975

  12. Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons.

    PubMed

    Widera, Darius; Klenke, Christin; Nair, Deepak; Heidbreder, Meike; Malkusch, Sebastian; Sibarita, Jean-Baptiste; Choquet, Daniel; Kaltschmidt, Barbara; Heilemann, Mike; Kaltschmidt, Christian

    2016-10-01

    Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The caliber of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. We quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient ([Formula: see text]) in neurites from [Formula: see text] to [Formula: see text] after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from [Formula: see text] to [Formula: see text], whereas a velocity increase from [Formula: see text] to [Formula: see text] was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.

  13. Berberine Inhibits the Release of Glutamate in Nerve Terminals from Rat Cerebral Cortex

    PubMed Central

    Lu, Cheng-Wei; Huang, Shu-Kuei; Wang, Su-Jane

    2013-01-01

    Berberine, an isoquinoline plant alkaloid, protects neurons against neurotoxicity. An excessive release of glutamate is considered to be one of the molecular mechanisms of neuronal damage in several neurological diseases. In this study, we investigated whether berberine could affect endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes) and explored the possible mechanism. Berberine inhibited the release of glutamate evoked by the K+ channel blocker 4-aminopyridine (4-AP), and this phenomenon was prevented by the chelating extracellular Ca2+ ions and the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate. Inhibition of glutamate release by berberine was not due to it decreasing synaptosomal excitability, because berberine did not alter 4-AP-mediated depolarization. The inhibitory effect of berberine on glutamate release was associated with a reduction in the depolarization-induced increase in cytosolic free Ca2+ concentration. Involvement of the Cav2.1 (P/Q-type) channels in the berberine action was confirmed by blockade of the berberine-mediated inhibition of glutamate release by the Cav2.1 (P/Q-type) channel blocker ω-agatoxin IVA. In addition, the inhibitory effect of berberine on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Berberine decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synapsin I, the main presynaptic target of ERK; this decrease was also blocked by the MEK inhibition. Moreover, the inhibitory effect of berberine on evoked glutamate release was prevented in nerve terminals from mice lacking synapsin I. Together, these results indicated that berberine inhibits glutamate release from rats cortical synaptosomes, through the suppression of presynaptic Cav2.1 channels and ERK/synapsin I signaling

  14. COMPARISON OF THE ONTOGENY OF THE VESICULAR GLUTAMATE TRANSPORTER 3 (VGLUT3) WITH VGLUT1 AND VGLUT2 IN THE RAT RETINA

    PubMed Central

    Stella, Salvatore L.; Li, Stefanie; Sabatini, Andrea; Vila, Alejandro; Brecha, Nicholas C.

    2008-01-01

    Glutamate is the major excitatory neurotransmitter in the retina, and most glutamatergic neurons express one of the three known vesicular glutamate transporters (VGLUT1, 2, or 3). However, the expression profiles of these transporters vary greatly in the retina. VGLUT1 is expressed by photoreceptor and bipolar cell terminals, and VGLUT2 appears to be predominately expressed by ganglion cells, and perhaps Müller cells, cone photoreceptor terminals, and horizontal cells in some species. The discovery of a third vesicular glutamate transporter, VGLUT3, has brought about speculation concerning its role and function based on its expression in amacrine cells. To address this we studied the postnatal development of VGLUT3 from day 0 through adult in the rat retina, and compared this with the expression patterns of VGLUT1 and VGLUT2. VGLUT3 expression was restricted to a population of amacrine cells. Expression of VGLUT3 was first observed at postnatal day 10 (P10) in the soma and some processes, which extensively arborized in both the ON and OFF sublamina of the IPL by P15. In contrast, VGLUT1 and VGLUT2 expression appeared earlier than VGLUT3; with VGLUT1 initially detected at P5 in photoreceptor terminals and P6 in bipolar terminals, and VGLUT2 immunoreactivity initially detected at P0 in ganglion cell bodies, and remained prominent throughout all stages of development. Interestingly, VGLUT3 has extensive somatic expression throughout development, which could be involved in non-synaptic modulation by glutamate in developing retina, and could influence trophic and extra-synaptic neuronal signaling by glutamate in the inner retina. PMID:18482716

  15. The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2.

    PubMed

    Simonin, Alexandre; Montalbetti, Nicolas; Gyimesi, Gergely; Pujol-Giménez, Jonai; Hediger, Matthias A

    2015-12-18

    Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous GltPh coordinates L-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na(+) over Li(+). S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for L-aspartate over D-aspartate and L-glutamate, and lost their selectivity for Na(+) over Li(+). Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.

  16. Motor neuron-specific overexpression of the presynaptic choline transporter: impact on motor endurance and evoked muscle activity.

    PubMed

    Lund, D; Ruggiero, A M; Ferguson, S M; Wright, J; English, B A; Reisz, P A; Whitaker, S M; Peltier, A C; Blakely, R D

    2010-12-29

    The presynaptic, hemicholinium-3 sensitive, high-affinity choline transporter (CHT) supplies choline for acetylcholine (ACh) synthesis. In mice, a homozygous deletion of CHT (CHT-/-) leads to premature cessation of spontaneous or evoked neuromuscular signaling and is associated with perinatal cyanosis and lethality within 1 h. Heterozygous (CHT+/-) mice exhibit diminished brain ACh levels and demonstrate an inability to sustain vigorous motor activity. We sought to explore the contribution of CHT gene dosage to motor function in greater detail using transgenic mice where CHT is expressed under control of the motor neuron promoter Hb9 (Hb9:CHT). On a CHT-/- background, the Hb9:CHT transgene conferred mice with the ability to move and breath for a postnatal period of ∼24 h, thus increasing survival. Conversely, Hb9:CHT expression on a wild-type background (CHT+/+;Hb9:CHT) leads to an increased capacity for treadmill running compared to wild-type littermates. Analysis of the stimulated compound muscle action potential (CMAP) in these animals under basal conditions established that CHT+/+;Hb9:CHT mice display an unexpected, bidirectional change, producing either elevated or reduced CMAP amplitude, relative to CHT+/+ animals. To examine whether these two groups arise from underlying changes in synaptic properties, we used high-frequency stimulation of motor axons to assess CMAP recovery kinetics. Although CHT+/+; Hb9:CHT mice in the two groups display an equivalent, time-dependent reduction in CMAP amplitude, animals with a higher basal CMAP amplitude demonstrate a significantly enhanced rate of recovery. To explain our findings, we propose a model whereby CHT support for neuromuscular signaling involves contributions to ACh synthesis as well as cholinergic synaptic vesicle availability.

  17. Stimulation of α7 nicotinic acetylcholine receptor regulates glutamate transporter GLAST via basic fibroblast growth factor production in cultured cortical microglia.

    PubMed

    Morioka, Norimitsu; Harano, Sakura; Tokuhara, Masato; Idenoshita, Yuko; Zhang, Fang Fang; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

    2015-11-01

    The α7 nicotinic acetylcholine (nACh) receptor expressed in microglia has a crucial role in neuroprotection. Simulation of α7 nACh receptor leads to increased expression of glutamate/aspartate transporter (GLAST), which in turn decreases synaptic glutamate levels. However, the upregulation of GLAST in cultured rat cortical microglia appears long after (over 18 h) stimulation of the α7 nACh receptor with nicotine. Thus, the current study elucidated the pathway responsible for the induction of GLAST expression in cultured cortical microglia. Nicotine-induced GLAST mRNA expression was significantly inhibited by cycloheximide pretreatment, indicating that a protein intermediary, such as a growth factor, is required for GLAST expression. The expression of fibroblast growth factor-2 (FGF-2) mRNA in cortical microglia was significantly increased 6 and 12h after treatment with nicotine, and this increase was potently inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The treatment with nicotine also significantly increased FGF-2 protein expression. Furthermore, treatment with recombinant FGF-2 increased GLAST mRNA, protein expression and (14)C-glutamate uptake, a functional measurement of GLAST activity. Conversely, pretreatment with PD173074, an inhibitor of FGF receptor (FGFR) tyrosine kinase, significantly prevented the nicotine-induced expression of GLAST mRNA, its protein and (14)C-glutamate uptake. Reverse transcription polymerase chain reaction confirmed FGFR1 mRNA expression was confined to cultured cortical microglia. Together, the current findings demonstrate that the neuroprotective effect of activation of microglial α7 nACh receptors could be due to the expression of FGF-2, which in turn increases GLAST expression, thereby clearing glutamate from synapse and decreasing glutamate neurotransmission.

  18. Gabapentin loses efficacy over time after nerve injury in rats: role of glutamate transporter-1 in the locus coeruleus.

    PubMed

    Kimura, Masafumi; Eisenach, James C; Hayashida, Ken-Ichiro

    2016-09-01

    Despite being one of the first-choice analgesics for chronic neuropathic pain, gabapentin sometimes fails to provide analgesia, but the mechanisms for this lack of efficacy is unclear. Rats with nerve injury including L5-L6 spinal nerve ligation (SNL) respond uniformly and well to gabapentin, but many of these studies are performed within just a few weeks of injury, questioning their relevance to chronic neuropathic pain. In this study, intraperitoneal gabapentin showed a time-dependently reduction in antihypersensitivity after SNL, associated with downregulation of astroglial glutamate transporter-1 (GLT-1) in the locus coeruleus (LC). Consistently, SNL also time-dependently increased basal but masked gabapentin-induced noradrenergic neuronal activity in the LC. In rats 2 weeks after SNL, knock-down of GLT-1 in the LC reduced the antihypersensitivity effect of gabapentin. In rats 8 weeks after SNL, increasing GLT-1 expression by histone deacetylase inhibitor valproate restored the antihypersensitivity effect of gabapentin, associated with restored gabapentin-induced noradrenergic neuronal activity in the LC and subsequent spinal noradrenaline release. Knock-down of GLT-1 in the LC reversed the effect of valproate to restore gabapentin-induced antihypersensitivity. In addition, the antihypersensitivity effect of the intrathecal α2-adrenoceptor agonist clonidine also decreased with time after SNL injury. These results suggest that downregulation of GLT-1 in the LC and reduced spinal noradrenergic inhibition contribute to impaired analgesic efficacy from gabapentin in chronic neuropathic pain and that valproate can rescue this impaired efficacy.

  19. The importance of glutamate, glycine, and {gamma}-aminobutyric acid transport and regulation in manganese, mercury and lead neurotoxicity

    SciTech Connect

    Fitsanakis, Vanessa A.; Aschner, Michael . E-mail: michael.aschner@vanderbilt.edu

    2005-05-01

    Historically, amino acids were studied in the context of their importance in protein synthesis. In the 1950s, the focus of research shifted as amino acids were recognized as putative neurotransmitters. Today, many amino acids are considered important neurochemicals. Although many amino acids play a role in neurotransmission, glutamate (Glu), glycine (Gly), and {gamma}-aminobutyric acid (GABA) are among the more prevalent and better understood. Glu, the major excitatory neurotransmitter, and Gly and GABA, the major inhibitory neurotransmitters, in the central nervous system, are known to be tightly regulated. Prolonged exposure to environmental toxicants, such as manganese (Mn), mercury (Hg), or lead (Pb), however, can lead to dysregulation of these neurochemicals and subsequent neurotoxicity. While the ability of these metals to disrupt the regulation of Glu, Gly and GABA have been studied, few articles have examined the collective role of these amino acids in the respective metal's mechanism of toxicity. For each of the neurotransmitters above, we will provide a brief synopsis of their regulatory function, including the importance of transport and re-uptake in maintaining their optimal function. Additionally, the review will address the hypothesis that aberrant homeostasis of any of these amino acids, or a combination of the three, plays a role in the neurotoxicity of Mn, Hg, or Pb.

  20. Chicago sky blue 6B, a vesicular glutamate transporters inhibitor, attenuates methamphetamine-induced hyperactivity and behavioral sensitization in mice.

    PubMed

    He, Zongsheng; Yan, Lingdi; Yong, Zheng; Dong, Zhaoqi; Dong, Huajin; Gong, Zehui

    2013-02-15

    Several lines of evidence demonstrate that glutamatergic system plays an important role in drug addiction. The present study was designed to investigate the effects of Chicago sky blue 6B (CSB6B), a vesicular glutamate transporters (VGLUTs) inhibitor, on methamphetamine (METH)-induced behaviors in mice. Mice were induced behavioral sensitization to METH by subcutaneous injection of 1mg/kg METH once daily for 7 days and then challenged with 1mg/kg METH in 14th day. Intracerebroventricular administration of CSB6B (7.5μg) 2.5h prior to METH was to observe its effects on METH -induced behavioral sensitization. Our results showed that the expressions of behavioral sensitization were significantly attenuated by intracerebroventricular administration of CSB6B 2.5h prior to METH either during the development period or before methamphetamine challenge in mice, while CSB6B itself had no effect on locomotor activity. Meanwhile, pretreatment of CSB6B also attenuated hyperactivity caused by a single injection of METH in mice. These results demonstrated that CSB6B, a VGLUTs inhibitor, attenuated acute METH-induced hyperactivity and chronic METH-induced behavioral sensitization, which indicated that VGLUTs were involved in the effect of chronic METH-induced behavioral sensitization and may be a new target against the addiction of METH.

  1. Glutamic acid as anticancer agent: An overview.

    PubMed

    Dutta, Satyajit; Ray, Supratim; Nagarajan, K

    2013-10-01

    The objective of the article is to highlight various roles of glutamic acid like endogenic anticancer agent, conjugates to anticancer agents, and derivatives of glutamic acid as possible anticancer agents. Besides these emphases are given especially for two endogenous derivatives of glutamic acid such as glutamine and glutamate. Glutamine is a derivative of glutamic acid and is formed in the body from glutamic acid and ammonia in an energy requiring reaction catalyzed by glutamine synthase. It also possesses anticancer activity. So the transportation and metabolism of glutamine are also discussed for better understanding the role of glutamic acid. Glutamates are the carboxylate anions and salts of glutamic acid. Here the roles of various enzymes required for the metabolism of glutamates are also discussed.

  2. Astroglial FMRP-dependent translational down-regulation of mGluR5 underlies glutamate transporter GLT1 dysregulation in the fragile X mouse

    PubMed Central

    Higashimori, Haruki; Morel, Lydie; Huth, James; Lindemann, Lothar; Dulla, Chris; Taylor, Amaro; Freeman, Mike; Yang, Yongjie

    2013-01-01

    Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by the loss-of-function of fragile X mental retardation protein (FMRP). The loss of FMRP function in neurons abolishes its suppression on mGluR1/5-dependent dendritic protein translation, enhancing mGluR1/5-dependent synaptic plasticity and other disease phenotypes in FXS. In this study, we describe a new activation function of FMRP in regulating protein expression in astroglial cells. We found that astroglial glutamate transporter subtype glutamate transporter 1 (GLT1) and glutamate uptake is significantly reduced in the cortex of fmr1−/− mice. Correspondingly, neuronal excitability is also enhanced in acute fmr1−/− (but not in fmr1+/+ control) cortical slices treated with low doses (10 μm) of the GLT1-specific inhibitor dihydrokainate (DHK). Using mismatched astrocyte and neuron co-cultures, we demonstrate that the loss of astroglial (but not neuronal) FMRP particularly reduces neuron-dependent GLT1 expression and glutamate uptake in co-cultures. Interestingly, protein (but not mRNA) expression and the (S)-3,5-dihydroxyphenylglycine-dependent Ca2+ responses of astroglial mGluR5 receptor are also selectively reduced in fmr1−/− astrocytes and brain slices, attenuating neuron-dependent GLT1 expression. Subsequent FMRP immunoprecipitation and QRT–PCR analysis showed that astroglial mGluR5 (but not GLT1) mRNA is associated with FMRP. In summary, our results provide evidence that FMRP positively regulates translational expression of mGluR5 in astroglial cells, and FMRP-dependent down-regulation of mGluR5 underlies GLT1 dysregulation in fmr1−/− astrocytes. The dysregulation of GLT1 and reduced glutamate uptake may potentially contribute to enhanced neuronal excitability observed in the mouse model of FXS. PMID:23396537

  3. Neurotoxic Potential of Lunar and Martian Dust: Influence on Em, Proton Gradient, Active Transport, and Binding of Glutamate in Rat Brain Nerve Terminals

    PubMed Central

    Krisanova, Natalia; Kasatkina, Ludmila; Sivko, Roman; Borysov, Arseniy; Nazarova, Anastasiya; Slenzka, Klaus; Borisova, Tatiana

    2013-01-01

    Abstract The harmful effects of lunar dust (LD) on directly exposed tissues are documented in the literature, whereas researchers are only recently beginning to consider its effects on indirectly exposed tissues. During inhalation, nano-/microsized particles are efficiently deposited in nasal, tracheobronchial, and alveolar regions and transported to the central nervous system. The neurotoxic potential of LD and martian dust (MD) has not yet been assessed. Glutamate is the main excitatory neurotransmitter involved in most aspects of normal brain function, whereas disturbances in glutamate homeostasis contribute to the pathogenesis of major neurological disorders. The research was focused on the analysis of the effects of LD/MD simulants (JSC-1a/JSC, derived from volcanic ash) on the key characteristics of glutamatergic neurotransmission. The average size of LD and MD particles (even minor fractions) before and after sonication was determined by dynamic light scattering. With the use of radiolabeled l-[14C]glutamate, it was shown that there is an increase in l-[14C]glutamate binding to isolated rat brain nerve terminals (synaptosomes) in low [Na+] media and at low temperature in the presence of LD. MD caused significantly lesser changes under the same conditions, whereas nanoparticles of magnetite had no effect at all. Fluorimetric experiments with potential-sensitive dye rhodamine 6G and pH-sensitive dye acridine orange showed that the potential of the plasma membrane of the nerve terminals and acidification of synaptic vesicles were not altered by LD/MD (and nanoparticles of magnetite). Thus, the unique effect of LD to increase glutamate binding to the nerve terminals was shown. This can have deleterious effects on extracellular glutamate homeostasis in the central nervous system and cause alterations in the ambient level of glutamate, which is extremely important for proper synaptic transmission. During a long-term mission, a combination of constant irritation

  4. Glutamate excitotoxicity and Ca2+-regulation of respiration: Role of the Ca2+ activated mitochondrial transporters (CaMCs).

    PubMed

    Rueda, Carlos B; Llorente-Folch, Irene; Traba, Javier; Amigo, Ignacio; Gonzalez-Sanchez, Paloma; Contreras, Laura; Juaristi, Inés; Martinez-Valero, Paula; Pardo, Beatriz; Del Arco, Araceli; Satrustegui, Jorgina

    2016-08-01

    Glutamate elicits Ca(2+) signals and workloads that regulate neuronal fate both in physiological and pathological circumstances. Oxidative phosphorylation is required in order to respond to the metabolic challenge caused by glutamate. In response to physiological glutamate signals, cytosolic Ca(2+) activates respiration by stimulation of the NADH malate-aspartate shuttle through Ca(2+)-binding to the mitochondrial aspartate/glutamate carrier (Aralar/AGC1/Slc25a12), and by stimulation of adenine nucleotide uptake through Ca(2+) binding to the mitochondrial ATP-Mg/Pi carrier (SCaMC-3/Slc25a23). In addition, after Ca(2+) entry into the matrix through the mitochondrial Ca(2+) uniporter (MCU), it activates mitochondrial dehydrogenases. In response to pathological glutamate stimulation during excitotoxicity, Ca(2+) overload, reactive oxygen species (ROS), mitochondrial dysfunction and delayed Ca(2+) deregulation (DCD) lead to neuronal death. Glutamate-induced respiratory stimulation is rapidly inactivated through a mechanism involving Poly (ADP-ribose) Polymerase-1 (PARP-1) activation, consumption of cytosolic NAD(+), a decrease in matrix ATP and restricted substrate supply. Glutamate-induced Ca(2+)-activation of SCaMC-3 imports adenine nucleotides into mitochondria, counteracting the depletion of matrix ATP and the impaired respiration, while Aralar-dependent lactate metabolism prevents substrate exhaustion. A second mechanism induced by excitotoxic glutamate is permeability transition pore (PTP) opening, which critically depends on ROS production and matrix Ca(2+) entry through the MCU. By increasing matrix content of adenine nucleotides, SCaMC-3 activity protects against glutamate-induced PTP opening and lowers matrix free Ca(2+), resulting in protracted appearance of DCD and protection against excitotoxicity in vitro and in vivo, while the lack of lactate protection during in vivo excitotoxicity explains increased vulnerability to kainite-induced toxicity in Aralar

  5. Concise Asymmetric Synthesis and Pharmacological Characterization of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes.

    PubMed

    Leuenberger, Michele; Ritler, Andreas; Simonin, Alexandre; Hediger, Matthias A; Lochner, Martin

    2016-05-18

    Glutamate is the major excitatory neurotransmitter in the mammalian brain. Its rapid clearance after the release into the synaptic cleft is vital in order to avoid toxic effects and is ensured by several transmembrane transport proteins, so-called excitatory amino acid transporters (EAATs). Impairment of glutamate removal has been linked to several neurodegenerative diseases and EAATs have therefore received increased attention as therapeutic targets. O-Benzylated l-threo-β-hydroxyaspartate derivatives have been developed previously as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino-3-((3-(4-(trifluoromethyl)benzamido)benzyl)oxy)succinic acid) standing out as low-nanomolar inhibitor. We report the stereoselective synthesis of all four stereoisomers of TFB-TBOA in less than a fifth of synthetic steps than the published route. For the first time, the inhibitory activity and isoform selectivity of these TFB-TBOA enantio- and diastereomers were assessed on human glutamate transporters EAAT1-3. Furthermore, we synthesized potent photoaffinity probes based on TFB-TBOA using our novel synthetic strategy. PMID:26918289

  6. II. Glutamine and glutamate.

    PubMed

    Tapiero, H; Mathé, G; Couvreur, P; Tew, K D

    2002-11-01

    Glutamine and glutamate with proline, histidine, arginine and ornithine, comprise 25% of the dietary amino acid intake and constitute the "glutamate family" of amino acids, which are disposed of through conversion to glutamate. Although glutamine has been classified as a nonessential amino acid, in major trauma, major surgery, sepsis, bone marrow transplantation, intense chemotherapy and radiotherapy, when its consumption exceeds its synthesis, it becomes a conditionally essential amino acid. In mammals the physiological levels of glutamine is 650 micromol/l and it is one of the most important substrate for ammoniagenesis in the gut and in the kidney due to its important role in the regulation of acid-base homeostasis. In cells, glutamine is a key link between carbon metabolism of carbohydrates and proteins and plays an important role in the growth of fibroblasts, lymphocytes and enterocytes. It improves nitrogen balance and preserves the concentration of glutamine in skeletal muscle. Deamidation of glutamine via glutaminase produces glutamate a precursor of gamma-amino butyric acid, a neurotransmission inhibitor. L-Glutamic acid is a ubiquitous amino acid present in many foods either in free form or in peptides and proteins. Animal protein may contain from 11 to 22% and plants protein as much as 40% glutamate by weight. The sodium salt of glutamic acid is added to several foods to enhance flavor. L-Glutamate is the most abundant free amino acid in brain and it is the major excitatory neurotransmitter of the vertebrate central nervous system. Most free L-glutamic acid in brain is derived from local synthesis from L-glutamine and Kreb's cycle intermediates. It clearly plays an important role in neuronal differentiation, migration and survival in the developing brain via facilitated Ca++ transport. Glutamate also plays a critical role in synaptic maintenance and plasticity. It contributes to learning and memory through use-dependent changes in synaptic efficacy and

  7. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter.

    PubMed

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P; Geller, Alfred I

    2007-05-01

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an approximately 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported approximately 90% glutamatergic neuron-specific expression. The GAD67 promoter supported approximately 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine

  8. Strain-dependent variations in visceral sensitivity: relationship to stress, anxiety and spinal glutamate transporter expression.

    PubMed

    Moloney, R D; Dinan, T G; Cryan, J F

    2015-04-01

    Responses to painful stimuli differ between populations, ethnic groups, sexes and even among individuals of a family. However, data regarding visceral pain are still lacking. Thus, we investigated differences in visceral nociception across inbred and outbred mouse strains using colorectal distension. Anxiety and depression-like behaviour were assessed using the open field and forced swim test as well as the corticosterone stress response. Possible mechanistic targets [excitatory amino acid transporter (EAAT-1), brain-derived neurotrophic factor (BDNF) and 5HT1A receptor] were also assessed using quantitative real-time polymerase chain reaction. Adult, male, inbred and outbred mouse strains were used in all assays (inbred strains; CBA/J Hsd, C3H/HeNHsd, BALB/c OlaHsd, C57 BL/6JOlaHsd, DBA/2J RccHsd, CAST/EiJ, SM/J, A/J OlaHsd, 129P2/OlaHsd, FVB/NHan Hsd and outbred strains: Swiss Webster, CD-1). mRNA expression levels of EAAT-1, BDNF and 5HT1A receptor (HTR1A) were quantified in the lumbosacral spinal cord, amygdala and hippocampus. A significant effect of strain was found in visceral sensitivity, anxiety and depressive-like behaviours. Strain differences were also seen in both baseline and stress-induced corticosterone levels. CBA/J mice consistently exhibited heightened visceral sensitivity, anxiety behaviour and depression-like behaviour which were associated with decreased spinal EAAT-1 and hippocampal BDNF and HTR1A. Our results show the CBA/J mouse strain as a novel mouse model to unravel the complex mechanisms of brain-gut axis disorders such as irritable bowel syndrome, in particular the underlying mechanisms of visceral hypersensitivity, for which there is great need. Furthermore, this study highlights the importance of genotype and the consequences for future development of transgenic strains in pain research.

  9. Strain-dependent variations in visceral sensitivity: relationship to stress, anxiety and spinal glutamate transporter expression.

    PubMed

    Moloney, R D; Dinan, T G; Cryan, J F

    2015-04-01

    Responses to painful stimuli differ between populations, ethnic groups, sexes and even among individuals of a family. However, data regarding visceral pain are still lacking. Thus, we investigated differences in visceral nociception across inbred and outbred mouse strains using colorectal distension. Anxiety and depression-like behaviour were assessed using the open field and forced swim test as well as the corticosterone stress response. Possible mechanistic targets [excitatory amino acid transporter (EAAT-1), brain-derived neurotrophic factor (BDNF) and 5HT1A receptor] were also assessed using quantitative real-time polymerase chain reaction. Adult, male, inbred and outbred mouse strains were used in all assays (inbred strains; CBA/J Hsd, C3H/HeNHsd, BALB/c OlaHsd, C57 BL/6JOlaHsd, DBA/2J RccHsd, CAST/EiJ, SM/J, A/J OlaHsd, 129P2/OlaHsd, FVB/NHan Hsd and outbred strains: Swiss Webster, CD-1). mRNA expression levels of EAAT-1, BDNF and 5HT1A receptor (HTR1A) were quantified in the lumbosacral spinal cord, amygdala and hippocampus. A significant effect of strain was found in visceral sensitivity, anxiety and depressive-like behaviours. Strain differences were also seen in both baseline and stress-induced corticosterone levels. CBA/J mice consistently exhibited heightened visceral sensitivity, anxiety behaviour and depression-like behaviour which were associated with decreased spinal EAAT-1 and hippocampal BDNF and HTR1A. Our results show the CBA/J mouse strain as a novel mouse model to unravel the complex mechanisms of brain-gut axis disorders such as irritable bowel syndrome, in particular the underlying mechanisms of visceral hypersensitivity, for which there is great need. Furthermore, this study highlights the importance of genotype and the consequences for future development of transgenic strains in pain research. PMID:25851919

  10. Neurotoxic potential of lunar and martian dust: influence on em, proton gradient, active transport, and binding of glutamate in rat brain nerve terminals.

    PubMed

    Krisanova, Natalia; Kasatkina, Ludmila; Sivko, Roman; Borysov, Arseniy; Nazarova, Anastasiya; Slenzka, Klaus; Borisova, Tatiana

    2013-08-01

    The harmful effects of lunar dust (LD) on directly exposed tissues are documented in the literature, whereas researchers are only recently beginning to consider its effects on indirectly exposed tissues. During inhalation, nano-/microsized particles are efficiently deposited in nasal, tracheobronchial, and alveolar regions and transported to the central nervous system. The neurotoxic potential of LD and martian dust (MD) has not yet been assessed. Glutamate is the main excitatory neurotransmitter involved in most aspects of normal brain function, whereas disturbances in glutamate homeostasis contribute to the pathogenesis of major neurological disorders. The research was focused on the analysis of the effects of LD/MD simulants (JSC-1a/JSC, derived from volcanic ash) on the key characteristics of glutamatergic neurotransmission. The average size of LD and MD particles (even minor fractions) before and after sonication was determined by dynamic light scattering. With the use of radiolabeled l-[(14)C]glutamate, it was shown that there is an increase in l-[(14)C]glutamate binding to isolated rat brain nerve terminals (synaptosomes) in low [Na(+)] media and at low temperature in the presence of LD. MD caused significantly lesser changes under the same conditions, whereas nanoparticles of magnetite had no effect at all. Fluorimetric experiments with potential-sensitive dye rhodamine 6G and pH-sensitive dye acridine orange showed that the potential of the plasma membrane of the nerve terminals and acidification of synaptic vesicles were not altered by LD/MD (and nanoparticles of magnetite). Thus, the unique effect of LD to increase glutamate binding to the nerve terminals was shown. This can have deleterious effects on extracellular glutamate homeostasis in the central nervous system and cause alterations in the ambient level of glutamate, which is extremely important for proper synaptic transmission. During a long-term mission, a combination of constant irritation due

  11. Neurotoxic potential of lunar and martian dust: influence on em, proton gradient, active transport, and binding of glutamate in rat brain nerve terminals.

    PubMed

    Krisanova, Natalia; Kasatkina, Ludmila; Sivko, Roman; Borysov, Arseniy; Nazarova, Anastasiya; Slenzka, Klaus; Borisova, Tatiana

    2013-08-01

    The harmful effects of lunar dust (LD) on directly exposed tissues are documented in the literature, whereas researchers are only recently beginning to consider its effects on indirectly exposed tissues. During inhalation, nano-/microsized particles are efficiently deposited in nasal, tracheobronchial, and alveolar regions and transported to the central nervous system. The neurotoxic potential of LD and martian dust (MD) has not yet been assessed. Glutamate is the main excitatory neurotransmitter involved in most aspects of normal brain function, whereas disturbances in glutamate homeostasis contribute to the pathogenesis of major neurological disorders. The research was focused on the analysis of the effects of LD/MD simulants (JSC-1a/JSC, derived from volcanic ash) on the key characteristics of glutamatergic neurotransmission. The average size of LD and MD particles (even minor fractions) before and after sonication was determined by dynamic light scattering. With the use of radiolabeled l-[(14)C]glutamate, it was shown that there is an increase in l-[(14)C]glutamate binding to isolated rat brain nerve terminals (synaptosomes) in low [Na(+)] media and at low temperature in the presence of LD. MD caused significantly lesser changes under the same conditions, whereas nanoparticles of magnetite had no effect at all. Fluorimetric experiments with potential-sensitive dye rhodamine 6G and pH-sensitive dye acridine orange showed that the potential of the plasma membrane of the nerve terminals and acidification of synaptic vesicles were not altered by LD/MD (and nanoparticles of magnetite). Thus, the unique effect of LD to increase glutamate binding to the nerve terminals was shown. This can have deleterious effects on extracellular glutamate homeostasis in the central nervous system and cause alterations in the ambient level of glutamate, which is extremely important for proper synaptic transmission. During a long-term mission, a combination of constant irritation due

  12. The initial stage of reversal learning is impaired in mice hemizygous for the vesicular glutamate transporter (VGluT1).

    PubMed

    Granseth, B; Andersson, F K; Lindström, S H

    2015-07-01

    Behavioral flexibility is a complex cognitive function that is necessary for survival in changeable environments. Patients with schizophrenia or Parkinson's disease often suffer from cognitive rigidity, reducing their capacity to function in society. Patients and rodent models with focal lesions in the prefrontal cortex (PFC) show similar rigidity, owing to the loss of PFC regulation of subcortical reward circuits involved in behavioral flexibility. The vesicular glutamate transporter (VGluT1) is preferentially expressed at modulatory synapses, including PFC neurons that project to components of the reward circuit (such as the nucleus accumbens, NAc). VGluT1(+/-) mice display behavioral phenotypes matching many symptoms of schizophrenia, and VGluT1 expression is reduced in the PFC of patients with schizophrenia and Parkinson's disease. Thus, it appears likely that VGluT1-expressing synapses from PFC play a key role in behavioral flexibility. To examine this hypothesis, we studied behavioral flexibility in VGluT1(+/-) mice by testing reversal learning in a visual discrimination task. Here, we show that VGluT1(+/-) mice acquired the initial visual discrimination at the same rate as controls. However, they failed to suppress responses to the previously rewarded stimulus following reversal of reward contingencies. Thus, our genetic disruption of modulatory glutamatergic signaling, including that arising from PFC, appears to have impaired the first stage of reversal learning (extinguishing responses to previously rewarded stimuli). Our data show that this deficit stems from a preservative phenotype. These findings suggest that glutamatergic regulation from the cortex is important for behavioral flexibility and the disruption of this pathway may be relevant in diseases such as schizophrenia. PMID:26113146

  13. Chlordecone impairs Na(+)-stimulated L-( sup 3 H)glutamate transport and mobility of 16-doxyl stearate in rat liver plasma membrane vesicles

    SciTech Connect

    Rochelle, L.G.; Miller, T.L.; Curtis, L.R. )

    1990-09-01

    Chlordecone (CD) treatment of rat liver plasma membranes (LPM) provided in vitro evidence for mechanisms of in vivo liver dysfunction caused by CD. LPM preparations enriched 14- to 19-fold in the bile canalicular markers gamma-glutamyl transpeptidase, alkaline phosphatase, and leucine aminopeptidase were isolated from male Sprague-Dawley rats. CD inhibited the bile canalicular-specific active transport of Na(+)-stimulated L-({sup 3}H)glutamate in LPM vesicles. CD (0.08 and 0.5 mumol/mg protein) reduced both the initial velocity and the maximum level of Na(+)-stimulated L-(3H)glutamate uptake without significantly reducing Na(+)-independent uptake. In vitro treatment of LPM with CD (0.2-1.0 mumols/mg protein) also reduced the mobility of a 16-doxyl stearate spin label probe in a concentration-dependent manner. No change in mobility was apparent at CD concentrations below 0.2 mumol/mg protein. These results demonstrated that CD impaired a bile canalicular-specific transport system and induced liver plasma membrane perturbation. Na(+)-stimulated L-({sup 3}H)glutamate uptake was more sensitive to CD than was detectable immobilization of the spin label probe.

  14. Effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine by their transporters heterologously expressed in COS cells and in rat brain synaptosomes.

    PubMed

    Sugimura, M; Kitayama, S; Morita, K; Irifune, M; Takarada, T; Kawahara, M; Dohi, T

    2001-08-01

    Although the neurotransmitter uptake system is considered a possible target for the presynaptic action of anesthetic agents, observations are inconsistent concerning effects on the transporter and their clinical relevance. The present study examined the effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine in COS cells heterologously expressing the transporters for these neurotransmitters and in the rat brain synaptosomes. Halothane and isoflurane, but not thiamylal or thiopental, significantly inhibited uptake by COS cell systems of GABA, dopamine and glutamic acid in a concentration-dependent manner within clinically relevant ranges for anesthesia induced by these agents. Similarly, in synaptosomes halothane and isoflurane but not thiopental significantly suppressed the uptake of GABA and glutamic acid, respectively. These results do not support the hypothesis that volatile and intravenous anesthetics exert their action via specific inhibition of GABA uptake to enhance inhibitory GABAergic neuronal activity. Rather, they suggest that presynaptic uptake systems for various neurotransmitters including GABA may be the molecular targets for volatile anesthetic agents.

  15. Ceftriaxone upregulates the glutamate transporter in medial prefrontal cortex and blocks reinstatement of methamphetamine seeking in a condition place preference paradigm

    PubMed Central

    Abulseoud, Osama A.; Miller, Joseph D.; Wu, Jinhua; Choi, Doo-Sup; Holschneider, Daniel P.

    2014-01-01

    Glutamate signaling plays an essential role in drug-seeking behavior. Using reinstatement of conditioned place preference (CPP), we determined whether ceftriaxone, a β-lactam antibiotic known to increase the expression and activity of the glutamate transporter (EAAT2) on glial cells, blocks methamphetamine-triggered reinstatement of CPP. Rats acquired methamphetamine CPP following 7 consecutive days of conditioning, during which each animal received pairings of alternating morning methamphetamine (2.5 mg/kg, IP) and afternoon saline (IP). Animals showing CPP were successfully extinguished with repeated twice daily saline administration over a 7-day period. Ceftriaxone (200 mg/kg, IP) was administered (vs. saline) once a day for 7 days during the extinction period. Upon successful extinction, animals received a single dose of methamphetamine (2.5 mg/kg, IP) for reinstatement and were tested for CPP one day later. Using real time PCR, EAAT2 mRNA levels in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) were quantified in response to ceftriaxone. Ceftriaxone blocked methamphetamine-triggered reinstatement of CPP and significantly increased EAAT2 mRNA levels in the mPFC, with a trend towards significance in the NAc. In conclusion, Ceftriaxone modulated the expression of the glutamate transporter in a critical region of the cortico-striatal addiction circuitry and attenuated drug-seeking behavior in rats. Further research is needed to test the efficacy of compounds targeting the EAAT2 in human methamphetamine-dependent users. PMID:22521042

  16. In vivo expression of polyglutamine-expanded huntingtin by mouse striatal astrocytes impairs glutamate transport: a correlation with Huntington's disease subjects.

    PubMed

    Faideau, Mathilde; Kim, Jinho; Cormier, Kerry; Gilmore, Richard; Welch, Mackenzie; Auregan, Gwennaelle; Dufour, Noelle; Guillermier, Martine; Brouillet, Emmanuel; Hantraye, Philippe; Déglon, Nicole; Ferrante, Robert J; Bonvento, Gilles

    2010-08-01

    Huntington's disease (HD) is a neurodegenerative disorder previously thought to be of primary neuronal origin, despite ubiquitous expression of mutant huntingtin (mHtt). We tested the hypothesis that mHtt expressed in astrocytes may contribute to the pathogenesis of HD. To better understand the contribution of astrocytes in HD in vivo, we developed a novel mouse model using lentiviral vectors that results in selective expression of mHtt into striatal astrocytes. Astrocytes expressing mHtt developed a progressive phenotype of reactive astrocytes that was characterized by a marked decreased expression of both glutamate transporters, GLAST and GLT-1, and of glutamate uptake. These effects were associated with neuronal dysfunction, as observed by a reduction in DARPP-32 and NR2B expression. Parallel studies in brain samples from HD subjects revealed early glial fibrillary acidic protein expression in striatal astrocytes from Grade 0 HD cases. Astrogliosis was associated with morphological changes that increased with severity of disease, from Grades 0 through 4 and was more prominent in the putamen. Combined immunofluorescence showed co-localization of mHtt in astrocytes in all striatal HD specimens, inclusive of Grade 0 HD. Consistent with the findings from experimental mice, there was a significant grade-dependent decrease in striatal GLT-1 expression from HD subjects. These findings suggest that the presence of mHtt in astrocytes alters glial glutamate transport capacity early in the disease process and may contribute to HD pathogenesis. PMID:20494921

  17. Frequent coexpression of the vesicular glutamate transporter 1 and 2 genes, as well as coexpression with genes for choline acetyltransferase or glutamic acid decarboxylase in neurons of rat brain.

    PubMed

    Danik, Marc; Cassoly, Estelle; Manseau, Frédéric; Sotty, Florence; Mouginot, Didier; Williams, Sylvain

    2005-08-15

    It is widely believed that expression of the vesicular glutamate transporter genes VGLUT1 and VGLUT2 is restricted to glutamatergic neurons and that the two transporters segregate in different sets of neurons. Using single-cell multiplex RT-PCR (sc-RT-mPCR), we show that VGLUT1 and VGLUT2 mRNAs were coexpressed in most of the sampled neurons from the rat hippocampus, cortex, and cerebellum at postnatal Day (P)14 but not P60. In accordance, changes in VGLUT1 and VGLUT2 mRNA concentrations were found to occur in these and other brain areas between P14 and P60, as revealed by semiquantitative RT-PCR and quantitated by ribonuclease protection assay. VGLUT1 and -2 coexpression in the hippocampal formation is supported further by in situ hybridization data showing that virtually all cells in the CA1-CA3 pyramidal and granule cell layers were highly positive for both transcripts until P14. It was revealed using sc-RT-mPCR that transcripts for VGLUT1 and VGLUT2 were also present in neurons of the cerebellum, striatum, and septum that expressed markers for gamma-aminobutyric acid (GABA)ergic or cholinergic phenotypes, as well as in hippocampal cells containing transcripts for the glial fibrillary acidic protein. Our study suggests that VGLUT1 and VGLUT2 proteins may often transport glutamate into vesicles within the same neuron, especially during early postnatal development, and that they are expressed widely in presumed glutamatergic, GABAergic, and cholinergic neurons, as well as in astrocytes. Furthermore, our study shows that such coexpressing neurons remain in the adult brain and identifies several areas that contain them in both young and adult rats. PMID:15983996

  18. The development of benzo- and naphtho-fused quinoline-2,4-dicarboxylic acids as vesicular glutamate transporter (VGLUT) inhibitors reveals a possible role for neuroactive steroids.

    PubMed

    Carrigan, Christina N; Patel, Sarjubhai A; Cox, Holly D; Bolstad, Erin S; Gerdes, John M; Smith, Wesley E; Bridges, Richard J; Thompson, Charles M

    2014-02-01

    Substituted quinoline-2,4-dicarboxylates (QDCs) are conformationally-restricted mimics of glutamate that were previously reported to selectively block the glutamate vesicular transporters (VGLUTs). We find that expanding the QDC scaffold to benzoquinoline dicarboxylic acids (BQDC) and naphthoquinoline dicarboxylic acids (NQDCs) improves inhibitory activity with the NQDCs showing IC50∼70 μM. Modeling overlay studies showed that the polycyclic QDCs resembled steroid structures and led to the identification and testing of estrone sulfate, pregnenolone sulfate and pregnanolone sulfate that blocked the uptake of l-Glu by 50%, 70% and 85% of control, respectively. Pregnanolone sulfate was further characterized by kinetic pharmacological determinations that demonstrated competitive inhibition and a Ki of ≈20 μM. PMID:24424130

  19. Vesicular glutamate transporter 2 (VGLUT2) is co-stored with PACAP in projections from the rat melanopsin-containing retinal ganglion cells.

    PubMed

    Engelund, Anna; Fahrenkrug, Jan; Harrison, Adrian; Hannibal, Jens

    2010-05-01

    The retinal ganglion cell layer of the eye comprises a subtype of cells characterized by their intrinsic photosensitivity and expression of melanopsin (ipRGCs). These cells regulate a variety of non-image-forming (NIF) functions such as light entrainment of circadian rhythms, acute suppression of locomotor activity (masking), and pupillary light reflex. Two neurotransmitters have been identified in ipRGCs, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP). To date, little is known about their release and interplay. Here, we describe the presence and co-localization of vesicular glutamate transporter 2 (VGLUT2; a marker of glutamate signaling) and PACAP in ipRGCs and their projections in the brain. Nine adult male Wistar rats were assigned to one of three groups; anterograde tracing (n = 3), eye enucleation (n = 3), and untreated (n = 3). Under anaesthesia, rats were transcardially perfusion-fixated, after which the brains and eyes were removed for double immunohistochemical staining using a polyclonal anti-VGLUT2 antibody and a mouse monoclonal anti-PACAP antibody. Results revealed that VGLUT2- and PACAP-immunoreactivity (-ir) were present in ipRGCs and co-localized in their projections in the suprachiasmatic nucleus, the intergeniculate leaflet, and the olivary pretectal nucleus. We conclude that there is evidence to support the use of glutamate and PACAP as neurotransmitters in NIF photoperception by rat ipRGCs, and that these neurotransmitters are co-stored and probably released from the same nerve terminals. Furthermore, we conclude that VGLUT2 is the preferred subtype of vesicular transporter used by these cells.

  20. /sup 15/N-Ammonia assimilation, 2-oxoglutarate transport, and glutamate export in spinach chloroplasts in the presence of dicarboxylates in the light

    SciTech Connect

    Woo, K.C.; Boyle, F.A.; Flugge, I.U.; Heldt, H.W.

    1987-11-01

    The direct incorporation of /sup 15/NH/sub 4/Cl into amino acids in illuminated spinach (Spinacia oleracea L.) chloroplasts in the presence of 2-oxoglutarate plus malate was determined. The amido-N of glutamine was the most highly labeled N-atom during /sup 15/NH/sub 4/ assimilation in the presence of malate. In 4 minutes the /sup 15/N-label of the amido-N of glutamine was 37% enriched. In contrast, values obtained for both the N-atom of glutamate and the amino-N of glutamine were only about 20% while that of the N-atom of aspartate was only 3%. The addition of malate during the assimilation of /sup 15/NH/sub 4/Cl and Na/sup 15/NO/sub 2/ greatly increased the /sup 15/N-label into glutamine but did not qualitatively change the order of the incorporation of /sup 15/N-label into all the amino acids examined. This evidence indicates the direct involvement of the glutamine synthetase/glutamate synthase pathway for ammonia and nitrite assimilation in isolated chloroplasts. The addition of malate or succinate during ammonia assimilation also led to more than 3-fold increase in (/sup 14/C)2-oxoglutarate transport into the chloroplast as well as an increase in the export of (/sup 14/C)glutamate out of the chloroplast. Little (/sup 14/C)glutamine was detected in the medium of the chloroplast preparations. The stimulation of /sup 15/N-incorporation and (/sup 14/C)glutamate export by malate could be directly attributed to the increase in 2-oxoglutarate transport activity (via the 2-oxoglutarate translocator) observed in the presence of exogenous malate.

  1. Differential Regulation of Two Isoforms of the Glial Glutamate Transporter EAAT2 by DLG1 and CaMKII

    PubMed Central

    Wheeler, David S.; Amara, Susan G.

    2015-01-01

    The gene for EAAT2, the major astrocytic glutamate transporter, generates two carrier isoforms (EAAT2a and EAAT2b) that vary at their C termini as a consequence of alternative RNA splicing. The EAAT2b cytoplasmic C terminus contains a postsynaptic density-95/Discs large/zona occludens-1 (PDZ) ligand, which is absent in EAAT2a. To understand how the distinct C termini might affect transporter trafficking and surface localization, we generated Madin-Darby canine kidney (MDCK) cells that stably express EGFP-EAAT2a or EGFP-EAAT2b and found robust basolateral membrane expression of the EAAT2b isoform. In contrast, EAAT2a displayed a predominant distribution within intracellular vesicle compartments, constitutively cycling to and from the membrane. Addition of the PDZ ligand to EAAT2a as well as its deletion from EAAT2b confirmed the importance of the motif for cell-surface localization. Using EAAT2 constructs with an extracellular biotin acceptor tag to directly assess surface proteins, we observed significant PDZ ligand-dependent EAAT2b surface expression in cultured astrocytes, consistent with observations in cell lines. Discs large homolog 1 (DLG1; SAP97), a PDZ protein prominent in both astrocytes and MDCK cells, colocalized and coimmunoprecipitated with EAAT2b. shRNA knockdown of DLG1 expression decreased surface EAAT2b in both MDCK cells and cultured astrocytes, suggesting that the DLG scaffolding protein stabilizes EAAT2b at the surface. DLG1 can be phosphorylated by Ca2+/calmodulin-dependent protein kinase (CaMKII), resulting in disruption of its PDZ-mediated interaction. In murine astrocytes and acute brain slices, activation of CaMKII decreases EAAT2b surface expression but does not alter the distribution of EAAT2a. These data indicate that the surface expression and function of EAAT2b can be rapidly modulated through the disruption of its interaction with DLG1 by CaMKII activation. PMID:25834051

  2. Novel N-1,2-dihydroxypropyl analogs of lobelane inhibit vesicular monoamine transporter-2 function and methamphetamine-evoked dopamine release.

    PubMed

    Horton, David B; Siripurapu, Kiran B; Zheng, Guangrong; Crooks, Peter A; Dwoskin, Linda P

    2011-10-01

    Lobelane, a chemically defunctionalized saturated analog of lobeline, has increased selectivity for the vesicular monoamine transporter 2 (VMAT2) compared with the parent compound. Lobelane inhibits methamphetamine-evoked dopamine (DA) release and decreases methamphetamine self-administration. Unfortunately, tolerance develops to the ability of lobelane to decrease these behavioral effects of methamphetamine. Lobelane has low water solubility, which is problematic for drug development. The aim of the current study was to determine the pharmacological effect of replacement of the N-methyl moiety with a chiral N-1,2-dihydroxypropyl (N-1,2-diol) moiety, which enhances water solubility, altering the configuration of the N-1,2-diol moiety and incorporating phenyl ring substituents into the analogs. To determine VMAT2 selectivity, structure-activity relationships also were generated for inhibition of DA and serotonin transporters. Analogs with the highest potency for inhibiting DA uptake at VMAT2 and at least 10-fold selectivity were evaluated further for ability to inhibit methamphetamine-evoked DA release from superfused striatal slices. (R)-3-[2,6-cis-di(4-methoxyphenethyl)piperidin-1-yl]propane-1,2-diol (GZ-793A), the (R)-4-methoxyphenyl-N-1,2-diol analog, and (R)-3-[2,6-cis-di(1-naphthylethyl)piperidin-1-yl]propane-1,2-diol (GZ-794A), the (R)-1-naphthyl-N-1,2-diol analog, exhibited the highest potency (K(i) ∼30 nM) inhibiting VMAT2, and both analogs inhibited methamphetamine-evoked endogenous DA release (IC(50) = 10.6 and 0.4 μM, respectively). Thus, the pharmacophore for VMAT2 inhibition accommodates the N-1,2-diol moiety, which improves drug-likeness and enhances the potential for the development of these clinical candidates as treatments for methamphetamine abuse.

  3. Role of glutamic acid decarboxylase 67 in regulating cortical parvalbumin and GABA membrane transporter 1 expression: Implications for schizophrenia

    PubMed Central

    Curley, Allison A.; Eggan, Stephen M.; Lazarus, Matt S.; Huang, Z. Josh; Volk, David W.; Lewis, David A.

    2012-01-01

    Markers of GABA neurotransmission are altered in multiple regions of the neocortex in individuals with schizophrenia. Lower levels of glutamic acid decarboxylase 67 (GAD67) mRNA and protein, which is responsible for most cortical GABA synthesis, are accompanied by lower levels of GABA membrane transporter 1 (GAT1) mRNA. These alterations are thought to be most prominent in the parvalbumin (PV)-containing subclass of interneurons, which also contain lower levels of PV mRNA. Since GAT1 and PV each reduce the availability of GABA at postsynaptic receptors, lower levels of GAT1 and PV mRNAs have been hypothesized to represent compensatory responses to an upstream reduction in cortical GABA synthesis in schizophrenia. However, such cause-and-effect hypotheses cannot be directly tested in a human illness. Consequently, we used two mouse models with reduced GAD67 expression specifically in PV neurons (PVGAD67+/−) or in all interneurons (GABAGAD67+/−) and quantified GAD67, GAT1 and PV mRNA levels using methods identical to those employed in studies of schizophrenia. Cortical levels of PV or GAT1 mRNAs were not altered in PVGAD67+/− mice during postnatal development or in adulthood. Furthermore, cellular analyses confirmed the predicted reduction in GAD67 mRNA, but failed to show a deficit in PV mRNA in these animals. Levels of PV and GAT1 mRNAs were also unaltered in GABAGAD67+/− mice. Thus, mouse lines with cortical reductions in GAD67 mRNA that match or exceed those present in schizophrenia, and that differ in the developmental timing and cell typespecificity of the GAD67 deficit, failed to provide proof-of-concept evidence that lower PV and GAT1 expression in schizophrenia are a consequence of lower GAD67 expression. Together, these findings suggest that the correlated decrements in cortical GAD67, PV and GAT1 mRNAs in schizophrenia may be a common consequence of some other upstream factor. PMID:23103418

  4. Genetic predisposition and early life experience interact to determine glutamate transporter (GLT1) and solute carrier family 12 member 5 (KCC2) levels in rat hippocampus.

    PubMed

    Sterley, Toni-Lee; Howells, Fleur M; Dimatelis, Jacqueline J; Russell, Vivienne A

    2016-02-01

    Attention-deficit/hyperactivity disorder (ADHD) is one of the most common child psychiatric disorders. While it is typically treated with medications that target dopamine and norepinephrine transmission, there is increasing evidence that other neurotransmitter systems, such as glutamate and GABA, may be involved. The aetiology of ADHD is unknown; however, there is evidence that early life stress may contribute to the development of the disorder. In the present study we used proteomic analysis (iTRAQ) followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis to investigate hippocampal protein profiles of three rat strains: an animal model of ADHD, spontaneously hypertensive rats (SHR), their control Wistar-Kyoto rats (WKY), and Sprague-Dawley rats (SD). We additionally investigated how these protein profiles are affected by maternal separation, a model of early life stress. Our findings show that solute carrier family 12 member 5 (KCC2) is increased in SHR hippocampus. The glutamate transporter GLT1 splice variant, GLT1b, was increased (proteomic analysis) while total GLT1 (comprised mostly of GLT1a splice variant) was reduced (Western blot analysis) in SHR hippocampus, compared to WKY and SD--a pattern that is consistent with elevated extracellular glutamate levels. Maternal separation increased total GLT1 in hippocampi of SHR, WKY, and SD, and reduced GLT1b in SHR hippocampus. Together these findings provide evidence for disturbed glutamatergic and GABAergic transmission in SHR hippocampus, maternal separation effects on glutamate uptake in hippocampi of all three strains, as well a unique effect of maternal separation on GLT1b levels in SHR hippocampus. These data suggest significant involvement of glutamatergic and GABAergic transmission in the neuropathophysiology of ADHD, and implicates changes in glutamatergic transmission as a result of early life stress. PMID:26464063

  5. Music evokes vivid autobiographical memories.

    PubMed

    Belfi, Amy M; Karlan, Brett; Tranel, Daniel

    2016-08-01

    Music is strongly intertwined with memories-for example, hearing a song from the past can transport you back in time, triggering the sights, sounds, and feelings of a specific event. This association between music and vivid autobiographical memory is intuitively apparent, but the idea that music is intimately tied with memories, seemingly more so than other potent memory cues (e.g., familiar faces), has not been empirically tested. Here, we compared memories evoked by music to those evoked by famous faces, predicting that music-evoked autobiographical memories (MEAMs) would be more vivid. Participants listened to 30 songs, viewed 30 faces, and reported on memories that were evoked. Memories were transcribed and coded for vividness as in Levine, B., Svoboda, E., Hay, J. F., Winocur, G., & Moscovitch, M. [2002. Aging and autobiographical memory: Dissociating episodic from semantic retrieval. Psychology and Aging, 17, 677-689]. In support of our hypothesis, MEAMs were more vivid than autobiographical memories evoked by faces. MEAMs contained a greater proportion of internal details and a greater number of perceptual details, while face-evoked memories contained a greater number of external details. Additionally, we identified sex differences in memory vividness: for both stimulus categories, women retrieved more vivid memories than men. The results show that music not only effectively evokes autobiographical memories, but that these memories are more vivid than those evoked by famous faces.

  6. Patchy distributions of myelin and vesicular glutamate transporter 2 align with cytochrome oxidase blobs and interblobs in the superficial layers of the primary visual cortex

    PubMed Central

    Rockoff, Emily C; Balaram, Pooja; Kaas, Jon H

    2015-01-01

    Blobs are a modular component of the primary visual cortex (area 17) of all primates, but not of other mammals closely related to primates. They are characterized as an even distribution of patches, puffs, or blobs of dense cytochrome oxidase (CO) expression in layer III of area 17, and are now known to differ from surrounding, nonblob cortex in thalamic, intrinsic, and extrastriate connections. Previous studies have also recognized a blob-like pattern of myelin-dense patches in layer III of area 17 of primates, and more recently the vesicular glutamate transporter (VGLUT)-2 isoform of the VGLUT family has been found to selectively distribute to layer III patches in a similar blob-like pattern. Here, we sought to determine if the blob-like patterns all identify the same modular structures in area 17 of primates by staining alternate brain sections cut parallel to the surface of area 17 of a prosimian primate (Otolemur garnettii) for CO, myelin, and VGLUT2. By aligning the sections from the three preparations, we provide clear evidence that the three preparations all identify the same modular blob structures. The results provide a further understanding of the functional nature of the blobs by demonstrating that their higher level of CO activity is related to thalamic inputs from the lateral geniculate nucleus that use VGLUT2 as their main glutamate transporter, and via myelinated axons. PMID:26097384

  7. Synaptic glutamate spillover due to impaired glutamate uptake mediates heroin relapse.

    PubMed

    Shen, Hao-wei; Scofield, Michael D; Boger, Heather; Hensley, Megan; Kalivas, Peter W

    2014-04-16

    Reducing the enduring vulnerability to relapse is a therapeutic goal in treating drug addiction. Studies with animal models of drug addiction show a marked increase in extrasynaptic glutamate in the core subcompartment of the nucleus accumbens (NAcore) during reinstated drug seeking. However, the synaptic mechanisms linking drug-induced changes in extrasynaptic glutamate to relapse are poorly understood. Here, we discovered impaired glutamate elimination in rats extinguished from heroin self-administration that leads to spillover of synaptically released glutamate into the nonsynaptic extracellular space in NAcore and investigated whether restoration of glutamate transport prevented reinstated heroin seeking. Through multiple functional assays of glutamate uptake and analyzing NMDA receptor-mediated currents, we show that heroin self-administration produced long-lasting downregulation of glutamate uptake and surface expression of the transporter GLT-1. This downregulation was associated with spillover of synaptic glutamate to extrasynaptic NMDA receptors within the NAcore. Ceftriaxone restored glutamate uptake and prevented synaptic glutamate spillover and cue-induced heroin seeking. Ceftriaxone-induced inhibition of reinstated heroin seeking was blocked by morpholino-antisense targeting GLT-1 synthesis. These data reveal that the synaptic glutamate spillover in the NAcore results from reduced glutamate transport and is a critical pathophysiological mechanism underling reinstated drug seeking in rats extinguished from heroin self-administration.

  8. Synaptic Glutamate Spillover Due to Impaired Glutamate Uptake Mediates Heroin Relapse

    PubMed Central

    Scofield, Michael D.; Boger, Heather; Hensley, Megan; Kalivas, Peter W.

    2014-01-01

    Reducing the enduring vulnerability to relapse is a therapeutic goal in treating drug addiction. Studies with animal models of drug addiction show a marked increase in extrasynaptic glutamate in the core subcompartment of the nucleus accumbens (NAcore) during reinstated drug seeking. However, the synaptic mechanisms linking drug-induced changes in extrasynaptic glutamate to relapse are poorly understood. Here, we discovered impaired glutamate elimination in rats extinguished from heroin self-administration that leads to spillover of synaptically released glutamate into the nonsynaptic extracellular space in NAcore and investigated whether restoration of glutamate transport prevented reinstated heroin seeking. Through multiple functional assays of glutamate uptake and analyzing NMDA receptor-mediated currents, we show that heroin self-administration produced long-lasting downregulation of glutamate uptake and surface expression of the transporter GLT-1. This downregulation was associated with spillover of synaptic glutamate to extrasynaptic NMDA receptors within the NAcore. Ceftriaxone restored glutamate uptake and prevented synaptic glutamate spillover and cue-induced heroin seeking. Ceftriaxone-induced inhibition of reinstated heroin seeking was blocked by morpholino-antisense targeting GLT-1 synthesis. These data reveal that the synaptic glutamate spillover in the NAcore results from reduced glutamate transport and is a critical pathophysiological mechanism underling reinstated drug seeking in rats extinguished from heroin self-administration. PMID:24741055

  9. Impairment of SLC17A8 Encoding Vesicular Glutamate Transporter-3, VGLUT3, Underlies Nonsyndromic Deafness DFNA25 and Inner Hair Cell Dysfunction in Null Mice

    PubMed Central

    Ruel, Jérôme; Emery, Sarah; Nouvian, Régis; Bersot, Tiphaine; Amilhon, Bénédicte; Van Rybroek, Jana M.; Rebillard, Guy; Lenoir, Marc; Eybalin, Michel; Delprat, Benjamin; Sivakumaran, Theru A.; Giros, Bruno; El Mestikawy, Salah; Moser, Tobias; Smith, Richard J.H.; Lesperance, Marci M.; Puel, Jean-Luc

    2008-01-01

    Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, the most common sensory defect associated with aging in humans. We have identified SLC17A8, which encodes the vesicular glutamate transporter-3 (VGLUT3), as the gene responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. In two unrelated families, a heterozygous missense mutation, c.632C→T (p.A211V), was found to segregate with DFNA25 deafness and was not present in 267 controls. Linkage-disequilibrium analysis suggested that the families have a distant common ancestor. The A211 residue is conserved in VGLUT3 across species and in all human VGLUT subtypes (VGLUT1-3), suggesting an important functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs) before releasing it onto receptors of auditory-nerve terminals. Null mice with a targeted deletion of Slc17a8 exon 2 lacked auditory-nerve responses to acoustic stimuli, although auditory brainstem responses could be elicited by electrical stimuli, and robust otoacoustic emissions were recorded. Ca2+-triggered synaptic-vesicle turnover was normal in IHCs of Slc17a8 null mice when probed by membrane capacitance measurements at 2 weeks of age. Later, the number of afferent synapses, spiral ganglion neurons, and lateral efferent endings below sensory IHCs declined. Ribbon synapses remaining by 3 months of age had a normal ultrastructural appearance. We conclude that deafness in Slc17a8-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential for auditory coding at the IHC synapse. PMID:18674745

  10. Sex-dependent role of vesicular glutamate transporter 3 in stress-regulation and related anxiety phenotype during the early postnatal period.

    PubMed

    Balázsfi, Diána; Farkas, Lívia; Csikota, Péter; Fodor, Anna; Zsebők, Sándor; Haller, József; Zelena, Dóra

    2016-07-01

    Stress and related disorders are in the focus of interest and glutamate is one of the most important neurotransmitters that can affect these processes. Glutamatergic neurons are characterized by vesicular glutamate transporters (VGluT1-3) among which vGluT3 is unique contributing to the non-canonical, neuromodulatory effect of glutamate. We aimed to study the role of vGluT3 in stress axis regulation and related anxiety during the early postnatal period using knockout (KO) mice with special focus on sex differences. Anxiety was explored on postnatal day (PND) 7-8 by maternal separation-induced ultrasonic vocalization (USV). Stress-hormone levels were detected 60 min after intraperitoneal lipopolysaccharide (LPS) injection 7 days later. Both genotypes gained weight, but on PND 14-15 KO mice pups had smaller body weight compared to wild type (WT). vGluT3 KO mice reacted to an immune stressor with enhanced adrenocorticotropin (ACTH) and corticosterone secretion compared to WT. Although there was a tendency for enhanced anxiety measured by more emitted USV, this did not reach the level of significance. The only sex-related effect was the enhanced corticosterone reactivity in male pups. For the HPA axis regulation in neonates vGluT3 expression seems to be dispensable under basal conditions, but is required for optimal response to immune stressors, most probably through an interaction with other neurotransmitters. Disturbance of the fine balance between these systems may result in a borderline enhanced anxiety-like behavior in vGluT3 KO pups.

  11. Sex-dependent role of vesicular glutamate transporter 3 in stress-regulation and related anxiety phenotype during the early postnatal period.

    PubMed

    Balázsfi, Diána; Farkas, Lívia; Csikota, Péter; Fodor, Anna; Zsebők, Sándor; Haller, József; Zelena, Dóra

    2016-07-01

    Stress and related disorders are in the focus of interest and glutamate is one of the most important neurotransmitters that can affect these processes. Glutamatergic neurons are characterized by vesicular glutamate transporters (VGluT1-3) among which vGluT3 is unique contributing to the non-canonical, neuromodulatory effect of glutamate. We aimed to study the role of vGluT3 in stress axis regulation and related anxiety during the early postnatal period using knockout (KO) mice with special focus on sex differences. Anxiety was explored on postnatal day (PND) 7-8 by maternal separation-induced ultrasonic vocalization (USV). Stress-hormone levels were detected 60 min after intraperitoneal lipopolysaccharide (LPS) injection 7 days later. Both genotypes gained weight, but on PND 14-15 KO mice pups had smaller body weight compared to wild type (WT). vGluT3 KO mice reacted to an immune stressor with enhanced adrenocorticotropin (ACTH) and corticosterone secretion compared to WT. Although there was a tendency for enhanced anxiety measured by more emitted USV, this did not reach the level of significance. The only sex-related effect was the enhanced corticosterone reactivity in male pups. For the HPA axis regulation in neonates vGluT3 expression seems to be dispensable under basal conditions, but is required for optimal response to immune stressors, most probably through an interaction with other neurotransmitters. Disturbance of the fine balance between these systems may result in a borderline enhanced anxiety-like behavior in vGluT3 KO pups. PMID:27442776

  12. Hippocampal glutamate transporter 1 (GLT-1) complex levels are paralleling memory training in the Multiple T-maze in C57BL/6J mice.

    PubMed

    Heo, Seok; Jung, Gangsoo; Beuk, Tamara; Höger, Harald; Lubec, Gert

    2012-04-01

    The glutamate transporter 1 (GLT-1) is essential for glutamate uptake in the brain and associated with various psychiatric and neurological disorders. Pharmacological inhibition of GLT-1 results in memory deficits, but no study linking native GLT-1 complexes was published so far. It was therefore the aim of the study to associate this highly hydrophobic, eight transmembrane spanning domains containing transporter to memory training in the Multiple T-maze (MTM). C57BL/6J mice were used for the spatial memory training experiments, and trained mice were compared to untrained (yoked) animals. Mouse hippocampi were dissected out 6 h after training on day 4, and a total enriched membrane fraction was prepared by ultracentrifugation. Membrane proteins were separated by blue native polyacrylamide gel electrophoresis (BN-PAGE) with subsequent Western blotting against GLT-1 on these native gels. Moreover, GLT-1 complexes were identified by mass spectrometry (nano-LC-ESI-MS/MS). Animals learned the MTM task and multiple GLT-1 complexes were detected at apparent molecular weights of 242, 480 and 720 kDa on BN-PAGE Western blotting. GLT-1 complex levels were significantly higher in the trained group as compared to yoked controls, and antibody specificity was verified by immunoblotting on multidimensional gels. Hippocampal GLT-1 was unambiguously identified by mass spectrometry with high sequence coverage, and glycosylation was observed. It is revealed that increased GLT-1 complex levels are paralleling and are linked to spatial memory training. We provide evidence that signal termination, represented by the excitatory amino acid transporter GLT-1 complexes, is involved in spatial memory mechanisms.

  13. STEREOLOGICAL ESTIMATES OF THE BASAL FOREBRAIN CELL POPULATION IN THE RAT, INCLUDING NEURONS CONTAINING CHOLINE ACETYLTRANSFERASE (ChAT), GLUTAMIC ACID DECARBOXYLASE (GAD) OR PHOSPHATE-ACTIVATED GLUTAMINASE (PAG) AND COLOCALIZING VESICULAR GLUTAMATE TRANSPORTERS (VGluTs)

    PubMed Central

    GRITTI, I.; HENNY, P.; GALLONI, F.; MAINVILLE, L.; MARIOTTI, M.; JONES, B. E.

    2006-01-01

    The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and are comprised by GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as ~355,000 and the numbers of ChAT-immuno-positive (+) as ~22,000, GAD+ ~119,000 and PAG+ ~316,000, corresponding to ~5%, ~35% and ~90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also have the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, VGluT3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or

  14. Increased Glutamate Receptor and Transporter Expression in the Cerebral Cortex and Striatum of Gcdh-/- Mice: Possible Implications for the Neuropathology of Glutaric Acidemia Type I

    PubMed Central

    Lagranha, Valeska Lizzi; Matte, Ursula; de Carvalho, Talita Giacomet; Seminotti, Bianca; Pereira, Carolina Coffi; Koeller, David M.; Woontner, Michael; Goodman, Stephen I.; de Souza, Diogo Onofre Gomes; Wajner, Moacir

    2014-01-01

    We determined mRNA expression of the ionotropic glutamate receptors NMDA (NR1, NR2A and NR2B subunits), AMPA (GluR2 subunit) and kainate (GluR6 subunit), as well as of the glutamate transporters GLAST and GLT1 in cerebral cortex and striatum of wild type (WT) and glutaryl-CoA dehydrogenase deficient (Gchh-/-) mice aged 7, 30 and 60 days. The protein expression levels of some of these membrane proteins were also measured. Overexpression of NR2A and NR2B in striatum and of GluR2 and GluR6 in cerebral cortex was observed in 7-day-old Gcdh-/-. There was also an increase of mRNA expression of all NMDA subunits in cerebral cortex and of NR2A and NR2B in striatum of 30-day-old Gcdh-/- mice. At 60 days of life, all ionotropic receptors were overexpressed in cerebral cortex and striatum of Gcdh-/- mice. Higher expression of GLAST and GLT1 transporters was also verified in cerebral cortex and striatum of Gcdh-/- mice aged 30 and 60 days, whereas at 7 days of life GLAST was overexpressed only in striatum from this mutant mice. Furthermore, high lysine intake induced mRNA overexpression of NR2A, NR2B and GLAST transcripts in striatum, as well as of GluR2 and GluR6 in both striatum and cerebral cortex of Gcdh-/- mice. Finally, we found that the protein expression of NR2A, NR2B, GLT1 and GLAST were significantly greater in cerebral cortex of Gcdh-/- mice, whereas NR2B and GLT1 was similarly enhanced in striatum, implying that these transcripts were translated into their products. These results provide evidence that glutamate receptor and transporter expression is higher in Gcdh-/- mice and that these alterations may be involved in the pathophysiology of GA I and possibly explain, at least in part, the vulnerability of striatum and cerebral cortex to injury in patients affected by GA I. PMID:24594605

  15. Fangchinoline inhibits glutamate release from rat cerebral cortex nerve terminals (synaptosomes).

    PubMed

    Lin, Tzu-Yu; Lu, Cheng-Wei; Tien, Lu-Tai; Chuang, Shu-Han; Wang, Yu-Ru; Chang, Wen-Hsuan; Wang, Su-Jane

    2009-07-01

    Fangchinoline, an active component of radix stephaniae tetrandrinea, has been shown to possess neuroprotective properties. It has been reported that excessive glutamate release has been proposed to be involved in the pathogenesis of several neurological diseases. The primary purpose of the present study was to investigate the effect of fangchinoline on glutamate release in rat cerebral cortex nerve terminals and to explore the possible mechanism. Fangchinoline inhibited the release of glutamate evoked by 4-aminopyridine (4-AP) in a concentration-dependent manner, and this phenomenon resulted from a reduction of vesicular exocytosis but not from an inhibition of Ca(2+)-independent efflux via glutamate transporter. Fangchinoline did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization, but significantly reduced depolarization-induced increase in [Ca(2+)](C). Fangchinoline-mediated inhibition of glutamate release was significantly prevented by the N- and P/Q-type Ca(2+) channel blocker omega-conotoxin MVIIC, and by the PKC inhibitors, GF109203X and Ro318220. In addition, the glutamate release mediated by direct Ca(2+) entry with Ca(2+) ionophore (ionomycin) was unaffected by fangchinoline, which suggests that the inhibitory effect of fangchinoline is not due to directly interfering with the release process at some point subsequent to Ca(2+) influx. These results suggest that fangchinoline inhibits glutamate release from the rat cortical synaptosomes through the suppression of voltage-dependent Ca(2+) channel activity and subsequent reduces Ca(2+) entry into nerve terminals, rather than any upstream effect on nerve terminal excitability. This inhibition appears to involve the suppression of PKC signal transduction pathway. This finding may explain the neuroprotective effects of fangchinoline against neurotoxicity. PMID:19428795

  16. Effects of Cocaine-Kindling on the Expression of NMDA Receptors and Glutamate Levels in Mouse Brain

    PubMed Central

    Núñez-Taltavull, Juan F.; Budziszewska, Bogusława; Lasoń, Władysław; Gasior, Maciej; Zapata, Agustin; Shippenberg, Toni S.; Witkin, Jeffrey M.

    2010-01-01

    In the present study we examined the effects of cocaine seizure kindling on the expression of NMDA receptors and levels of extracellular glutamate in mouse brain. Quantitative autoradiography did not reveal any changes in binding of [3H] MK-801 to NMDA receptors in several brain regions. Likewise, in situ hybridization and Western blotting revealed no alteration in expression of the NMDA receptor subunits, NR1 and NR2B. Basal overflow of glutamate in the ventral hippocampus determined by microdialysis in freely moving animals also did not differ between cocaine-kindled and control groups. Perfusion with the selective excitatory amino acid transporter inhibitor, pyrrolidine-2,4-dicarboxylic acid (tPDC, 0.6 mM), increased glutamate overflow confirming transport inhibition. Importantly, KCl-evoked glutamate overflow under tPDC perfusion was significantly higher in cocaine-kindled mice than in control mice. These data suggest that enhancement of depolarization stimulated glutamate release may be one of the mechanisms underlying the development of increased seizure susceptibility after cocaine kindling. PMID:20927585

  17. Glutamate transporter gene (SLC1A1) single nucleotide polymorphism (rs301430) and repetitive behaviors and anxiety in children with autism spectrum disorder.

    PubMed

    Gadow, Kenneth D; Roohi, Jasmin; DeVincent, Carla J; Kirsch, Sarah; Hatchwell, Eli

    2010-09-01

    Investigated association of single nucleotide polymorphism (SNP) rs301430 in glutamate transporter gene (SLC1A1) with severity of repetitive behaviors (obsessive-compulsive behaviors, tics) and anxiety in children with autism spectrum disorder (ASD). Mothers and/or teachers completed a validated DSM-IV-referenced rating scale for 67 children with autism spectrum disorder. Although analyses were not significant for repetitive behaviors, youths homozygous for the high expressing C allele had more severe anxiety than carriers of the T allele. Allelic variation in SLC1A1 may be a biomarker for or modifier of anxiety symptom severity in children with ASD, but study findings are best conceptualized as tentative pending replication with larger independent samples.

  18. Bilateral enhancement of excitation via up-regulation of vesicular glutamate transporter subtype 1, not subtype 2, immunoreactivity in the unilateral hypoxic epilepsy model.

    PubMed

    Kim, Duk-Soo; Kwak, Sung-Eun; Kim, Ji-Eun; Won, Moo Ho; Choi, Hui-Chul; Song, Hong-Ki; Kwon, Oh-Shin; Kim, Yeong-In; Choi, Soo-Young; Kang, Tae-Cheon

    2005-09-01

    In the present study, the change of vesicular glutamate transporter (VGLUT) immunoreactivity on long-term impaired excitability in the hippocampus after recovery from unilateral hypoxic-ischemic insult was investigated in order to extend our understanding of the mechanism of epileptogenesis using unilateral hypoxic epilepsy models. Both the lesioned (submitted to ischemia) and the unlesioned hippocampi exhibited the frequent occurrence of interictal spikes and occasionally the sustained ictal discharges. However, paired-pulse inhibition was significantly reduced in the unlesioned dentate gyrus, not in the lesioned dentate gyrus. VGLUT1 immunoreactivity was significantly elevated in both hippocampi following hypoxic ischemia, although VGLUT2 immunodensity was unaltered. These findings suggest that the enhancement of VGLUT1 immunoreactivity in both hippocampi after unilateral hypoxic ischemia may contribute to the hyperexcitability, which may play an important role in the epileptogenesis (presumably accompanied by altered inhibitory transmission) after neurodegeneration.

  19. Decreased expression of glutamate transporter GLAST in Bergmann glia is associated with the loss of Purkinje neurons in the spinocerebellar ataxia type 1.

    PubMed

    Cvetanovic, Marija

    2015-02-01

    Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease of the cerebellum caused by a polyglutamine-repeat expansion in the protein ATXN1. We have previously demonstrated that astrocytic activation occurs early in pathogenesis, correlates with disease progression, and can occur when mutant ATXN1 expression is limited to Purkinje neurons. We now show that expression of glutamate and aspartate transporter, GLAST, is decreased in cerebellar astrocytes in a mouse model of SCA1. This decrease occurs in non-cell autonomous manner late in disease and correlates well with the loss of Purkinje neurons. Astrogliosis or decreased neuronal activity does not correlate with diminished GLAST expression. In addition, Bergmann glia remain capable of transcriptional upregulation of GLAST in response to improvement in Purkinje neurons supporting the notion of active neuron-glia crosstalk in disease.

  20. Glycogen synthase kinase 3 beta regulates glial glutamate transporter protein expression in the spinal dorsal horn in rats with neuropathic pain

    PubMed Central

    Weng, Han-Rong; Gao, Mei; Maixner, Dylan W.

    2014-01-01

    Dysfunctional glial glutamate transporters and over production of pro-inflammatory cytokines (including interleukin-1β, IL-1β) are two prominent mechanisms by which glial cells enhance neuronal activities in the spinal dorsal horn in neuropathic pain conditions. Endogenous molecules regulating production of IL-1β and glial glutamate functions remain poorly understood. In this study, we revealed a dynamic alteration of GSK3β activities and its role in regulating glial glutamate transporter 1 (GLT-1) protein expression in the spinal dorsal horn and nociceptive behaviors following the nerve injury. Specifically, GSK3β was expressed in both neurons and astrocytes in the spinal dorsal horn. GSK3β activities were suppressed on day 3 but increased on day 10 following the nerve injury. In parallel, protein expression of GLT-1 in the spinal dorsal horn was enhanced on day 3 but reduced on day 10. In contrast to these time-dependent changes, the activation of astrocytes and over-production of IL-1β were found on both day 3 and day 10. Meanwhile, thermal hyperalgesia was observed from day 2 through day 10 and mechanical allodynia from day 4 through day 10. Pre-emptive pharmacological inhibition of GSK3β activities significantly ameliorated thermal hyperalgesia and mechanical allodynia at the late stage but did not have effects at the early stage. These were accompanied with the suppression of GSK3β activities, prevention of decreased GLT-1 protein expression, inhibition of astrocytic activation, and reduction of IL-1β in the spinal dorsal horn on day 10. These data indicate that the increased GSK3β activity in the spinal dorsal horn is attributable to the downregulation of GLT-1 protein expression in neuropathic rats at the late stage. Further, we also demonstrated that the nerve-injury-induced thermal hyperalgesia on day 10 was transiently suppressed by pharmacological inhibition of GSK3β. Our study suggests that GSK3β may be a potential target for the

  1. Glutamate Receptor Stimulation Up-Regulates Glutamate Uptake in Human Müller Glia Cells.

    PubMed

    López-Colomé, Ana María; López, Edith; Mendez-Flores, Orquidia G; Ortega, Arturo

    2016-07-01

    Glutamate, the main excitatory amino acid in the vertebrate retina, is a well know activator of numerous signal transduction pathways, and has been critically involved in long-term synaptic changes acting through ionotropic and metabotropic glutamate receptors. However, recent findings underlining the importance of intensity and duration of glutamate stimuli for specific neuronal responses, including excitotoxicity, suggest a crucial role for Na(+)-dependent glutamate transporters, responsible for the removal of this neurotransmitter from the synaptic cleft, in the regulation of glutamate-induced signaling. Transporter proteins are expressed in neurons and glia cells, albeit most of glutamate uptake occurs in the glial compartment. Within the retina, Müller glia cells are in close proximity to glutamatergic synapses and participate in the recycling of glutamate through the glutamate/glutamine shuttle. In this context, we decided to investigate a plausible role of glutamate as a regulatory signal for its own transport in human retinal glia cells. To this end, we determined [(3)H]-D-aspartate uptake in cultures of spontaneously immortalized human Müller cells (MIO-M1) exposed to distinct glutamatergic ligands. A time and dose-dependent increase in the transporter activity was detected. This effect was dependent on the activation of the N-methyl D-aspartate subtype of glutamate receptors, due to a dual effect: an increase in affinity and an augmented expression of the transporter at the plasma membrane, as established via biotinylation experiments. Furthermore, a NMDA-dependent association of glutamate transporters with the cystoskeletal proteins ezrin and glial fibrillary acidic protein was also found. These results add a novel mediator of the glutamate transporter modulation and further strengthen the notion of the critical involvement of glia cells in synaptic function. PMID:27017513

  2. Glutamate-based antidepressants: preclinical psychopharmacology.

    PubMed

    Pilc, Andrzej; Wierońska, Joanna M; Skolnick, Phil

    2013-06-15

    Over the past 20 years, converging lines of evidence have both linked glutamatergic dysfunction to the pathophysiology of depression and demonstrated that the glutamatergic synapse presents multiple targets for developing novel antidepressants. The robust antidepressant effects of the N-methyl-D-aspartate receptor antagonists ketamine and traxoprodil provide target validation for this family of ionotropic glutamate receptors. This article reviews the preclinical evidence that it may be possible to develop glutamate-based antidepressants by not only modulating ionotropic (N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) and metabotropic glutamate (mGlu) receptors, including mGlu2/3, mGLu5 and mGlu7 receptors, but also by altering synaptic concentrations of glutamate via specialized transporters such as glial glutamate transporter 1 (excitatory amino-acid transporter 2).

  3. Regulation of the glutamate transporter by amino acid deprivation and associated effects on the level of EAAC1 mRNA in the renal epithelial cell line NBL-I.

    PubMed Central

    Plakidou-Dymock, S; McGivan, J D

    1993-01-01

    The glutamate transport system of the bovine renal epithelial cell line NBL-1 was studied. The Km for Na(+)-dependent glutamate transport was found to be 13.8 +/- 2.4 microM (Vmax. 365 +/- 19.2 pmol/3 min per mg) and for Na(+)-dependent aspartate transport 4.5 +/- 1.1 microM (Vmax. 108 +/- 6.3 pmol/3 min per mg). The Km values are in close agreement with those expected for high-affinity Na(+)-dependent glutamate transport by System XAG-. Upon deprivation of amino acids, the Vmax. for Na+/aspartate co-transport rose to 203 +/- 6.0 pmol/3 min per mg (Km 3.8 +/- 0.5 microns). A probe was constructed to the high-affinity excitatory amino acid carrier (EAAC1) [Kanai and Hediger (1992) Nature (London) 360, 467-471]. The probe hybridized to a 3.5 kb transcript. On deprivation of amino acids, the level of EAAC1 mRNA decreased sharply before the measurable increase in transport levels, but was subsequently restored to control levels. A motif, which we propose is linked to amino acid deprivation, was found in the EAAC1 primary sequence. Images Figure 6 PMID:8240287

  4. Stress-induced inhibition of nonsense mediated RNA decay regulates intracellular cystine transport and intracellular glutathione through regulation of the cystine/glutamate exchanger SLC7A11

    PubMed Central

    Martin, Leenus; Gardner, Lawrence B.

    2014-01-01

    SLC7A11 encodes a subunit of the xCT cystine/glutamate amino acid transport system and plays a critical role in the generation of glutathione and the protection of cells from oxidative stress. Expression of SLC7A11 promotes tumorigenesis and chemotherapy resistance, but while SLC7A11 has been previously noted to be upregulated in hypoxic cells its regulation has not been fully delineated. We have recently shown that nonsense mediated RNA decay (NMD) is inhibited by cellular stresses generated by the tumor microenvironment, including hypoxia, and augments tumorigenesis. Here we demonstrate that the inhibition of NMD by various cellular stresses leads to the stabilization and upregulation of SLC7A11 mRNA and protein. The inhibition of NMD and upregulation of SLC7A11 augments intracellular cystine transport, and increases intracellular levels of cysteine and glutathione. Accordinglyy, the inhibition of NMD protects cells against oxidative stress via SLC7A11 upregulation. Together our studies identify a mechanism for the dynamic regulation of SLC7A11, through the stress-inhibited regulation of NMD, and add to the growing evidence that the inhibition of NMD is an adaptive response. PMID:25399695

  5. Electrogenic uptake of sulphur-containing analogues of glutamate and aspartate by Müller cells from the salamander retina.

    PubMed Central

    Bouvier, M; Miller, B A; Szatkowski, M; Attwell, D

    1991-01-01

    1. The effect of sulphur-containing analogues of glutamate and aspartate on the membrane current of glial cells was studied by whole-cell clamping Müller cells isolated from the salamander retina. 2. L-Cysteic acid (CA), L-cysteinesulphinic acid (CSA), L-homocysteic acid (HCA), L-homocysteinesulphinic acid (HCSA) and S-sulpho-L-cysteine (SC) all evoked an inward membrane current that was large at negative potentials, and was smaller (but did not reverse) at more positive potentials up to +30 mV. 3. Removal of external sodium ions abolished the amino acid-evoked currents. Whole-cell clamping with pipettes containing no potassium led to a rapid suppression of the currents, that did not occur when potassium was included in the pipette. 4. The dependence of the currents on sulphur-containing amino acid concentration obeyed first-order Michaelis-Menten kinetics. The current evoked by co-application of L-glutamate and a sulphur-containing analogue was smaller than the sum of the currents produced by glutamate alone and by the sulphur analogue alone. 5. These data are consistent with the sulphur amino acid-evoked current being caused by uptake on the electrogenic glutamate uptake carrier, which co-transports an excess of Na+ ions into the cell, and counter-transports one K+ ion out of the cell. 6. The apparent Km (Michaelis-Menten constant) values for activation of uptake by CA (6 microM) and by CSA (60 microM) are low enough for uptake on the glutamate uptake carrier to be a plausible mechanism for terminating the postulated neurotransmitter action of these agents. However, the apparent Km values for uptake of HCA (2.95 mM), HCSA (1.65 mM) and SC (greater than 1 mM) are much higher than the EC50 (half-maximal effective concentration) concentrations for these agents' activation of NMDA (N-methyl-D-aspartate) channels. 7. Comparing the concentrations of sulphur amino acids needed to activate NMDA channels with their rate of uptake suggests that their potency for causing

  6. Isoflurane unveils a critical role of glutamate transporter type 3 in regulating hippocampal GluR1 trafficking and context-related learning and memory in mice.

    PubMed

    Cao, J; Wang, Z; Mi, W; Zuo, Z

    2014-07-11

    Glutamate transporter type 3 (EAAT3) may play a role in cognition. Isoflurane enhances EAAT3 trafficking to the plasma membrane. Thus, we used isoflurane to determine how EAAT3 might regulate learning and memory and the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, such as GluR1, to the plasma membrane, a fundamental biochemical process for learning and memory. Here, isoflurane increased EAAT3 but did not change GluR1 levels in the plasma membrane of wild-type mouse hippocampus. Isoflurane increased protein phosphatase activity in the wild-type and EAAT3(-/-) mouse hippocampus. Also, isoflurane reduced GluR1 in the plasma membrane and decreased phospho-GluR1 in EAAT3(-/-) mice. The phosphatase inhibitor okadaic acid attenuated these effects. Finally, isoflurane inhibited context-related fear conditioning in EAAT3(-/-) mice but not in wild-type mice. Thus, isoflurane may increase GluR1 trafficking to the plasma membrane via EAAT3 and inhibit GluR1 trafficking via protein phosphatase. Lack of EAAT3 effects leads to decreased GluR1 trafficking and impaired cognition after isoflurane exposure in EAAT3(-/-) mice.

  7. Genomic organization, promoter analysis, and chromosomal localization of the gene for the mouse glial high-affinity glutamate transporter Slc1a3

    SciTech Connect

    Hagiwara, Tatsuya; Tanaka, Kohichi; Maeno-Hikichi, Yuka

    1996-05-01

    The mouse gene encoding glial high-affinity, Na -dependent glutamate transporter Slcla3 (GluT-1/GLAST) was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the mouse genome and comprised 10 exons spanning more than 56 kilobases. The transcription initiation sites were mapped to positions 503, which is the first transcriptional point (defined as +1), 128 (+376), and 64 (+440) basepairs upstream of the 3{prime}-end of exon 1 by primer extension. The 5{prime}-flanking region of the mouse GluT-1 gene had a typical CCAAT box and a GC box but lacked at TATA box. These features of the promoter region were characteristic of housekeeping genes. The fusion plasmids containing approximately 4 kb of the 5{prime}-flanking region (-3830 to +450) and the firefly luciferase gene induced a significant luciferase activity when transfected into COS-1 cells. Distal deletion of the 5{prime}-flanking region, leaving 619 bp (-169 to +450), resulted in a marked decrease in luciferase activity in COS-1 cells, suggesting that a CCAAT box, which was positioned at -200, is necessary for the expression of this gene. In situ hybridization localized this gene. In situ hybridization localized this gene to mouse chromosome 15A2. These structural features will lead to a better understanding of the regulatory mechanism of the expression of the GluT-1 gene by ischemia and will also provide a basis for future evolutionary comparisons with other neurotransmitter transporters. 40 refs., 6 figs., 1 tab.

  8. EVALUATING THE NMDA-GLUTAMATE RECEPTOR AS A SITE OF ACTION FOR TOLUENE, IN VIVO

    EPA Science Inventory

    In vitro, toluene disrupts the function of NMDA-glutamate receptors, indicating that effects on NMDA receptor function may contribute to toluene neurotoxicity. NMDA-glutamate receptors are widely present in the visual system and contribute to pattern-elicited visual evoked potent...

  9. Immunohistochemical localization of vesicular glutamate transporter 2 (vGluT2) in the central nervous system of the pigeon (Columba livia).

    PubMed

    Atoji, Yasuro

    2011-10-01

    Glutamatergic neurons are distributed widely in the telencephalic pallium of birds, but their targets are unknown. In the present study, a polyclonal antibody was produced against pigeon vesicular glutamate transporter 2 (vGluT2) and was used for Western blot and immunohistochemistry to detect projection targets of glutamatergic neurons in the pigeon central nervous system. The molecular weight of vGluT2 was measured at 65 kDa. vGluT2 immunoreactivity was observed in neuropil, but not in neuronal cell bodies or glia. Immunoreactive neuropil generally appeared homogeneous, but fine granules or puncta were found in many areas or nuclei. The telencephalon showed strong immunoreactivity, except for the globus pallidus. In particular, the mesopallium and hippocampal formation revealed the most intense immunostaining. In the diencephalon, vGluT2 immunoreactivity was intense in the dorsal thalamus and hypothalamus. In the midbrain, strong immunostaining was seen in the periaqueductal gray, the dorsal part of the lateral mesencephalic nucleus, and the isthmo-optic nucleus. The optic tectum showed moderate immunoreactivity. In the cerebellar cortex, glomeruli in the granular layer were intensely immunoreactive, and the molecular layer showed intensely homogeneous immunostaining. In the caudal brainstem, the cochlear magnocellular and angular nuclei showed strongly immunoreactive puncta around neuronal cell bodies. In the spinal cord, the dorsal horn revealed moderate immunoreactivity and the marginal nucleus was strongly immunoreactive. Ultrastructural observations revealed that vGluT2 immunoreactivity is localized in asymmetric, presynaptic terminals. The present results indicate an extensive distribution of glutamatergic projections and circuits in the avian central nervous system.

  10. Effect of insulin on muscle glutamate uptake

    PubMed Central

    Aoki, T. T.; Brennan, M. F.; Müller, W. A.; Moore, F. D.; Cahill, G. F.

    1972-01-01

    For decades, investigators concerned with protein metabolism in man have performed detailed amino acid analyses of human plasma obtained under a wide range of experimental situations. A large body of information has been used to calculated rates of protein synthesis and proteolysis. During the course of an investigation of the effect of intrabrachial artery infusion of insulin (70 μU/min per kg body weight) on glutamate uptake by human forearm muscle, it was discovered that plasma arterio-deep venous glutamate difference analysis failed to document any increase in the uptake of this amino acid, suggesting that insulin had little influence on glutamate uptake by muscle. However, whole blood glutamate analyses, performed on the same blood samples, revealed that (a) the resting muscle uptake of glutamate is smaller than previously reported and (b) insulin is capable of markedly increasing glutamate uptake by muscle from whole blood. Since the hematocrit was obtained on all samples, detailed analyses of the various compartments in which glutamate could be found were performed. It was determined that circulating blood cells have a dynamic role in glutamate transport. These data underscore the need for both whole blood and plasma amino acid analysis in investigations concerned with protein synthesis and/or amino acid flux, for analysis of plasma samples alone could be misleading as illustrated in the present study. Images PMID:5080414

  11. Chemoenzymatic synthesis of new 2,4-syn-functionalized (S)-glutamate analogues and structure-activity relationship studies at ionotropic glutamate receptors and excitatory amino acid transporters.

    PubMed

    Assaf, Zeinab; Larsen, Anja P; Venskutonytė, Raminta; Han, Liwei; Abrahamsen, Bjarke; Nielsen, Birgitte; Gajhede, Michael; Kastrup, Jette S; Jensen, Anders A; Pickering, Darryl S; Frydenvang, Karla; Gefflaut, Thierry; Bunch, Lennart

    2013-02-28

    In the mammalian central nervous system, (S)-glutamate (Glu) is released from the presynaptic neuron where it activates a plethora of pre- and postsynaptic Glu receptors. The fast acting ionotropic Glu receptors (iGluRs) are ligand gated ion channels and are believed to be involved in a vast number of neurological functions such as memory and learning, synaptic plasticity, and motor function. The synthesis of 14 enantiopure 2,4-syn-Glu analogues 2b-p is accessed by a short and efficient chemoenzymatic approach starting from readily available cyclohexanone 3. Pharmacological characterization at the iGluRs and EAAT1-3 subtypes revealed analogue 2i as a selective GluK1 ligand with low nanomolar affinity. Two X-ray crystal structures of the key analogue 2i in the ligand-binding domain (LBD) of GluA2 and GluK3 were determined. Partial domain closure was seen in the GluA2-LBD complex with 2i comparable to that induced by kainate. In contrast, full domain closure was observed in the GluK3-LBD complex with 2i, similar to that of GluK3-LBD with glutamate bound.

  12. 3-Aminoglutarate is a "silent" false transmitter for glutamate neurons.

    PubMed

    Foster, Alan C; Chen, June; Runyan, Stephen; Dinh, Tim; Venadas, Steven; Ehring, George R; Li, Yong-Xin; Staubli, Ursula

    2015-10-01

    Understanding the storage and release of the excitatory neurotransmitter, L-glutamate by synaptic vesicles has lagged behind receptor characterizations due to a lack of pharmacological agents. We report that the glutamate analog, 3-aminoglutarate (3-AG) is a "silent" false transmitter for glutamate neurons that may be a useful tool to study storage and release mechanisms. Like L-glutamate itself, 3-AG is a high-affinity substrate for both the plasma membrane (EAATs) and vesicular (vGLUT) glutamate transporters. As such, EAATs facilitate 3-AG entry into neuronal cytoplasm allowing 3-AG to compete with L-glutamate for transport into vesicles thus reducing glutamate content. In a synaptosomal preparation, 3-AG inhibited calcium-dependent endogenous L-glutamate release. Unlike L-glutamate, 3-AG had low affinity for both ionotropic (NMDA and AMPA) and G-protein coupled (mGlu1-8) receptors. Consequently, 3-AG behaves as a "silent" false transmitter that may be used in physiological experiments to probe synaptic vesicle storage and release mechanisms for L-glutamate. The companion paper by Wu et al. (2015) describes initial experiments that explore the effects of 3-AG on glutamate synaptic transmission under physiological and pathophysiological conditions.

  13. Understanding safety of glutamate in food and brain.

    PubMed

    Mallick, H N

    2007-01-01

    Glutamate is ubiquitous in nature and is present in all living organisms. It is the principal excitatory neurotransmitter in central nervous system. Glutamate is being used as food additive for enhancing flavour for over last 1200 years imparting a unique taste known as "umami" in Japanese. It is being marketed for about last 100 years. The taste of umami is now recognized as the fifth basic taste. Many of the foods used in cooking for enhancing flavour contain high amount of glutamate. Breast milk has the highest concentration of glutamate amongst all amino acids. Glutamate in high doses as gavage or parenteral injection have been reported to produce neurodegeneration in infant rodents. The neurodegeneration was not produced when gluamate was given with food. The Joint FAO/WHO Expert Committee on Food Additives, based on enumerable scientific evidence, has declared that, "glutamate as an additive in food" is not an health hazard to human being. Glutamate is used as signaling molecule not only in neuronal but also in non-neuronal tissues. Excessive accumulation of glutamate in the synaptic cleft has been associated with excitotoxicty and glutamate is implicated in number of neurological disorders. Excessive accumulation could be attributed to increase release, failure of transport system for uptake mechanism, neuronal injury due to hypoxia-ischemia, trauma and associated metabolic failures. The role blood brain barrier, vesicular glutamate and sodium dependent excitatory amino acid transporters in glutamate homeostasis are emphasized in the review.

  14. Electrogenic glutamate uptake in glial cells is activated by intracellular potassium

    NASA Astrophysics Data System (ADS)

    Barbour, Boris; Brew, Helen; Attwell, David

    1988-09-01

    Uptake of glutamate into glial cells in the CNS maintains the extracellular glutamate concentration below neurotoxic levels and helps terminate its action as a neurotransmitter 1. The co-transport of two sodium ions on the glutamate carrier is thought to provide the energy needed to transport glutamate into cells2,3. We have shown recently that glutamate uptake can be detected electrically because the excess of Na+ ions transported with each glutamate anion results in a net current flow into the cell4. We took advantage of the control of the environment, both inside and outside the cell, provided by whole-cell patch-clamping and now report that glutamate uptake is activated by intracellular potassium and inhibited by extracellular potassium. Our results indicate that one K+ ion is transported out of the cell each time a glutamate anion and three Na+ ions are transported in. A carrier with this stoichiometry can accumulate glutamate against a much greater concentration gradient than a carrier co-transporting one glutamate anion and two Na+ ions. Pathological rises in extracellular potassium concentration will inhibit glutamate uptake by depolarizing glial cells and by preventing the loss of K+ from the glutamate carrier. This will facilitate a rise in the extracellular glutamate concentration to neurotoxic levels and contribute to the neuronal death occurring in brain anoxia and ischaemia.

  15. Effects of γ-Aminobutyric acid transporter 1 inhibition by tiagabine on brain glutamate and γ-Aminobutyric acid metabolism in the anesthetized rat In vivo.

    PubMed

    Patel, Anant B; de Graaf, Robin A; Rothman, Douglas L; Behar, Kevin L

    2015-07-01

    γ-Aminobutyric acid (GABA) clearance from the extracellular space after release from neurons involves reuptake into terminals and astrocytes through GABA transporters (GATs). The relative flows through these two pathways for GABA released from neurons remains unclear. This study determines the effect of tiagabine, a selective inhibitor of neuronal GAT-1, on the rates of glutamate (Glu) and GABA metabolism and GABA resynthesis via the GABA-glutamine (Gln) cycle. Halothane-anesthetized rats were administered tiagabine (30 mg/kg, i.p.) and 45 min later received an intravenous infusion of either [1,6-(13)C2]glucose (in vivo) or [2-(13)C]acetate (ex vivo). Nontreated rats served as controls. Metabolites and (13)C enrichments were measured with (1)H-[(13)C]-nuclear magnetic resonance spectroscopy and referenced to their corresponding endpoint values measured in extracts from in situ frozen brain. Metabolic flux estimates of GABAergic and glutamatergic neurons were determined by fitting a metabolic model to the (13)C turnover data measured in vivo during [1,6-(13)C2]glucose infusion. Tiagabine-treated rats were indistinguishable (P > 0.05) from controls in tissue amino acid levels and in (13)C enrichments from [2-(13)C]acetate. Tiagabine reduced average rates of glucose oxidation and neurotransmitter cycling in both glutamatergic neurons (↓18%, CMR(glc(ox)Glu): control, 0.27 ± 0.05 vs. tiagabine, 0.22 ± 0.04 µmol/g/min; ↓11%, V(cyc(Glu-Gln)): control 0.23 ± 0.05 vs. tiagabine 0.21 ± 0.04 µmol/g/min and GABAergic neurons (↓18-25%, CMR(glc(ox)GABA): control 0.09 ± 0.02 vs. tiagabine 0.07 ± 0.03 µmol/g/min; V(cyc(GABA-Gln)): control 0.08 ± 0.02 vs. tiagabine 0.07 ± 0.03 µmol/g/min), but the changes in glutamatergic and GABAergic fluxes were not significant (P > 0.10). The results suggest that any reduction in GABA metabolism by tiagabine might be an indirect response to reduced glutamatergic drive rather than direct compensatory effects.

  16. Astroglial potassium clearance contributes to short-term plasticity of synaptically evoked currents at the tripartite synapse

    PubMed Central

    Sibille, Jérémie; Pannasch, Ulrike; Rouach, Nathalie

    2014-01-01

    Abstract Astroglial processes enclose ∼60% of CA1 hippocampal synapses to form the tripartite synapse. Although astrocytes express ionic channels, neurotransmitter receptors and transporters to detect neuronal activity, the nature, plasticity and impact of the currents induced by neuronal activity on short-term synaptic plasticity remain elusive in hippocampal astrocytes. Using simultaneous electrophysiological recordings of astrocytes and neurons, we found that single stimulation of Schaffer collaterals in hippocampal slices evokes in stratum radiatum astrocytes a complex prolonged inward current synchronized to synaptic and spiking activity in CA1 pyramidal cells. The astroglial current is composed of three components sensitive to neuronal activity, i.e. a long-lasting potassium current mediated by Kir4.1 channels, a transient glutamate transporter current and a slow residual current, partially mediated by GABA transporters and Kir4.1-independent potassium channels. We show that all astroglial membrane currents exhibit activity-dependent short-term plasticity. However, only the astroglial glutamate transporter current displays neuronal-like dynamics and plasticity. As Kir4.1 channel-mediated potassium uptake contributes to 80% of the synaptically evoked astroglial current, we investigated in turn its impact on short-term synaptic plasticity. Using glial conditional Kir4.1 knockout mice, we found that astroglial potassium uptake reduces synaptic responses to repetitive stimulation and post-tetanic potentiation. These results show that astrocytes integrate synaptic activity via multiple ionic channels and transporters and contribute to short-term plasticity in part via potassium clearance mediated by Kir4.1 channels. PMID:24081156

  17. Evoked and spontaneous transmission favored by distinct sets of synapses

    PubMed Central

    Peled, Einat S.; Newman, Zachary L.; Isacoff, Ehud Y.

    2014-01-01

    Summary Background Spontaneous “miniature” transmitter release takes place at low rates at all synapses. Long thought as an unavoidable leak, spontaneous release has recently been suggested to be mediated by distinct pre- and post-synaptic molecular machineries and to have a specialized role in setting up and adjusting neuronal circuits. It remains unclear how spontaneous and evoked transmission are related at individual synapses, how they are distributed spatially when an axon makes multiple contacts with a target and whether they are commonly regulated. Results Electrophysiological recordings in the Drosophila larval neuromuscular junction, in the presence of the use-dependent glutamate receptor (GluR) blocker Philanthotoxin, indicated that spontaneous and evoked transmission employ distinct sets of GluRs. In vivo imaging of transmission using synaptically-targeted GCaMP3 to detect Ca2+ influx through the GluRs revealed little spatial overlap between synapses participating in spontaneous and evoked transmission. Spontaneous and evoked transmission were oppositely correlated with presynaptic levels of the protein Brp: synapses with high Brp favored evoked transmission, whereas synapses with low Brp were more active spontaneously. High frequency stimulation did not increase the overlap between evoked and spontaneous transmission, and instead decreased the rate of spontaneous release from synapses that were highly active in evoked transmission. Conclusions While individual synapses can participate in both evoked and spontaneous transmission, highly-active synapses show a preference for one mode of transmission. The presynaptic protein Brp promotes evoked transmission and suppresses spontaneous release. These findings suggest the existence of presynaptic mechanisms that promote synaptic specialization to either evoked or spontaneous transmission. PMID:24560571

  18. HIV-1, Methamphetamine and Astrocyte Glutamate Regulation: Combined Excitotoxic Implications for Neuro-AIDS

    PubMed Central

    Cisneros, Irma E; Ghorpade, Anuja

    2012-01-01

    Glutamate, the most abundant excitatory transmitter in the brain can lead to neurotoxicity when not properly regulated. Excitotoxicity is a direct result of abnormal regulation of glutamate concentrations in the synapse, and is a common neurotoxic mediator associated with neurodegenerative disorders. It is well accepted that methamphetamine (METH), a potent central nervous stimulant with high abuse potential, and human immunodeficiency virus (HIV)-1 are implicated in the progression of neurocognitive malfunction. Both have been shown to induce common neurodegenerative effects such as astrogliosis, compromised blood brain barrier integrity, and excitotoxicity in the brain. Reduced glutamate uptake from neuronal synapses likely leads to the accumulation of glutamate in the extracellular spaces. Astrocytes express the glutamate transporters responsible for majority of the glutamate uptake from the synapse, as well as for vesicular glutamate release. However, the cellular and molecular mechanisms of astrocyte-mediated excitotoxicity in the context of METH and HIV-1 are undefined. Topics reviewed include dysregulation of the glutamate transporters, specifically excitatory amino acid transporter-2, metabotropic glutamate receptor(s) expression and the release of glutamate by vesicular exocytosis. We also discuss glutamate concentration dysregulation through astrocytic expression of enzymes for glutamate synthesis and metabolism. Lastly, we discuss recent evidence of various astrocyte and neuron crosstalk mechanisms implicated in glutamate regulation. Astrocytes play an essential role in the neuropathologies associated with METH/HIV-1-induced excitotoxicity. We hope to shed light on common cellular and molecular pathways astrocytes share in glutamate regulation during drug abuse and HIV-1 infection. PMID:22591363

  19. HIV-1, methamphetamine and astrocyte glutamate regulation: combined excitotoxic implications for neuro-AIDS.

    PubMed

    Cisneros, Irma E; Ghorpade, Anuja

    2012-07-01

    Glutamate, the most abundant excitatory transmitter in the brain can lead to neurotoxicity when not properly regulated. Excitotoxicity is a direct result of abnormal regulation of glutamate concentrations in the synapse, and is a common neurotoxic mediator associated with neurodegenerative disorders. It is well accepted that methamphetamine (METH), a potent central nervous stimulant with high abuse potential, and human immunodeficiency virus (HIV)-1 are implicated in the progression of neurocognitive malfunction. Both have been shown to induce common neurodegenerative effects such as astrogliosis, compromised blood brain barrier integrity, and excitotoxicity in the brain. Reduced glutamate uptake from neuronal synapses likely leads to the accumulation of glutamate in the extracellular spaces. Astrocytes express the glutamate transporters responsible for majority of the glutamate uptake from the synapse, as well as for vesicular glutamate release. However, the cellular and molecular mechanisms of astrocyte-mediated excitotoxicity in the context of METH and HIV-1 are undefined. Topics reviewed include dysregulation of the glutamate transporters, specifically excitatory amino acid transporter-2, metabotropic glutamate receptor(s) expression and the release of glutamate by vesicular exocytosis. We also discuss glutamate concentration dysregulation through astrocytic expression of enzymes for glutamate synthesis and metabolism. Lastly, we discuss recent evidence of various astrocyte and neuron crosstalk mechanisms implicated in glutamate regulation. Astrocytes play an essential role in the neuropathologies associated with METH/HIV-1-induced excitotoxicity. We hope to shed light on common cellular and molecular pathways astrocytes share in glutamate regulation during drug abuse and HIV-1 infection.

  20. Extracellular glutamate alters mature osteoclast and osteoblast functions.

    PubMed

    Seidlitz, Eric P; Sharma, Mohit K; Singh, Gurmit

    2010-09-01

    Glutamatergic intercellular communication is involved in many aspects of metabolic homeostasis in normal bone. In bone metastasis, the balance between bone formation and degradation is disrupted. Although the responsible mechanisms are not clear, we have previously identified that cancer cell lines used in bone tumour models secrete glutamate, suggesting that tumour-derived glutamate may disrupt sensitive signalling systems in bone. This study examines the role of glutamate in mature osteoclastic bone resorption, osteoblast differentiation, and bone nodule formation. Glutamate was found to have no effect on the survival or activity of mature osteoclasts, although glutamate transporter inhibition and receptor blockade increased the number of bone resorption pits. Furthermore, transporter inhibition increased the area of resorbed bone while significantly decreasing the number of osteoclasts. Alkaline phosphatase activity and extracellular matrix mineralization were used as measurements of osteoblast differentiation. Glutamate significantly increased osteoblast differentiation and mineralization, but transport inhibitors had no effect. These studies support earlier findings suggesting that glutamate may be more important for osteoclastogenesis than for osteoclast proliferation or functions. Since glutamate is capable of changing the differentiation and activities of both osteoclast and osteoblast cell types in bone, it is reasonable to postulate that tumour-derived glutamate may impact bone homeostasis in bone metastasis.

  1. Spatiotemporal resolution of BDNF neuroprotection against glutamate excitotoxicity in cultured hippocampal neurons.

    PubMed

    Melo, C V; Okumoto, S; Gomes, J R; Baptista, M S; Bahr, B A; Frommer, W B; Duarte, C B

    2013-05-01

    Brain-derived neurotrophic factor (BDNF) protects hippocampal neurons from glutamate excitotoxicity as determined by analysis of chromatin condensation, through activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways. However, it is still unknown whether BDNF also prevents the degeneration of axons and dendrites, and the functional demise of synapses, which would be required to preserve neuronal activity. Herein, we have studied the time-dependent changes in several neurobiological markers, and the regulation of proteolytic mechanisms in cultured rat hippocampal neurons, through quantitative western blot and immunocytochemistry. Calpain activation peaked immediately after the neurodegenerative input, followed by a transient increase in ubiquitin-conjugated proteins and increased abundance of cleaved-caspase-3. Proteasome and calpain inhibition did not reproduce the protective effect of BDNF and caspase inhibition in preventing chromatin condensation. However, proteasome and calpain inhibition did protect the neuronal markers for dendrites (MAP-2), axons (Neurofilament-H) and the vesicular glutamate transporters (VGLUT1-2), whereas caspase inhibition was unable to mimic the protective effect of BDNF on neurites and synaptic markers. BDNF partially prevented the downregulation of synaptic activity measured by the KCl-evoked glutamate release using a Förster (Fluorescence) resonance energy transfer (FRET) glutamate nanosensor. These results translate a time-dependent activation of proteases and spatial segregation of these mechanisms, where calpain activation is followed by proteasome deregulation, from neuronal processes to the soma, and finally by caspase activation in the cell body. Moreover, PI3-K and PLCγ small molecule inhibitors significantly blocked the protective action of BDNF, suggesting an activity-dependent mechanism of neuroprotection. Ultimately, we hypothesize that neuronal repair after a

  2. Adenylyl cyclase activation underlies intracellular cyclic AMP accumulation, cyclic AMP transport, and extracellular adenosine accumulation evoked by beta-adrenergic receptor stimulation in mixed cultures of neurons and astrocytes derived from rat cerebral cortex.

    PubMed

    Rosenberg, P A; Li, Y

    1995-09-18

    We have previously shown that stimulation of cortical cultures containing both neurons and astrocytes with the beta-adrenergic agonist isoproterenol (ISO) results in transport of cAMP from astrocytes followed by extracellular hydrolysis to adenosine [Rosenberg et al. J. Neurosci. 14 (1994) 2953-2965]. In this study we found that the endogenous catecholamines epinephrine (EPI) and norepinephrine (NE), but not dopamine, serotonin, or histamine, all at 10 microM, significantly stimulated intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation in cortical cultures. Detailed dose-response experiments were performed for NE and EPI, as well as ISO. For each catecholamine, the potencies in evoking intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation were similar. These data provide additional evidence that a single common mechanism, namely beta-adrenergic mediated activation of adenylyl cyclase, underlies intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation. It appears that regulation of extracellular adenosine levels via cAMP transport and extracellular hydrolysis to adenosine may be a final common pathway of neuromodulation in cerebral cortex for catecholamines, and, indeed, any substance whose receptors are coupled to adenylyl cyclase.

  3. Glutamate signalling in roots.

    PubMed

    Forde, Brian G

    2014-03-01

    As a signalling molecule, glutamate is best known for its role as a fast excitatory neurotransmitter in the mammalian nervous system, a role that requires the activity of a family of ionotropic glutamate receptors (iGluRs). The unexpected discovery in 1998 that Arabidopsis thaliana L. possesses a family of iGluR-related (GLR) genes laid the foundations for an assessment of glutamate's potential role as a signalling molecule in plants that is still in progress. Recent advances in elucidating the function of Arabidopsis GLR receptors has revealed similarities with iGluRs in their channel properties, but marked differences in their ligand specificities. The ability of plant GLR receptors to act as amino-acid-gated Ca(2+) channels with a broad agonist profile, combined with their expression throughout the plant, makes them strong candidates for a multiplicity of amino acid signalling roles. Although root growth is inhibited in the presence of a number of amino acids, only glutamate elicits a specific sequence of changes in growth, root tip morphology, and root branching. The recent finding that the MEKK1 gene is a positive regulator of glutamate sensitivity at the root tip has provided genetic evidence for the existence in plants of a glutamate signalling pathway analogous to those found in animals. This short review will discuss the most recent advances in understanding glutamate signalling in roots, considering them in the context of previous work in plants and animals.

  4. Sodium-Dependent Glutamate Uptake by an Alkaliphilic, Thermophilic Bacillus Strain, TA2.A1

    PubMed Central

    Peddie, Catherine J.; Cook, Gregory M.; Morgan, Hugh W.

    1999-01-01

    A strain of Bacillus designated TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70°C. Bacillus strain TA2.A1 utilized glutamate as a sole carbon and energy source for growth, and sodium chloride (>5 mM) was an obligate requirement for growth. Growth on glutamate was inhibited by monensin and amiloride, both inhibitors that collapse the sodium gradient (ΔpNa) across the cell membrane. N,N-Dicyclohexylcarbodiimide inhibited the growth of Bacillus strain TA2.A1, suggesting that an F1F0-ATPase (H type) was being used to generate cellular ATP needed for anabolic reactions. Vanadate, an inhibitor of V-type ATPases, did not affect the growth of Bacillus strain TA2.A1. Glutamate transport by Bacillus strain TA2.A1 could be driven by an artificial membrane potential (ΔΨ), but only when sodium was present. In the absence of sodium, the rate of ΔΨ-driven glutamate uptake was fourfold lower. No glutamate transport was observed in the presence of ΔpNa alone (i.e., no ΔΨ). Glutamate uptake was specifically inhibited by monensin, and the Km for sodium was 5.6 mM. The Hill plot had a slope of approximately 1, suggesting that sodium binding was noncooperative and that the glutamate transporter had a single binding site for sodium. Glutamate transport was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that the transmembrane pH gradient was not required for glutamate transport. The rate of glutamate transport increased with increasing glutamate concentration; the Km for glutamate was 2.90 μM, and the Vmax was 0.7 nmol · min−1 mg of protein. Glutamate transport was specifically inhibited by glutamate analogues. PMID:10322019

  5. Glutamate-dependent transcriptional regulation of GLAST: role of PKC.

    PubMed

    López-Bayghen, Esther; Ortega, Arturo

    2004-10-01

    The Na+-dependent glutamate/aspartate transporter GLAST plays a major role in the removal of glutamate from the synaptic cleft. Short-term, as well as long-term changes in transporter activity are triggered by glutamate. An important locus of regulation is the density of transporter molecules present at the plasma membrane. A substrate-dependent change in the translocation rate of the transporter molecules accounts for the short-term effect, whereas the long-term modulation apparently involves transcriptional regulation. Using cultured chick cerebellar Bergmann glial cells, we report here that glutamate receptors activation mediate a substantial reduction in the transcriptional activity of the chglast promoter through the Ca2+/diacylglicerol-dependent protein kinase (PKC) signaling cascade. Overexpression of constitutive active PKC isoforms of mimic the glutamate effect. Accordingly, increased levels of c-Jun or c-Fos, but not Jun-B, Jun-D or Fos-B, lower the chglast promoter activity. Serial deletions and electrophorectic mobility shift assays were used to define a specific region within the 5' proximal region of the chglast promoter, associated with transcriptional repression. A putative glutamate response element could be defined in the proximal promoter stretch more likely between nts -40 and -78. These results demonstrate that GLAST is under glutamate-dependent transcriptional control through PKC, and support the notion of a pivotal role of this neurotransmitter in the regulation of its own removal from the synaptic cleft, thereby modulating, mainly in the long term, glutamatergic transmission.

  6. Shifted pallidal co-release of GABA and glutamate in habenula drives cocaine withdrawal and relapse.

    PubMed

    Meye, Frank J; Soiza-Reilly, Mariano; Smit, Tamar; Diana, Marco A; Schwarz, Martin K; Mameli, Manuel

    2016-08-01

    Cocaine withdrawal produces aversive states and vulnerability to relapse, hallmarks of addiction. The lateral habenula (LHb) encodes negative stimuli and contributes to aversive withdrawal symptoms. However, it remains unclear which inputs to LHb promote this and what the consequences are for relapse susceptibility. We report, using rabies-based retrolabeling and optogenetic mapping, that the entopeduncular nucleus (EPN, the mouse equivalent of the globus pallidus interna) projects to an LHb neuronal subset innervating aversion-encoding midbrain GABA neurons. EPN-to-LHb excitatory signaling is limited by GABAergic cotransmission. This inhibitory component decreases during cocaine withdrawal as a result of reduced presynaptic vesicular GABA transporter (VGAT). This shifts the EPN-to-LHb GABA/glutamate balance, disinhibiting EPN-driven LHb activity. Selective virally mediated VGAT overexpression at EPN-to-LHb terminals during withdrawal normalizes GABAergic neurotransmission. This intervention rescues cocaine-evoked aversive states and prevents stress-induced reinstatement, used to model relapse. This identifies diminished inhibitory transmission at EPN-to-LHb GABA/glutamate synapses as a mechanism contributing to the relapsing feature of addictive behavior. PMID:27348214

  7. Changes in NAD(P)H fluorescence and membrane current produced by glutamate uptake into salamander Müller cells.

    PubMed Central

    Barbour, B; Magnus, C; Szatkowski, M; Gray, P T; Attwell, D

    1993-01-01

    1. Glutamate uptake into isolated, whole-cell patch-clamped glial cells was studied by monitoring the increase of cell fluorescence generated as glutamate and NAD(P) were converted into alpha-ketoglutarate and NAD(P)H by glutamate dehydrogenase. The current generated by the glutamate uptake carrier was recorded simultaneously. 2. L-Glutamate evoked an increase of cell fluorescence and an inward uptake current. L- and D-aspartate generated an uptake current but no fluorescence response, consistent with the amino acid specificity of glutamate dehydrogenase. 3. In the absence of external sodium the glutamate-evoked fluorescence response and uptake current were abolished, showing that there is no sodium-independent glutamate uptake across the cell membrane. 4. Varying the glutamate concentration altered both the fluorescence response and the uptake current. The fluorescence response saturated at a lower glutamate concentration than the uptake current, and depended in a Michaelis-Menten fashion on the uptake current. 5. The fluorescence response and the uptake current were reduced by membrane depolarization, and also by removal of intracellular potassium. 6. The dependence of the fluorescence response on uptake current when membrane potential was altered or intracellular potassium was removed was the same as that seen when the external glutamate concentration was altered. 7. These fluorescence studies show that glutamate uptake is inhibited by depolarization and by removal of intracellular potassium, consistent with the conclusion of earlier work in which uptake was monitored solely as a membrane current. The data are consistent with high-affinity electrogenic sodium- and potassium-dependent glutamate uptake with fixed stoichiometry being the only significant influx route for glutamate. Other possible interpretations of the data are also discussed. PMID:8105078

  8. Neuronal vs glial glutamate uptake: Resolving the conundrum.

    PubMed

    Danbolt, N C; Furness, D N; Zhou, Y

    2016-09-01

    Neither normal brain function nor the pathological processes involved in neurological diseases can be adequately understood without knowledge of the release, uptake and metabolism of glutamate. The reason for this is that glutamate (a) is the most abundant amino acid in the brain, (b) is at the cross-roads between several metabolic pathways, and (c) serves as the major excitatory neurotransmitter. In fact most brain cells express glutamate receptors and are thereby influenced by extracellular glutamate. In agreement, brain cells have powerful uptake systems that constantly remove glutamate from the extracellular fluid and thereby limit receptor activation. It has been clear since the 1970s that both astrocytes and neurons express glutamate transporters. However the relative contribution of neuronal and glial transporters to the total glutamate uptake activity, however, as well as their functional importance, has been hotly debated ever since. The present short review provides (a) an overview of what we know about neuronal glutamate uptake as well as an historical description of how we got there, and (b) a hypothesis reconciling apparently contradicting observations thereby possibly resolving the paradox. PMID:27235987

  9. Amperometric Measurement of Glutamate Release Modulation by Gabapentin and Pregabalin in Rat Neocortical Slices: Role of Voltage-Sensitive Ca2+ α2δ-1 Subunit

    PubMed Central

    Dooley, David J.; Pomerleau, François; Huettl, Peter; Gerhardt, Greg A.

    2011-01-01

    Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinically to treat several disorders associated with excessive or inappropriate excitability, including epilepsy; pain from diabetic neuropathy, postherpetic neuralgia, and fibromyalgia; and generalized anxiety disorder. The molecular basis for these drugs' therapeutic effects are believed to involve the interaction with the auxiliary α2δ subunit of voltage-sensitive Ca2+ channel (VSCC) translating into a modulation of pathological neurotransmitter release. Glutamate as the primary excitatory neurotransmitter in the mammalian central nervous system contributes, under conditions of excessive glutamate release, to neurological and psychiatric disorders. This study used enzyme-based microelectrode arrays to directly measure extracellular glutamate release in rat neocortical slices and determine the modulation of this release by GBP and PGB. Both drugs attenuated K+-evoked glutamate release without affecting basal glutamate levels. PGB (0.1–100 μM) exhibited concentration-dependent inhibition of K+-evoked glutamate release with an IC50 value of 5.3 μM. R-(−)-3-Isobutylgaba, the enantiomer of PGB, did not significantly reduce K+-evoked glutamate release. The decrease of K+-evoked glutamate release by PGB was blocked by the l-amino acid l-isoleucine, a potential endogenous ligand of the α2δ subunit. In neocortical slices from transgenic mice having a point mutation (i.e., R217A) of the α2δ-1 (subtype) subunit of VSCC, PGB did not affect K+-evoked glutamate release yet inhibited this release in wild-type mice. The results show that GBP and PGB attenuated stimulus-evoked glutamate release in rodent neocortical slices and that the α2δ-1 subunit of VSCC appears to mediate this effect. PMID:21464332

  10. Central Role of Glutamate Metabolism in the Maintenance of Nitrogen Homeostasis in Normal and Hyperammonemic Brain

    PubMed Central

    Cooper, Arthur J. L.; Jeitner, Thomas M.

    2016-01-01

    Glutamate is present in the brain at an average concentration—typically 10–12 mM—far in excess of those of other amino acids. In glutamate-containing vesicles in the brain, the concentration of glutamate may even exceed 100 mM. Yet because glutamate is a major excitatory neurotransmitter, the concentration of this amino acid in the cerebral extracellular fluid must be kept low—typically µM. The remarkable gradient of glutamate in the different cerebral compartments: vesicles > cytosol/mitochondria > extracellular fluid attests to the extraordinary effectiveness of glutamate transporters and the strict control of enzymes of glutamate catabolism and synthesis in well-defined cellular and subcellular compartments in the brain. A major route for glutamate and ammonia removal is via the glutamine synthetase (glutamate ammonia ligase) reaction. Glutamate is also removed by conversion to the inhibitory neurotransmitter γ-aminobutyrate (GABA) via the action of glutamate decarboxylase. On the other hand, cerebral glutamate levels are maintained by the action of glutaminase and by various α-ketoglutarate-linked aminotransferases (especially aspartate aminotransferase and the mitochondrial and cytosolic forms of the branched-chain aminotransferases). Although the glutamate dehydrogenase reaction is freely reversible, owing to rapid removal of ammonia as glutamine amide, the direction of the glutamate dehydrogenase reaction in the brain in vivo is mainly toward glutamate catabolism rather than toward the net synthesis of glutamate, even under hyperammonemia conditions. During hyperammonemia, there is a large increase in cerebral glutamine content, but only small changes in the levels of glutamate and α-ketoglutarate. Thus, the channeling of glutamate toward glutamine during hyperammonemia results in the net synthesis of 5-carbon units. This increase in 5-carbon units is accomplished in part by the ammonia-induced stimulation of the anaplerotic enzyme pyruvate

  11. Central Role of Glutamate Metabolism in the Maintenance of Nitrogen Homeostasis in Normal and Hyperammonemic Brain.

    PubMed

    Cooper, Arthur J L; Jeitner, Thomas M

    2016-01-01

    Glutamate is present in the brain at an average concentration-typically 10-12 mM-far in excess of those of other amino acids. In glutamate-containing vesicles in the brain, the concentration of glutamate may even exceed 100 mM. Yet because glutamate is a major excitatory neurotransmitter, the concentration of this amino acid in the cerebral extracellular fluid must be kept low-typically µM. The remarkable gradient of glutamate in the different cerebral compartments: vesicles > cytosol/mitochondria > extracellular fluid attests to the extraordinary effectiveness of glutamate transporters and the strict control of enzymes of glutamate catabolism and synthesis in well-defined cellular and subcellular compartments in the brain. A major route for glutamate and ammonia removal is via the glutamine synthetase (glutamate ammonia ligase) reaction. Glutamate is also removed by conversion to the inhibitory neurotransmitter γ-aminobutyrate (GABA) via the action of glutamate decarboxylase. On the other hand, cerebral glutamate levels are maintained by the action of glutaminase and by various α-ketoglutarate-linked aminotransferases (especially aspartate aminotransferase and the mitochondrial and cytosolic forms of the branched-chain aminotransferases). Although the glutamate dehydrogenase reaction is freely reversible, owing to rapid removal of ammonia as glutamine amide, the direction of the glutamate dehydrogenase reaction in the brain in vivo is mainly toward glutamate catabolism rather than toward the net synthesis of glutamate, even under hyperammonemia conditions. During hyperammonemia, there is a large increase in cerebral glutamine content, but only small changes in the levels of glutamate and α-ketoglutarate. Thus, the channeling of glutamate toward glutamine during hyperammonemia results in the net synthesis of 5-carbon units. This increase in 5-carbon units is accomplished in part by the ammonia-induced stimulation of the anaplerotic enzyme pyruvate carboxylase

  12. Spontaneous and evoked synaptic rewiring in the neonatal neocortex.

    PubMed

    Le Bé, Jean-Vincent; Markram, Henry

    2006-08-29

    The local microcircuitry of the neocortex is structurally a tabula rasa, with the axon of each pyramidal neuron having numerous submicrometer appositions with the dendrites of all neighboring pyramidal neurons, but is functionally highly selective, with synapses formed onto only a small proportion of these targets. This design leaves a vast potential for the microcircuit to rewire without extensive axonal or dendritic growth. To examine whether rewiring does take place, we used multineuron patch-clamp recordings on 12- to 14-day-old rat neocortical slices and studied long-term changes in synaptic connectivity within clusters of neurons. We found pyramidal neurons spontaneously connecting and disconnecting from each other and that exciting the slice with glutamate greatly increases the number of new connections established. Evoked emergence of new synaptic connections requires action potential activity and activation of metabotropic glutamate receptor 5, but not NMDA receptor or group II or group III metabotropic glutamate receptor activation. We also found that it is the weaker connections that are selectively eliminated. These results provide direct evidence for spontaneous and evoked rewiring of the neocortical microcircuitry involving entire functional multisynaptic connections. We speculate that this form of microcircuit plasticity enables an evolution of the microcircuit connectivity by natural selection as a function of experience.

  13. Production of D-Glutamate from L-Glutamate with Glutamate Racemase and L-Glutamate Oxidase.

    PubMed

    Oikawa, T; Watanabe, M; Makiura, H; Kusakabe, H; Yamade, K; Soda, K

    1999-01-01

    We studied production of D-glutamate from L-glutamate using a bioreactor consisting of two columns of sequentially connected immobilized glutamate racemase (EC 5.1.1.3, from Bacillus subtilis IFO 3336) and L-glutamate oxidase (EC 1.4.3.11, from Streptomyces sp. X119-6): L-glutamate was racemized by the glutamate racemase column, and then L-glutamate was oxidized by the L-glutamate oxidase column. Consequently only D-glutamate remained, and was easily separated from the α-ketoglutarate formed by anion-exchange chromatography. Both enzymes were highly stabilized by immobilization. The pH and temperature optima of immobilized glutamate racemase (pH 8, 40°C) were similar to those of immobilized L-glutamate oxidase (pH 7, 50°C). Accordingly, we connected the two columns tandemly to do both enzyme reactions under the same conditions. Actually 4.5 μmol of D-glutamate was produced and isolated from 10 μmol of L-glutamate, about 90% of the theoretical yield. PMID:27373918

  14. Chronic Glutamate Toxicity in Neurodegenerative Diseases—What is the Evidence?

    PubMed Central

    Lewerenz, Jan; Maher, Pamela

    2015-01-01

    Together with aspartate, glutamate is the major excitatory neurotransmitter in the brain. Glutamate binds and activates both ligand-gated ion channels (ionotropic glutamate receptors) and a class of G-protein coupled receptors (metabotropic glutamate receptors). Although the intracellular glutamate concentration in the brain is in the millimolar range, the extracellular glutamate concentration is kept in the low micromolar range by the action of excitatory amino acid transporters that import glutamate and aspartate into astrocytes and neurons. Excess extracellular glutamate may lead to excitotoxicity in vitro and in vivo in acute insults like ischemic stroke via the overactivation of ionotropic glutamate receptors. In addition, chronic excitotoxicity has been hypothesized to play a role in numerous neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimer's disease and Huntington's disease. Based on this hypothesis, a good deal of effort has been devoted to develop and test drugs that either inhibit glutamate receptors or decrease extracellular glutamate. In this review, we provide an overview of the different pathways that are thought to lead to an over-activation of the glutamatergic system and glutamate toxicity in neurodegeneration. In addition, we summarize the available experimental evidence for glutamate toxicity in animal models of neurodegenerative diseases. PMID:26733784

  15. Protein kinase C -dependent regulation of synaptosomal glutamate uptake under conditions of hypergravity

    NASA Astrophysics Data System (ADS)

    Borisova, Tatiana; Krisanova, Natalia; Borisov, Arseniy; Sivko, Roman

    Glutamate is not only the main excitatory neurotransmitter in the mammalian CNS, but also a potent neurotoxin. Excessive concentration of ambient glutamate over activates glutamate receptors and causes neurotoxicity. Uptake of glutamate from the extracellular space into nerve cells was mediated by sodium-dependent glutamate transporters located in the plasma membrane. It was shown that the activity of glutamate transporters in rat brain nerve terminals was decreased after hypergravity (centrifugation of rats in special containers at 10 G for 1 hour). This decrease may result from the reduction in the number of glutamate transporters expressed in the plasma membrane of nerve terminals after hypergravity that was regulated by protein kinase C. The possibility of the involvement of protein kinase C in the regulation of the activity of glutamate transporters was assessed under conditions of hypergravity. The effect of protein kinase C inhibitor GF 109 203X on synaptosomal L-[14C]glutamate uptake was analysed. It was shown that the inhibitor decreased L-[14C]glutamate uptake by 15 % in control but did not influence it after hypergravity. In control, the initial velocity of L-[14C]glutamate uptake in the presence of the inhibitor decreased from 2.5 ± 0.2 nmol x min-1 x mg-1 of proteins to 2.17 ± 0.1 nmol x min-1 x mg-1 of proteins, whereas after hypergravity this value lowered from 2.05 ± 0.1 nmol x min-1 x mg-1 of proteins to 2.04 ± 0.1 nmol x min-1 x mg-1 of proteins. Thus, protein kinase C -dependent alteration in the cell surface expression of glutamate transporters may be one of the causes of a decrease in the activity of glutamate transporters after hypergravity.

  16. A review of glutamate's role in traumatic brain injury mechanisms

    NASA Astrophysics Data System (ADS)

    Good, Cameron H.

    2013-05-01

    Glutamate is the primary excitatory neurotransmitter used by the central nervous system (CNS) for synaptic communication, and its extracellular concentration is tightly regulated by glutamate transporters located on nearby astrocytes. Both animal models and human clinical studies have demonstrated elevated glutamate levels immediately following a traumatic brain event, with the duration and severity of the rise corresponding to prognosis. This rise in extracellular glutamate likely results from a combination of excessive neurotransmitter release from damaged neurons and down regulation of uptake mechanisms in local astrocytes. The immediate results of a traumatic event can lead to necrotic tissue in severely injured regions, while prolonged increases in excitatory transmission can cause secondary excitotoxic injury through activation of delayed apoptotic pathways. Initial TBI animal studies utilized a variety of broad glutamate receptor antagonists to successfully combat secondary injury mechanisms, but unfortunately this same strategy has proven inconclusive in subsequent human trials due to deleterious side effects and heterogeneity of injuries. More recent treatment strategies have utilized specific glutamate receptor subunit antagonists in an effort to minimize side effects and have shown promising results. Future challenges will be detecting the concentration and kinetics of the glutamate rise following injury, determining which patient populations could benefit from antagonist treatment based on their extracellular glutamate concentrations and when drugs should be administered to maximize efficacy.

  17. Effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by Corynebacterium glutamicum.

    PubMed

    Cao, Yan; Duan, Zuoying; Shi, Zhongping

    2014-02-01

    Biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic Corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with Tween 40 addition during fermentation. The transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (TP) of glutamate were investigated under the conditions of varied biotin contents and Tween 40 supplementation. When biotin was insufficient, the genes encoding key enzymes and TP were down-regulated in the early production phase, in particular, the transcription level of isocitrate dehydrogenase (ICDH) which was only 2% of that of control. Although the cells' morphology transformation and TP level were not affected, low transcription level of ICDH led to lower final glutamate concentration (64 g/L). When biotin was excessive, the transcription levels of key enzymes were at comparable levels as those of control with ICDH as an exception, which was only 3-22% of control level throughout production phase. In this case, little intracellular glutamate accumulation (1.5 mg/g DCW) and impermeable membrane resulted in non glutamate secretion into broth, even though the quantity of TP was more than 10-folds of control level. Addition of Tween 40 when biotin was excessive stimulated the expression of all key enzymes and TP, intracellular glutamate content was much higher (10-12 mg/g DCW), and final glutamate concentration reached control level (75-80 g/L). Hence, the membrane alteration and TP were indispensable in glutamate secretion. Biotin and Tween 40 influenced the expression level of ICDH and glutamate efflux, thereby influencing glutamate production.

  18. MPA-capped CdTe quantum dots exposure causes neurotoxic effects in nematode Caenorhabditis elegans by affecting the transporters and receptors of glutamate, serotonin and dopamine at the genetic level, or by increasing ROS, or both

    NASA Astrophysics Data System (ADS)

    Wu, Tianshu; He, Keyu; Zhan, Qinglin; Ang, Shengjun; Ying, Jiali; Zhang, Shihan; Zhang, Ting; Xue, Yuying; Tang, Meng

    2015-12-01

    As quantum dots (QDs) are widely used in biomedical applications, the number of studies focusing on their biological properties is increasing. While several studies have attempted to evaluate the toxicity of QDs towards neural cells, the in vivo toxic effects on the nervous system and the molecular mechanisms are unclear. The aim of the present study was to investigate the neurotoxic effects and the underlying mechanisms of water-soluble cadmium telluride (CdTe) QDs capped with 3-mercaptopropionic acid (MPA) in Caenorhabditis elegans (C. elegans). Our results showed that exposure to MPA-capped CdTe QDs induced behavioral defects, including alterations to body bending, head thrashing, pharyngeal pumping and defecation intervals, as well as impaired learning and memory behavior plasticity, based on chemotaxis or thermotaxis, in a dose-, time- and size-dependent manner. Further investigations suggested that MPA-capped CdTe QDs exposure inhibited the transporters and receptors of glutamate, serotonin and dopamine in C. elegans at the genetic level within 24 h, while opposite results were observed after 72 h. Additionally, excessive reactive oxygen species (ROS) generation was observed in the CdTe QD-treated worms, which confirmed the common nanotoxicity mechanism of oxidative stress damage, and might overcome the increased gene expression of neurotransmitter transporters and receptors in C. elegans induced by long-term QD exposure, resulting in more severe behavioral impairments.

  19. MPA-capped CdTe quantum dots exposure causes neurotoxic effects in nematode Caenorhabditis elegans by affecting the transporters and receptors of glutamate, serotonin and dopamine at the genetic level, or by increasing ROS, or both.

    PubMed

    Wu, Tianshu; He, Keyu; Zhan, Qinglin; Ang, Shengjun; Ying, Jiali; Zhang, Shihan; Zhang, Ting; Xue, Yuying; Tang, Meng

    2015-12-28

    As quantum dots (QDs) are widely used in biomedical applications, the number of studies focusing on their biological properties is increasing. While several studies have attempted to evaluate the toxicity of QDs towards neural cells, the in vivo toxic effects on the nervous system and the molecular mechanisms are unclear. The aim of the present study was to investigate the neurotoxic effects and the underlying mechanisms of water-soluble cadmium telluride (CdTe) QDs capped with 3-mercaptopropionic acid (MPA) in Caenorhabditis elegans (C. elegans). Our results showed that exposure to MPA-capped CdTe QDs induced behavioral defects, including alterations to body bending, head thrashing, pharyngeal pumping and defecation intervals, as well as impaired learning and memory behavior plasticity, based on chemotaxis or thermotaxis, in a dose-, time- and size-dependent manner. Further investigations suggested that MPA-capped CdTe QDs exposure inhibited the transporters and receptors of glutamate, serotonin and dopamine in C. elegans at the genetic level within 24 h, while opposite results were observed after 72 h. Additionally, excessive reactive oxygen species (ROS) generation was observed in the CdTe QD-treated worms, which confirmed the common nanotoxicity mechanism of oxidative stress damage, and might overcome the increased gene expression of neurotransmitter transporters and receptors in C. elegans induced by long-term QD exposure, resulting in more severe behavioral impairments.

  20. Glutamate dysfunction associated with developmental cerebellar damage: Relevance to autism spectrum disorders

    PubMed Central

    McKimm, Erik J.; Corkill, Beau; Goldowitz, Dan; Albritton, Lorraine M.; Homayouni, Ramin; Blaha, Charles D.; Mittleman, Guy

    2014-01-01

    Neural abnormalities commonly associated with autism spectrum disorders include prefrontal cortex (PFC) dysfunction and cerebellar pathology in the form of Purkinje cell loss and cerebellar hypoplasia. It has been reported that loss of cerebellar Purkinje cells results in aberrant dopamine neurotransmission in the PFC which occurs via dysregulation of multisynaptic efferents from the cerebellum to the PFC. Using a mouse model we investigated the possibility that developmental cerebellar Purkinje cell loss could disrupt glutamatergic cerebellar projections to the PFC that ultimately modulate DA release. We measured glutamate release evoked by local electrical stimulation using fixed potential amperometry in combination with glutamate selective enzyme-based recording probes in urethane anesthetized Lurcher mutant and wildtype mice. Target sites included the medio-dorsal and ventro-lateral thalamic nuclei, reticulo-tegmental nuclei, pedunculopontine nuclei, and ventral tegmental area. With the exception of the ventral tegmental area, results indicated that in comparison to wildtype mice, evoked glutamate release was reduced in Lurcher mutants by between 9% to 72% at all stimulated sites. These results are consistent with the notion that developmental loss of cerebellar Purkinje cells drives reductions in evoked glutamate release in cerebellar efferent pathways that ultimately influence PFC dopamine release. Possible mechanisms whereby reductions in glutamate release could occur are discussed. PMID:24307139

  1. Evoked Potential Variability

    PubMed Central

    Hu, Lingli; Boutros, Nash N.; Jansen, Ben H.

    2008-01-01

    An unsupervised correlation-based clustering method was developed to assess the trial-to-trial variability of auditory evoked potentials (AEPs). The method first decomposes single trials into three frequency bands, each containing activity primarily associated with one of the three major AEP components, i.e., P50, N100 and P200. Next, single-trial evoked potentials with similar post-stimulus characteristics are clustered and selectively averaged to determine the presence or absence of an AEP component. The method was evaluated on actual AEP and spontaneous EEG data collected from 25 healthy participants using a paradigm in which pairs of identical tones were presented, with the first stimulus (S1) presented 0.5 s before the second stimulus (S2). Homogeneous, well-separated clusters were obtained and substantial AEP variability was found. Also, there was a trend for S2 to produce fewer ‘complete’ (and significantly smaller) responses than S1. Tests conducted on spontaneous EEG produced similar clusters as obtained from EP data, but significantly fewer stimuli produced responses containing all three EP components than seen in AEP data. These findings suggest that the clustering method presented here performs adequately to assess trial-to-trial EP variability. Also, the results suggest that the sensory gating observed in normal controls may be caused by the fact that the second stimulus generates fewer ‘responsive’ trials than the first stimulus, thus resulting in smaller ensemble averages. PMID:19103222

  2. Evoked potentials in multiple sclerosis.

    PubMed

    Kraft, George H

    2013-11-01

    Before the development of magnetic resonance imaging (MRI), evoked potentials (EPs)-visual evoked potentials, somatosensory evoked potentials, and brain stem auditory evoked responses-were commonly used to determine a second site of disease in patients being evaluated for possible multiple sclerosis (MS). The identification of an area of the central nervous system showing abnormal conduction was used to supplement the abnormal signs identified on the physical examination-thus identifying the "multiple" in MS. This article is a brief overview of additional ways in which central nervous system (CNS) physiology-as measured by EPs-can still contribute value in the management of MS in the era of MRIs.

  3. Evidence for synergistic control of glutamate biosynthesis by glutamate dehydrogenases and glutamate in Bacillus subtilis.

    PubMed

    Stannek, Lorena; Thiele, Martin J; Ischebeck, Till; Gunka, Katrin; Hammer, Elke; Völker, Uwe; Commichau, Fabian M

    2015-09-01

    In the Gram-positive bacterium, Bacillus subtilis glutamate is synthesized by the glutamine synthetase and the glutamate synthase (GOGAT). During growth with carbon sources that exert carbon catabolite repression, the rocG glutamate dehydrogenase (GDH) gene is repressed and the transcription factor GltC activates the expression of the GOGAT encoding gltAB genes. In the presence of amino acids of the glutamate family, the GDH RocG is synthesized and the enzyme prevents GltC from binding to DNA. The dual control of glutamate biosynthesis allows the efficient utilization of the available nutrients. Here we provide genetic and biochemical evidence that, like RocG, also the paralogous GDH GudB can inhibit the transcription factor GltC, thereby controlling glutamate biosynthesis. Contradictory previous observations show that high level of GDH activity does not result in permanent inhibition of GltC. By controlling the intracellular levels of glutamate through feeding with exogenous arginine, we observed that the GDH-dependent control of GltC and thus expression of the gltAB genes inversely correlates with the glutamate pool. These results suggest that the B. subtilis GDHs RocG and GudB in fact act as glutamate sensors. In conclusion, the GDH-mediated control of glutamate biosynthesis seems to depend on the intracellular glutamate concentration. PMID:25711804

  4. The cystine/glutamate antiporter system xc− drives breast tumor cell glutamate release and cancer-induced bone pain

    PubMed Central

    Slosky, Lauren M.; BassiriRad, Neemah M.; Symons, Ashley M.; Thompson, Michelle; Doyle, Timothy; Forte, Brittany L.; Staatz, William D.; Bui, Lynn; Neumann, William L.; Mantyh, Patrick W.; Salvemini, Daniela; Largent-Milnes, Tally M.; Vanderah, Todd W.

    2016-01-01

    Abstract Bone is one of the leading sites of metastasis for frequently diagnosed malignancies, including those arising in the breast, prostate and lung. Although these cancers develop unnoticed and are painless in their primary sites, bone metastases result in debilitating pain. Deeper investigation of this pain may reveal etiology and lead to early cancer detection. Cancer-induced bone pain (CIBP) is inadequately managed with current standard-of-care analgesics and dramatically diminishes patient quality of life. While CIBP etiology is multifaceted, elevated levels of glutamate, an excitatory neurotransmitter, in the bone-tumor microenvironment may drive maladaptive nociceptive signaling. Here, we establish a relationship between the reactive nitrogen species peroxynitrite, tumor-derived glutamate, and CIBP. In vitro and in a syngeneic in vivo model of breast CIBP, murine mammary adenocarcinoma cells significantly elevated glutamate via the cystine/glutamate antiporter system xc−. The well-known system xc− inhibitor sulfasalazine significantly reduced levels of glutamate and attenuated CIBP-associated flinching and guarding behaviors. Peroxynitrite, a highly reactive species produced in tumors, significantly increased system xc− functional expression and tumor cell glutamate release. Scavenging peroxynitrite with the iron and mangano-based porphyrins, FeTMPyP and SRI10, significantly diminished tumor cell system xc− functional expression, reduced femur glutamate levels and mitigated CIBP. In sum, we demonstrate how breast cancer bone metastases upregulate a cystine/glutamate co-transporter to elevate extracellular glutamate. Pharmacological manipulation of peroxynitrite or system xc− attenuates CIBP, supporting a role for tumor-derived glutamate in CIBP and validating the targeting of system xc− as a novel therapeutic strategy for the management of metastatic bone pain. PMID:27482630

  5. Metabolic pathways and activity-dependent modulation of glutamate concentration in the human brain.

    PubMed

    Mangia, Silvia; Giove, Federico; Dinuzzo, Mauro

    2012-11-01

    Glutamate is one of the most versatile molecules present in the human brain, involved in protein synthesis, energy production, ammonia detoxification, and transport of reducing equivalents. Aside from these critical metabolic roles, glutamate plays a major part in brain function, being not only the most abundant excitatory neurotransmitter, but also the precursor for γ-aminobutyric acid, the predominant inhibitory neurotransmitter. Regulation of glutamate levels is pivotal for normal brain function, as abnormal extracellular concentration of glutamate can lead to impaired neurotransmission, neurodegeneration and even neuronal death. Understanding how the neuron-astrocyte functional and metabolic interactions modulate glutamate concentration during different activation status and under physiological and pathological conditions is a challenging task, and can only be tentatively estimated from current literature. In this paper, we focus on describing the various metabolic pathways which potentially affect glutamate concentration in the brain, and emphasize which ones are likely to produce the variations in glutamate concentration observed during enhanced neuronal activity in human studies.

  6. Adjective Metaphors Evoke Negative Meanings

    PubMed Central

    Sakamoto, Maki; Utsumi, Akira

    2014-01-01

    Previous metaphor studies have paid much attention to nominal metaphors and predicative metaphors, but little attention has been given to adjective metaphors. Although some studies have focused on adjective metaphors, they only examined differences in the acceptability of various types of adjective metaphors. This paper explores the cognitive effects evoked by adjective metaphors. Three psychological experiments revealed that (1) adjective metaphors, especially those modified by color adjectives, tend to evoke negative effect; (2) although the meanings of metaphors are basically affected by the meanings of their vehicles, when a vehicle has a neutral meaning, negative meanings are evoked most frequently for adjective metaphors compared to nominal and predicative metaphors; (3) negative meanings evoked by adjective metaphors are related to poeticness, and poetic metaphors evoke negative meanings more easily than less poetic metaphors. Our research sheds new light on studies of the use of metaphor, which is one of the most basic human cognitive abilities. PMID:24586480

  7. Adjective metaphors evoke negative meanings.

    PubMed

    Sakamoto, Maki; Utsumi, Akira

    2014-01-01

    Previous metaphor studies have paid much attention to nominal metaphors and predicative metaphors, but little attention has been given to adjective metaphors. Although some studies have focused on adjective metaphors, they only examined differences in the acceptability of various types of adjective metaphors. This paper explores the cognitive effects evoked by adjective metaphors. Three psychological experiments revealed that (1) adjective metaphors, especially those modified by color adjectives, tend to evoke negative effect; (2) although the meanings of metaphors are basically affected by the meanings of their vehicles, when a vehicle has a neutral meaning, negative meanings are evoked most frequently for adjective metaphors compared to nominal and predicative metaphors; (3) negative meanings evoked by adjective metaphors are related to poeticness, and poetic metaphors evoke negative meanings more easily than less poetic metaphors. Our research sheds new light on studies of the use of metaphor, which is one of the most basic human cognitive abilities.

  8. Coupling of glutamate and glucose uptake in cultured Bergmann glial cells.

    PubMed

    Mendez-Flores, Orquidia G; Hernández-Kelly, Luisa C; Suárez-Pozos, Edna; Najimi, Mustapha; Ortega, Arturo

    2016-09-01

    Glutamate, the main excitatory neurotransmitter in the vertebrate brain, exerts its actions through specific membrane receptors present in neurons and glial cells. Over-stimulation of glutamate receptors results in neuronal death, phenomena known as excitotoxicity. A family of sodium-dependent, glutamate uptake transporters mainly expressed in glial cells, removes the amino acid from the synaptic cleft preventing neuronal death. The sustained sodium influx associated to glutamate removal in glial cells, activates the sodium/potassium ATPase restoring the ionic balance, additionally, glutamate entrance activates glutamine synthetase, both events are energy demanding, therefore glia cells increase their ATP expenditure favouring glucose uptake, and triggering several signal transduction pathways linked to proper neuronal glutamate availability, via the glutamate/glutamine shuttle. To further characterize these complex transporters interactions, we used the well-established model system of cultured chick cerebellum Bergmann glia cells. A time and dose-dependent increase in the activity, plasma membrane localization and protein levels of glucose transporters was detected upon d-aspartate exposure. Interestingly, this increase is the result of a protein kinase C-dependent signaling cascade. Furthermore, a glutamate-dependent glucose and glutamate transporters co-immunoprecipitation was detected. These results favour the notion that glial cells are involved in glutamatergic neuronal physiology. PMID:27184733

  9. Peripherally restricted viral challenge elevates extracellular glutamate and enhances synaptic transmission in the hippocampus.

    PubMed

    Hunsberger, Holly C; Wang, Desheng; Petrisko, Tiffany J; Alhowail, Ahmad; Setti, Sharay E; Suppiramaniam, Vishnu; Konat, Gregory W; Reed, Miranda N

    2016-07-01

    Peripheral infections increase the propensity and severity of seizures in susceptible populations. We have previously shown that intraperitoneal injection of a viral mimic, polyinosinic-polycytidylic acid (PIC), elicits hypersusceptibility of mice to kainic acid (KA)-induced seizures. This study was undertaken to determine whether this seizure hypersusceptibility entails alterations in glutamate signaling. Female C57BL/6 mice were intraperitoneally injected with PIC, and after 24 h, glutamate homeostasis in the hippocampus was monitored using the enzyme-based microelectrode arrays. PIC challenge robustly increased the level of resting extracellular glutamate. While pre-synaptic potassium-evoked glutamate release was not affected, glutamate uptake was profoundly impaired and non-vesicular glutamate release was augmented, indicating functional alterations of astrocytes. Electrophysiological examination of hippocampal slices from PIC-challenged mice revealed a several fold increase in the basal synaptic transmission as compared to control slices. PIC challenge also increased the probability of pre-synaptic glutamate release as seen from a reduction of paired-pulse facilitation and synaptic plasticity as seen from an enhancement of long-term potentiation. Altogether, our results implicate a dysregulation of astrocytic glutamate metabolism and an alteration of excitatory synaptic transmission as the underlying mechanism for the development of hippocampal hyperexcitability, and consequently seizure hypersusceptibility following peripheral PIC challenge. Peripheral infections/inflammations enhance seizure susceptibility. Here, we explored the effect of peritoneal inflammation induced by a viral mimic on glutamate homeostasis and glutamatergic neurotransmission in the mouse hippocampus. We found that peritoneal inflammation elevated extracellular glutamate concentration and enhanced the probability of pre-synaptic glutamate release resulting in hyperexcitability of

  10. Reduction of glutamate content in rat superior colliculus after retino-tectal denervation.

    PubMed

    Sakurai, T; Miyamoto, T; Okada, Y

    1990-02-16

    The effect of afferent lesions on glutamate content was measured in the lamina of the superior colliculus (SC) in the rat. The analysis was performed 12 days after unilateral enucleation (left eye), or ablation of visual cortex (right), or both enucleation and ablation. The glutamate contained in the superficial grey layer (SGL) and deep layer was measured in the sectioned freeze-dried sample using an enzymatic cycling method of NAD-NADH. The upper layer of SGL contralateral to enucleation exhibited a significant reduction (23%) in glutamate content. Combining enucleation and ablation further decreased (35%) glutamate content. Additionally, the synaptic potential evoked in the SGL of SC slices after stimulation of optic layer was blocked by the application of kynurenic acid (3 mM) or DNQX (30 microM). These results indicate that the retino-tectal pathway in the rat can be glutamatergic in nature.

  11. Effect of carnitine on muscular glutamate uptake and intramuscular glutathione in malignant diseases.

    PubMed

    Breitkreutz, R; Babylon, A; Hack, V; Schuster, K; Tokus, M; Böhles, H; Hagmüller, E; Edler, L; Holm, E; Dröge, W

    2000-01-01

    Abnormally low intramuscular glutamate and glutathione (GSH) levels and/or a decreased muscular uptake of glutamate by the skeletal muscle tissue have previously been found in malignant diseases and simian immunodeficiency virus (SIV) infection and may contribute to the development of cachexia. We tested the hypothesis that an impaired mitochondrial energy metabolism may compromise the Na+-dependent glutamate transport. A randomized double-blind clinical trial was designed to study the effects of L-carnitine, i.e. an agent known to enhance mitochondrial integrity and function, on the glutamate transport and plasma glutamate level of cancer patients. The effect of carnitine on the intramuscular glutamate and GSH levels was examined in complementary experiments with tumour-bearing mice. In the mice, L-carnitine treatment ameliorated indeed the tumour-induced decrease in muscular glutamate and GSH levels and the increase in plasma glutamate levels. The carnitine-treated group in the randomized clinical study showed also a significant decrease in the plasma glutamate levels but only a moderate and statistically not significant increase in the relative glutamate uptake in the lower extremities. Further studies may be warranted to determine the effect of L-carnitine on the intramuscular GSH levels in cancer patients.

  12. Effect of carnitine on muscular glutamate uptake and intramuscular glutathione in malignant diseases

    PubMed Central

    Breitkreutz, R; Babylon, A; Hack, V; Schuster, K; Tokus, M; Böhles, H; Hagmüller, E; Edler, L; Holm, E; Dröge, W

    2000-01-01

    Abnormally low intramuscular glutamate and glutathione (GSH) levels and/or a decreased muscular uptake of glutamate by the skeletal muscle tissue have previously been found in malignant diseases and simian immunodeficiency virus (SIV) infection and may contribute to the development of cachexia. We tested the hypothesis that an impaired mitochondrial energy metabolism may compromise the Na+-dependent glutamate transport. A randomized double-blind clinical trial was designed to study the effects of L -carnitine, i.e. an agent known to enhance mitochondrial integrity and function, on the glutamate transport and plasma glutamate level of cancer patients. The effect of carnitine on the intramuscular glutamate and GSH levels was examined in complementary experiments with tumour-bearing mice. In the mice, L -carnitine treatment ameliorated indeed the tumour-induced decrease in muscular glutamate and GSH levels and the increase in plasma glutamate levels. The carnitine-treated group in the randomized clinical study showed also a significant decrease in the plasma glutamate levels but only a moderate and statistically not significant increase in the relative glutamate uptake in the lower extremities. Further studies may be warranted to determine the effect of L -carnitine on the intramuscular GSH levels in cancer patients. © 2000 Cancer Research Campaign PMID:10646895

  13. Dysfunctional TCA-Cycle Metabolism in Glutamate Dehydrogenase Deficient Astrocytes.

    PubMed

    Nissen, Jakob D; Pajęcka, Kamilla; Stridh, Malin H; Skytt, Dorte M; Waagepetersen, Helle S

    2015-12-01

    Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and α-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from α-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids.

  14. Echinacoside Inhibits Glutamate Release by Suppressing Voltage-Dependent Ca2+ Entry and Protein Kinase C in Rat Cerebrocortical Nerve Terminals

    PubMed Central

    Lu, Cheng Wei; Lin, Tzu Yu; Huang, Shu Kuei; Wang, Su Jane

    2016-01-01

    The glutamatergic system may be involved in the effects of neuroprotectant therapies. Echinacoside, a phenylethanoid glycoside extracted from the medicinal Chinese herb Herba Cistanche, has neuroprotective effects. This study investigated the effects of echinacoside on 4-aminopyridine-evoked glutamate release in rat cerebrocortical nerve terminals (synaptosomes). Echinacoside inhibited Ca2+-dependent, but not Ca2+-independent, 4-aminopyridine-evoked glutamate release in a concentration-dependent manner. Echinacoside also reduced the 4-aminopyridine-evoked increase in cytoplasmic free Ca2+ concentration but did not alter the synaptosomal membrane potential. The inhibitory effect of echinacoside on 4-aminopyridine-evoked glutamate release was prevented by ω-conotoxin MVIIC, a wide-spectrum blocker of Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but was insensitive to the intracellular Ca2+ release-inhibitors dantrolene and 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157). Furthermore, echinacoside decreased the 4-aminopyridine-induced phosphorylation of protein kinase C, and protein kinase C inhibitors abolished the effect of echinacoside on glutamate release. According to these results, we suggest that the inhibitory effect of echinacoside on evoked glutamate release is associated with reduced voltage-dependent Ca2+ entry and subsequent suppression of protein kinase C activity. PMID:27347934

  15. A role for preoptic glutamate in the regulation of male reproductive behavior.

    PubMed

    Dominguez, Juan M

    2009-02-01

    Although much progress has been made toward understanding the role of the medial preoptic area (MPOA) in the regulation of male reproductive behaviors, the precise mechanisms responsible for its activation during mating are largely unclear. Several studies implicate glutamate in this response. However, not until recently was there direct evidence supporting this hypothesis. Results obtained using in vivo microdialysis showed that levels of glutamate increased in the MPOA during mating, particularly with ejaculation. Levels then decreased rapidly following ejaculation, during a period of sexual quiescence. The magnitude of this decrease correlated with time spent in quiescence. Additionally, central administration of glutamate uptake inhibitors increased levels of glutamate and facilitated behavior. Glutamate activation of N-methyl-D-aspartate (NMDA) receptors in the MPOA is at least partly responsible for behavioral effects evoked by increase glutamate. This is evidenced by histological analysis of the MPOA, which shows that nearly all cells containing mating-induced Fos also contained NMDA receptors. Mating also increased phosphorylation of NMDA receptors, indicating receptor activation. Finally, bilateral microinjections of NMDA receptor antagonists inhibited copulation. This neurochemical, anatomical, and behavioral evidence points to a key role of preoptic glutamate in the regulation of sexual behavior in males. The implications of these findings are discussed.

  16. High-affinity glutamate transporter and glutamine synthetase content in longissimus dorsi and adipose tissues of growing Angus steers differs among suckling, weanling, backgrounding, and finishing production stages.

    PubMed

    Matthews, J C; Huang, J; Rentfrow, G

    2016-03-01

    Skeletal muscle and adipose tissues play important roles in maintaining whole-body Glu and N homeostasis by the uptake of Glu and release of Gln. To test the hypothesis that expression of high-affinity Glu transporters (GLAST1, EAAT4, EAAC1, GLT-1) and glutamine synthetase (GS) would increase in longissimus dorsi and adipose tissue of newborn Angus steers randomly assigned ( = 6) to develop through suckling (S; 32 d) and/or weanling (W; 184 d), backgrounding (B; 248 d), and finishing (F; 423 d) production stages. Carcass quality was determined at slaughter to verify shifts in adipose and lean deposition with development. Expression of mRNA (RT-PCR/Southern) and relative protein abundance (Western analysis) were determined in tissue homogenates isolated from longissimus dorsi, and kidney and subcutaneous adipose. The effect of production stage or tissue type on carcass and protein abundance was assessed by 1-way ANOVA using the GLM procedure of SAS, and Fisher's protected LSD procedure was used to separate data means. Neither GLAST1 nor EAAT4 mRNA or protein was detected. EAAC1, GLT-1, and GS mRNA were identified in all tissues, but GLT-1 and GS protein were not detected in kidney or subcutaneous adipose, and GS protein was not detected in longissimus dorsi. The EAAC1 content of subcutaneous ( = 0.06) and kidney ( = 0.02) adipose was 2 times greater in B and F than W steers, whereas GS was 5 times greater ( < 0.07) in B than F steers (B = W > F). For longissimus dorsi, EAAC1 ( < 0.01) and GLT-1 ( < 0.04) content decreased with development (S > W > B = F, S = W > B = F, respectively). Within F steers, EAAC1 and GLT-1 mRNA was expressed by subcutaneous, kidney, omental, mesenchymal, and intramuscular adipose tissues, whereas GS mRNA was expressed by all except for intramuscular. Only EAAC1 protein was detected in any adipose tissue, with EAAC1 content being 104% and 112% greater ( < 0.01) in intramuscular than in kidney or subcutaneous adipose, respectively, and not

  17. High-affinity glutamate transporter and glutamine synthetase content in longissimus dorsi and adipose tissues of growing Angus steers differs among suckling, weanling, backgrounding, and finishing production stages.

    PubMed

    Matthews, J C; Huang, J; Rentfrow, G

    2016-03-01

    Skeletal muscle and adipose tissues play important roles in maintaining whole-body Glu and N homeostasis by the uptake of Glu and release of Gln. To test the hypothesis that expression of high-affinity Glu transporters (GLAST1, EAAT4, EAAC1, GLT-1) and glutamine synthetase (GS) would increase in longissimus dorsi and adipose tissue of newborn Angus steers randomly assigned ( = 6) to develop through suckling (S; 32 d) and/or weanling (W; 184 d), backgrounding (B; 248 d), and finishing (F; 423 d) production stages. Carcass quality was determined at slaughter to verify shifts in adipose and lean deposition with development. Expression of mRNA (RT-PCR/Southern) and relative protein abundance (Western analysis) were determined in tissue homogenates isolated from longissimus dorsi, and kidney and subcutaneous adipose. The effect of production stage or tissue type on carcass and protein abundance was assessed by 1-way ANOVA using the GLM procedure of SAS, and Fisher's protected LSD procedure was used to separate data means. Neither GLAST1 nor EAAT4 mRNA or protein was detected. EAAC1, GLT-1, and GS mRNA were identified in all tissues, but GLT-1 and GS protein were not detected in kidney or subcutaneous adipose, and GS protein was not detected in longissimus dorsi. The EAAC1 content of subcutaneous ( = 0.06) and kidney ( = 0.02) adipose was 2 times greater in B and F than W steers, whereas GS was 5 times greater ( < 0.07) in B than F steers (B = W > F). For longissimus dorsi, EAAC1 ( < 0.01) and GLT-1 ( < 0.04) content decreased with development (S > W > B = F, S = W > B = F, respectively). Within F steers, EAAC1 and GLT-1 mRNA was expressed by subcutaneous, kidney, omental, mesenchymal, and intramuscular adipose tissues, whereas GS mRNA was expressed by all except for intramuscular. Only EAAC1 protein was detected in any adipose tissue, with EAAC1 content being 104% and 112% greater ( < 0.01) in intramuscular than in kidney or subcutaneous adipose, respectively, and not

  18. 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure influence the expression of glutamate transporter GLT-1 in C6 glioma cells via the Ca(2+) /protein kinase C pathway.

    PubMed

    Zhao, Jianya; Zhang, Yan; Zhao, Jianmei; Wang, Cheng; Mao, Jiamin; Li, Ting; Wang, Xiaoke; Nie, Xiaoke; Jiang, Shengyang; Wu, Qiyun

    2016-11-01

    The widespread environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is considered one of the most toxic dioxin-like compounds. Although epidemiological studies have shown that TCDD exposure is linked to some neurological and neurophysiological disorders, the underlying mechanism of TCDD-mediated neurotoxicity has remained unclear. Astrocytes are the most abundant cells in the nervous systems, and are recognized as the important mediators of normal brain functions as well as neurological, neurodevelopmental and neurodegenerative brain diseases. In this study, we investigated the role of TCDD in regulating the expression of glutamate transporter GLT-1 in astrocytes. TCDD, at concentrations of 0.1-100 nm, had no significantly harmful effect on the viability of C6 glioma cells. However, the expression of GLT-1 in C6 glioma cells was downregulated in a dose- and time-dependent manner. TCDD also caused activation of protein kinase C (PKC), as TCDD induced translocation of the PKC from the cytoplasm or perinuclear to the membrane. The translocation of PKC was inhibited by one Ca(2+) blocker, nifedipine, suggesting that the effects are triggered by the initial elevated intracellular concentration of free Ca(2+) . Finally, we showed that inhibition of the PKC activity reverses the TCDD-triggered reduction of GLT-1. In summary, our results suggested that TCDD exposure could downregulate the expression of GLT-1 in C6 via Ca(2+) /PKC pathway. The downregulation of GLT-1 might participate in TCDD-mediated neurotoxicity. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Proline-induced inhibition of glutamate release in hippocampal area CA1.

    PubMed

    Cohen, S M; Nadler, J V

    1997-09-26

    Concentrations of proline typical of human CSF have been shown to potentiate transmission at Schaffer collateral-commissural synapses on CA1 pyramidal cells of the rat hippocampus. This study tested the hypothesis that proline enhances excitatory synaptic transmission by increasing glutamate release. Two concentrations of proline were used: a concentration typical of normal human CSF (3 microM) and a concentration typical of CSF in persons with the genetic disorder hyperprolinemia type II (30 microM). Continuous exposure of hippocampal slices to either concentration of proline potentiated Schaffer collateral-commissural synaptic transmission. Proline shifted the plot of field EPSP slope against fiber volley amplitude upward. Contrary to the original hypothesis, neither concentration of proline reduced paired-pulse facilitation; 30 microM proline enhanced paired-pulse facilitation, whereas 3 microM proline had no effect. In line with its enhancement of paired-pulse facilitation, 30 microM proline reduced both the K+-evoked release of glutamate and aspartate from CA1 slices and the release of glutamate and aspartate from CA1 synaptosomes evoked by 4-aminopyridine. These results suggest that the proline-induced potentiation of Schaffer collateral-commissural synaptic transmission probably involves a postsynaptic, rather than a presynaptic, mechanism. Concentrations of proline normally found in human CSF little affect glutamate release. However, proline-induced inhibition of glutamate release may contribute to the neuropsychiatric disorders associated with hyperprolinemia type II.

  20. Complement selectively elicits glutamate release from nerve endings in different regions of mammal central nervous system.

    PubMed

    Merega, Elisa; Di Prisco, Silvia; Lanfranco, Massimiliano; Severi, Paolo; Pittaluga, Anna

    2014-05-01

    Our study was aimed at investigating whether complement, a complex of soluble and membrane-associated serum proteins, could, in addition to its well-documented post-synaptic activity, also pre-synaptically affect the release of classic neurotransmitters in central nervous system (CNS). Complement (dilution 1 : 10 to 1 : 10000) elicited the release of preloaded [(3) H]-d-aspartate ([(3) H]d-ASP) and endogenous glutamate from mouse cortical synaptosomes in a dilution-dependent manner. It also evoked [(3) H]d-ASP release from mouse hippocampal, cerebellar, and spinal cord synaptosomes, as well as from rat and human cortical nerve endings, but left unaltered the release of GABA, [(3) H]noradrenaline or [(3) H]acetylcholine. Lowering external Na(+) (from 140 to 40 mM) or Ca(2+) (from 1.2 to 0.1 mM) ions prevented the 1 : 300 complement-evoked [(3) H]d-ASP release from mouse cortical synaptosomes. Complement-induced releasing effect was unaltered in synaptosomes entrapped with the Ca(2+) ions chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N', tetra-acetic acid or with pertussis toxin. Nifedipine,/ω-conotoxin GVIA/ω-conotoxin MVIIC mixture as well as the vesicular ATPase blocker bafilomycin A1 were also inefficacious. The excitatory amino acid transporter blocker DL-threo-ß-benzyloxyaspartic acid, on the contrary, reduced the complement-evoked releasing effect in a concentration-dependent manner. We concluded that complement-induced releasing activity is restricted to glutamatergic nerve endings, where it was accounted for by carrier-mediated release. Our observations afford new insights into the molecular events accounting for immune and CNS crosstalk. We investigated whether complement, a complex of soluble and membrane-associated serum proteins, could pre-synaptically affect the release of classic neurotransmitters in the central nervous system (CNS). Our data provide evidence that complement-induced releasing activity is restricted to glutamatergic nerve endings

  1. Hesperidin inhibits glutamate release and exerts neuroprotection against excitotoxicity induced by kainic acid in the hippocampus of rats.

    PubMed

    Chang, Chia Ying; Lin, Tzu Yu; Lu, Cheng Wei; Huang, Shu Kuei; Wang, Ying Chou; Chou, Shang Shing Peter; Wang, Su Jane

    2015-09-01

    The citrus flavonoid hesperidin exerts neuroprotective effects and could cross the blood-brain barrier. Given the involvement of glutamate neurotoxicity in the pathogenesis of neurodegenerative disorders, this study was conducted to evaluate the potential role of hesperidin in glutamate release and glutamate neurotoxicity in the hippocampus of rats. In rat hippocampal nerve terminals (synaptosomes), hesperidin inhibited the release of glutamate and elevation of cytosolic free Ca(2+) concentration evoked by 4-aminopyridine (4-AP), but did not alter 4-AP-mediated depolarization. The inhibitory effect of hesperidin on evoked glutamate release was prevented by chelating the extracellular Ca(2+) ions and blocking the activity of Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels or protein kinase C. In hippocampal slice preparations, whole-cell patch clamp experiments showed that hesperidin reduced the frequency of spontaneous excitatory postsynaptic currents without affecting their amplitude, indicating the involvement of a presynaptic mechanism. In addition, intraperitoneal (i.p.) injection of kainic acid (KA, 15 mg/kg) elevated the extracellular glutamate levels and caused considerable neuronal loss in the hippocampal CA3 area. These KA-induced alterations were attenuated by pretreatment with hesperidin (10 or 50 mg/kg, i.p.) before administering the KA. These results demonstrate that hesperidin inhibits evoked glutamate release in vitro and attenuates in vivo KA-induced neuronal death in the hippocampus. Our findings indicate that hesperidin may be a promising candidate for preventing or treating glutamate excitotoxicity related brain disorders such as neurodegenerative diseases.

  2. Evoked emotions predict food choice.

    PubMed

    Dalenberg, Jelle R; Gutjar, Swetlana; Ter Horst, Gert J; de Graaf, Kees; Renken, Remco J; Jager, Gerry

    2014-01-01

    In the current study we show that non-verbal food-evoked emotion scores significantly improve food choice prediction over merely liking scores. Previous research has shown that liking measures correlate with choice. However, liking is no strong predictor for food choice in real life environments. Therefore, the focus within recent studies shifted towards using emotion-profiling methods that successfully can discriminate between products that are equally liked. However, it is unclear how well scores from emotion-profiling methods predict actual food choice and/or consumption. To test this, we proposed to decompose emotion scores into valence and arousal scores using Principal Component Analysis (PCA) and apply Multinomial Logit Models (MLM) to estimate food choice using liking, valence, and arousal as possible predictors. For this analysis, we used an existing data set comprised of liking and food-evoked emotions scores from 123 participants, who rated 7 unlabeled breakfast drinks. Liking scores were measured using a 100-mm visual analogue scale, while food-evoked emotions were measured using 2 existing emotion-profiling methods: a verbal and a non-verbal method (EsSense Profile and PrEmo, respectively). After 7 days, participants were asked to choose 1 breakfast drink from the experiment to consume during breakfast in a simulated restaurant environment. Cross validation showed that we were able to correctly predict individualized food choice (1 out of 7 products) for over 50% of the participants. This number increased to nearly 80% when looking at the top 2 candidates. Model comparisons showed that evoked emotions better predict food choice than perceived liking alone. However, the strongest predictive strength was achieved by the combination of evoked emotions and liking. Furthermore we showed that non-verbal food-evoked emotion scores more accurately predict food choice than verbal food-evoked emotions scores. PMID:25521352

  3. Evoked emotions predict food choice.

    PubMed

    Dalenberg, Jelle R; Gutjar, Swetlana; Ter Horst, Gert J; de Graaf, Kees; Renken, Remco J; Jager, Gerry

    2014-01-01

    In the current study we show that non-verbal food-evoked emotion scores significantly improve food choice prediction over merely liking scores. Previous research has shown that liking measures correlate with choice. However, liking is no strong predictor for food choice in real life environments. Therefore, the focus within recent studies shifted towards using emotion-profiling methods that successfully can discriminate between products that are equally liked. However, it is unclear how well scores from emotion-profiling methods predict actual food choice and/or consumption. To test this, we proposed to decompose emotion scores into valence and arousal scores using Principal Component Analysis (PCA) and apply Multinomial Logit Models (MLM) to estimate food choice using liking, valence, and arousal as possible predictors. For this analysis, we used an existing data set comprised of liking and food-evoked emotions scores from 123 participants, who rated 7 unlabeled breakfast drinks. Liking scores were measured using a 100-mm visual analogue scale, while food-evoked emotions were measured using 2 existing emotion-profiling methods: a verbal and a non-verbal method (EsSense Profile and PrEmo, respectively). After 7 days, participants were asked to choose 1 breakfast drink from the experiment to consume during breakfast in a simulated restaurant environment. Cross validation showed that we were able to correctly predict individualized food choice (1 out of 7 products) for over 50% of the participants. This number increased to nearly 80% when looking at the top 2 candidates. Model comparisons showed that evoked emotions better predict food choice than perceived liking alone. However, the strongest predictive strength was achieved by the combination of evoked emotions and liking. Furthermore we showed that non-verbal food-evoked emotion scores more accurately predict food choice than verbal food-evoked emotions scores.

  4. High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc-) Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells

    PubMed Central

    Thomas, Ajit G.; Sattler, Rita; Tendyke, Karen; Loiacono, Kara A.; Hansen, Hans; Sahni, Vishal; Hashizume, Yutaka; Rojas, Camilo; Slusher, Barbara S.

    2015-01-01

    The cystine-glutamate antiporter (system xc-) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc-. Human glioma cells were chosen based on their high system xc- activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of

  5. High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc-) Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells.

    PubMed

    Thomas, Ajit G; Sattler, Rita; Tendyke, Karen; Loiacono, Kara A; Hansen, Hans; Sahni, Vishal; Hashizume, Yutaka; Rojas, Camilo; Slusher, Barbara S

    2015-01-01

    The cystine-glutamate antiporter (system xc-) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc-. Human glioma cells were chosen based on their high system xc- activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of

  6. Glutamate-related gene expression changes with age in the mouse auditory midbrain.

    PubMed

    Tadros, Sherif F; D'Souza, Mary; Zettel, Martha L; Zhu, Xiaoxia; Waxmonsky, Nicole C; Frisina, Robert D

    2007-01-01

    Glutamate is the main excitatory neurotransmitter in both the peripheral and central auditory systems. Changes of glutamate and glutamate-related genes with age may be an important factor in the pathogenesis of age-related hearing loss-presbycusis. In this study, changes in glutamate-related mRNA gene expression in the CBA mouse inferior colliculus with age and hearing loss were examined and correlations were sought between these changes and functional hearing measures, such as the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs). Gene expression of 68 glutamate-related genes was investigated using both genechip microarray and real-time PCR (qPCR) molecular techniques for four different age/hearing loss CBA mouse subject groups. Two genes showed consistent differences between groups for both the genechip and qPCR. Pyrroline-5-carboxylate synthetase enzyme (Pycs) showed down-regulation with age and a high-affinity glutamate transporter (Slc1a3) showed up-regulation with age and hearing loss. Since Pycs plays a role in converting glutamate to proline, its deficiency in old age may lead to both glutamate increases and proline deficiencies in the auditory midbrain, playing a role in the subsequent inducement of glutamate toxicity and loss of proline neuroprotective effects. The up-regulation of Slc1a3 gene expression may reflect a cellular compensatory mechanism to protect against age-related glutamate or calcium excitoxicity.

  7. [Evoked potentials and inhalation anesthetics].

    PubMed

    Thiel, A; Russ, W; Hempelmann, G

    1988-01-01

    Intraoperative monitoring of evoked potentials can be affected by various factors including volatile anaesthetics. These effects have to be considered in order to give correct interpretations of the obtained data. Visual evoked potentials (VEP) and auditory evoked potentials (AEP) will show strong alterations under general anaesthesia whereas brainstem auditory evoked potentials (BAEP) are slightly affected. The effects of nitrous oxide, halothane, enflurane, and isoflurane on somatosensory evoked potentials (SEP) after median nerve stimulation were studied in 35 healthy adult patients. pCO2 and tympanic membrane temperature were held constant. Simultaneous cervical and cortical SEP recording was performed using surface electrodes. After induction of anaesthesia SEP were recorded during normoventilation with 100% oxygen and after inhalation of 66.6% nitrous oxide. 10 patients received halothane at inspired concentrations of 0.5, 1.0, 1.5, and 2.0%. After nitrous oxide had been replaced by oxygen, halothane was reduced in steps of 0.5%. SEP were recorded at the end of each period (15 min). Equipotent doses of enflurane or isoflurane were administered to 15 and 10 patients, respectively. Nitrous oxide depressed early cortical SEP amplitude. Halothane, enflurane, and isoflurane caused dose dependent increases of latencies. Reduction of amplitude was most pronounced with isoflurane. Using high doses of enflurane in oxygen cortical SEP showed unusual high amplitudes associated with marked increases of latencies. Even under high concentrations of volatile anaesthetics cervical SEP were minimally affected. The effects of anaesthetic gases have to be considered when SEP are recorded intraoperatively.

  8. The actions of L-glutamate at the postsynaptic membrane of the squid giant synapse.

    PubMed

    Adams, D J; Gillespie, J I

    1988-11-01

    The actions of L-glutamate on the postsynaptic membrane of the squid giant synapse were investigated using two methods of application: ionophoresis and bath perfusion. Bath perfusion of 10 mmoll-1 sodium glutamate did not produce an appreciable depolarization of the postsynaptic membrane but reversibly blocked the neurally evoked postsynaptic potential (PSP). The postsynaptic membrane depolarized when L-glutamate was applied ionophoretically. The sensitivity to glutamate application was not uniform, but sharply localized to sites which may correspond to synaptic contacts made by branching colaterals from the postsynaptic axon. The relationship between membrane potential and amplitude of the glutamate-activated postsynaptic potential (PSP) examined under current-clamp conditions was linear over the voltage range studied (-110 to -60 mV) with an extrapolated reversal potential of -36 mV. The amplitude of the glutamate-activated PSP was reduced either by replacing Na+ in the external solution with Tris+ (Na+-free) or by raising the extracellular K+ concentration to 20 mmoll-1 and was abolished by removing both Na+ and Ca2+ from the bath solution. The PSP amplitude was insensitive to changes in the extracellular Mg2+ concentration. The extrapolated reversal potential of the glutamate PSP was shifted to more positive potentials in both Na+-free and raised-K+ bathing solutions and was unchanged by anion substitution. The depolarization induced by L-glutamate increased with increasing ionophoretic current and reached a maximum with large pulses. Double logarithmic plots of the coulomb dose-response relationship gave a limiting slope in the range 1.7-2.2, suggesting that two glutamate molecules are required for receptor activation. The time course of desensitization of the glutamate response was studied using a double-pulse method. The initial decrease in the ratio, PSP2/PSP1, is followed by a slower time-dependent recovery of the postsynaptic response with a time constant

  9. Single rodent mesohabenular axons release glutamate and GABA

    PubMed Central

    Root, David H.; Mejias-Aponte, Carlos; Zhang, Shiliang; Wang, Huiling; Hoffman, Alexander F.; Lupica, Carl R.; Morales, Marisela

    2016-01-01

    The lateral habenula (LHb) is involved in reward, aversion, addiction, and depression, through descending interactions with several brain structures, including the ventral tegmental area (VTA). VTA provides reciprocal inputs to LHb, but their actions are unclear. Here we show that the majority of rat and mouse VTA neurons innervating LHb co-express markers for both glutamate-signaling (vesicular glutamate transporter 2, VGluT2) and GABA-signaling (glutamate decarboxylase, GAD; and vesicular GABA transporter, VGaT). A single axon from these mesohabenular neurons co-expresses VGluT2-protein and VGaT-protein, and surprisingly establishes symmetric and asymmetric synapses on LHb neurons. In LHb slices, light activation of mesohabenular fibers expressing channelrhodopsin-2 (ChR2) driven by VGluT2 or VGaT promoters elicits release of both glutamate and GABA onto single LHb neurons. In vivo light-activation of mesohabenular terminals inhibits or excites LHb neurons. Our findings reveal an unanticipated type of VTA neuron that co-transmits glutamate and GABA, and provides the majority of mesohabenular inputs. PMID:25242304

  10. Ethanol-Associated Changes in Glutamate Reward Neurocircuitry: A Minireview of Clinical and Preclinical Genetic Findings.

    PubMed

    Bell, Richard L; Hauser, Sheketha R; McClintick, Jeanette; Rahman, Shafiqur; Edenberg, Howard J; Szumlinski, Karen K; McBride, William J

    2016-01-01

    Herein, we have reviewed the role of glutamate, the major excitatory neurotransmitter in the brain, in a number of neurochemical, -physiological, and -behavioral processes mediating the development of alcohol dependence. The findings discussed include results from both preclinical as well as neuroimaging and postmortem clinical studies. Expression levels for a number of glutamate-associated genes and/or proteins are modulated by alcohol abuse and dependence. These changes in expression include metabotropic receptors and ionotropic receptor subunits as well as different glutamate transporters. Moreover, these changes in gene expression parallel the pharmacologic manipulation of these same receptors and transporters. Some of these gene expression changes may have predated alcohol abuse and dependence because a number of glutamate-associated polymorphisms are related to a genetic predisposition to develop alcohol dependence. Other glutamate-associated polymorphisms are linked to age at the onset of alcohol-dependence and initial level of response/sensitivity to alcohol. Finally, findings of innate and/or ethanol-induced glutamate-associated gene expression differences/changes observed in a genetic animal model of alcoholism, the P rat, are summarized. Overall, the existing literature indicates that changes in glutamate receptors, transporters, enzymes, and scaffolding proteins are crucial for the development of alcohol dependence and there is a substantial genetic component to these effects. This indicates that continued research into the genetic underpinnings of these glutamate-associated effects will provide important novel molecular targets for treating alcohol abuse and dependence. PMID:26809998

  11. Adenosine A2A receptors modulate glutamate uptake in cultured astrocytes and gliosomes.

    PubMed

    Matos, Marco; Augusto, Elisabete; Santos-Rodrigues, Alexandre Dos; Schwarzschild, Michael A; Chen, Jiang-Fan; Cunha, Rodrigo A; Agostinho, Paula

    2012-05-01

    Glutamate is the primary excitatory neurotransmitter in the central nervous system, where its toxic build-up leads to synaptic dysfunction and excitotoxic cell death that underlies many neurodegenerative diseases. Therefore, efforts have been made to understand the regulation of glutamate transporters, which are responsible for the clearance of extracellular glutamate. We now report that adenosine A(2A) receptors (A(2A) R) control the uptake of D-aspartate in primary cultured astrocytes as well as in an ex vivo preparation enriched in glial plasmalemmal vesicles (gliosomes) from adult rats, whereas A(1) R and A(3) R were devoid of effects. Thus, the acute exposure to the A(2A) R agonist, CGS 21680, inhibited glutamate uptake, an effect prevented by the A(2A) R antagonist, SCH 58261, and abbrogated in cultured astrocytes from A(2A) R knockout mice. Furthermore, the prolonged activation of A(2A) R lead to a cAMP/protein kinase A-dependent reduction of GLT-I and GLAST mRNA and protein levels, which leads to a sustained decrease of glutamate uptake. This dual mechanism of inhibition of glutamate transporters by astrocytic A(2A) R provides a novel candidate mechanism to understand the ability of A(2) (A) R to control synaptic plasticity and neurodegeneration, two conditions tightly associated with the control of extracellular glutamate levels by glutamate transporters.

  12. Ethanol-Associated Changes in Glutamate Reward Neurocircuitry: A Minireview of Clinical and Preclinical Genetic Findings.

    PubMed

    Bell, Richard L; Hauser, Sheketha R; McClintick, Jeanette; Rahman, Shafiqur; Edenberg, Howard J; Szumlinski, Karen K; McBride, William J

    2016-01-01

    Herein, we have reviewed the role of glutamate, the major excitatory neurotransmitter in the brain, in a number of neurochemical, -physiological, and -behavioral processes mediating the development of alcohol dependence. The findings discussed include results from both preclinical as well as neuroimaging and postmortem clinical studies. Expression levels for a number of glutamate-associated genes and/or proteins are modulated by alcohol abuse and dependence. These changes in expression include metabotropic receptors and ionotropic receptor subunits as well as different glutamate transporters. Moreover, these changes in gene expression parallel the pharmacologic manipulation of these same receptors and transporters. Some of these gene expression changes may have predated alcohol abuse and dependence because a number of glutamate-associated polymorphisms are related to a genetic predisposition to develop alcohol dependence. Other glutamate-associated polymorphisms are linked to age at the onset of alcohol-dependence and initial level of response/sensitivity to alcohol. Finally, findings of innate and/or ethanol-induced glutamate-associated gene expression differences/changes observed in a genetic animal model of alcoholism, the P rat, are summarized. Overall, the existing literature indicates that changes in glutamate receptors, transporters, enzymes, and scaffolding proteins are crucial for the development of alcohol dependence and there is a substantial genetic component to these effects. This indicates that continued research into the genetic underpinnings of these glutamate-associated effects will provide important novel molecular targets for treating alcohol abuse and dependence.

  13. Functional role for redox in the epileptogenesis: molecular regulation of glutamate in the hippocampus of FeCl3-induced limbic epilepsy model.

    PubMed

    Ueda, Yuto; Doi, Taku; Nagatomo, Keiko; Willmore, L James; Nakajima, Akira

    2007-08-01

    We used western blotting to measure the quantity of glutamate and gamma-aminobutyric acid (GABA) transporters proteins within hippocampal tissue obtained from rats who had undergone epileptogenesis. Chronic seizures were induced by amygdalar injection of FeCl(3). We found that the glial glutamate transporters GLAST and GLT-1 were down-regulated at 60 days after initiation of chronic and recurrent seizures. However, the neuronal glutamate transporter EAAC-1 and the GABA transporter GAT-3 were increased. We performed in vivo microdialysis in freely moving animals to estimate in vivo redox state. We found that the hippocampal tissues were oxidized, resulting in even further impairment of glutamate transport. Our data show that epileptogenesis in rats resulting in chronic and recurrent seizures is associated with collapse of glutamate regulation caused by both the molecular down-regulation of glial glutamate transporters combined with the functional failure due to oxidation.

  14. Mechanically evoked itch in humans.

    PubMed

    Fukuoka, Miyuki; Miyachi, Yoshiki; Ikoma, Akihiko

    2013-06-01

    When a newly developed experimental method to vibrate vellus hairs on human skin was applied to the face and arm in healthy subjects, intense itch was reproducibly induced on the face, but not on the arm, without any flare reactions. In contrast to histamine-induced itch, mechanically evoked itch was not characterized as burning or stinging by any subjects, and was resistant to histamine H1-receptor antagonists. When the stimulation was continued for 10 min, mechanically evoked itch reached the maximum intensity within 10 s, but gradually attenuated after 60 to 90 s and was rarely perceivable at the end of stimulation. When the stimulation was discontinued at 90 s, mechanically evoked itch rapidly attenuated after the end of stimulation, but took more than 10 min before it completely diminished. These results indicate a possible involvement of C-tactile neurons in mechanically evoked itch because they have consistent characteristics such as low mechanical thresholds, intermediate adaptation, after discharge, favorable response to slowly moving stimuli, and fatigue during repeated mechanical stimulation, although it needs to be confirmed by future microneurographic studies. Touch-alloknesis was present in the adjacent skin area until mechanically evoked itch completely diminished, supporting the hypothesis that itch sensitization can be caused by a continuous activation of peripheral itch neurons whether or not they are histamine-sensitive C nerves. In conclusion, this study provides direct evidence of mechanosensitive nerves involved in itch in human skin. The purity of mechanically evoked itch without any pain-related sensory components is a major advantage for investigating the differentiation of itch from pain.

  15. Vestibular-evoked myogenic potentials.

    PubMed

    Colebatch, J G; Rosengren, S M; Welgampola, M S

    2016-01-01

    The vestibular-evoked myogenic potential (VEMP) is a short-latency potential evoked through activation of vestibular receptors using sound or vibration. It is generated by modulated electromyographic signals either from the sternocleidomastoid muscle for the cervical VEMP (cVEMP) or the inferior oblique muscle for the ocular VEMP (oVEMP). These reflexes appear to originate from the otolith organs and thus complement existing methods of vestibular assessment, which are mainly based upon canal function. This review considers the basis, methodology, and current applications of the cVEMP and oVEMP in the assessment and diagnosis of vestibular disorders, both peripheral and central. PMID:27638068

  16. Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy.

    PubMed

    Feng, Shengjie; Ma, Shaorong; Jia, Caixia; Su, Yujuan; Yang, Shenglian; Zhou, Kechun; Liu, Yani; Cheng, Ju; Lu, Dunguo; Fan, Liu; Wang, Yizheng

    2016-05-01

    Sonic hedgehog (Shh), both as a mitogen and as a morphogen, plays an important role in cell proliferation and differentiation during early development. Here, we show that Shh inhibits glutamate transporter activities in neurons, rapidly enhances extracellular glutamate levels, and affects the development of epilepsy. Shh is quickly released in response to epileptic, but not physiological, stimuli. Inhibition of neuronal glutamate transporters by Shh depends on heterotrimeric G protein subunit Gαi and enhances extracellular glutamate levels. Inhibiting Shh signaling greatly reduces epileptiform activities in both cell cultures and hippocampal slices. Moreover, pharmacological or genetic inhibition of Shh signaling markedly suppresses epileptic phenotypes in kindling or pilocarpine models. Our results suggest that Shh contributes to the development of epilepsy and suppression of its signaling prevents the development of the disease. Thus, Shh can act as a modulator of neuronal activity, rapidly regulating glutamate levels and promoting epilepsy. PMID:27113760

  17. An Optimized Glutamate Receptor Photoswitch with Sensitized Azobenzene Isomerization.

    PubMed

    Gascón-Moya, Marta; Pejoan, Arnau; Izquierdo-Serra, Mercè; Pittolo, Silvia; Cabré, Gisela; Hernando, Jordi; Alibés, Ramon; Gorostiza, Pau; Busqué, Félix

    2015-10-16

    A new azobenzene-based photoswitch, 2, has been designed to enable optical control of ionotropic glutamate receptors in neurons via sensitized two-photon excitation with NIR light. In order to develop an efficient and versatile synthetic route for this molecule, a modular strategy is described which relies on the use of a new linear fully protected glutamate derivative stable in basic media. The resulting compound undergoes one-photon trans-cis photoisomerization via two different mechanisms: direct excitation of its azoaromatic unit and irradiation of the pyrene sensitizer, a well-known two-photon sensitive chromophore. Moreover, 2 presents large thermal stability of its cis isomer, in contrast to other two-photon responsive switches relying on the intrinsic nonlinear optical properties of push-pull substituted azobenzenes. As a result, the molecular system developed herein is a very promising candidate for evoking large photoinduced biological responses during the multiphoton operation of neuronal glutamate receptors with NIR light, which require accumulation of the protein-bound cis state of the switch upon repeated illumination.

  18. Bicyclic glutamic acid derivatives.

    PubMed

    Meyer, Udo; Bisel, Philippe; Weckert, Edgar; Frahm, August Wilhelm

    2006-05-15

    For the second-generation asymmetric synthesis of the trans-tris(homoglutamic) acids via Strecker reaction of chiral ketimines, the cyanide addition as the key stereodifferentiating step produces mixtures of diastereomeric alpha-amino nitrile esters the composition of which is independent of the reaction temperature and the type of the solvent, respectively. The subsequent hydrolysis is exclusively achieved with concentrated H(2)SO(4) yielding diastereomeric mixtures of three secondary alpha-amino alpha-carbamoyl-gamma-esters and two diastereomeric cis-fused angular alpha-carbamoyl gamma-lactams as bicyclic glutamic acid derivatives, gained from in situ stereomer differentiating cyclisation of the secondary cis-alpha-amino alpha-carbamoyl-gamma-esters. Separation was achieved by CC. The pure secondary trans-alpha-amino alpha-carbamoyl-gamma-esters cyclise on heating and treatment with concentrated H(2)SO(4), respectively, to diastereomeric cis-fused angular secondary alpha-amino imides. Their hydrogenolysis led to the enantiomeric cis-fused angular primary alpha-amino imides. The configuration of all compounds was completely established by NMR methods, CD-spectra, and by X-ray analyses of the (alphaR,1R,5R)-1-carbamoyl-2-(1-phenylethyl)-2-azabicyclo[3.3.0]octan-3-one and of the trans-alphaS,1S,2R-2-ethoxycarbonylmethyl-1-(1-phenylethylamino)cyclopentanecarboxamide. PMID:16596563

  19. Functional architecture of olfactory ionotropic glutamate receptors

    PubMed Central

    Abuin, Liliane; Bargeton, Benoîte; Ulbrich, Maximilian H.; Isacoff, Ehud Y.; Kellenberger, Stephan; Benton, Richard

    2010-01-01

    Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate chemical communication between neurons at synapses. A variant iGluR subfamily, the Ionotropic Receptors (IRs), was recently proposed to detect environmental volatile chemicals in olfactory cilia. Here we elucidate how these peripheral chemosensors have evolved mechanistically from their iGluR ancestors. Using a Drosophila model, we demonstrate that IRs act in combinations of up to three subunits, comprising individual odor-specific receptors and one or two broadly expressed co-receptors. Heteromeric IR complex formation is necessary and sufficient for trafficking to cilia and mediating odor-evoked electrophysiological responses in vivo and in vitro. IRs display heterogeneous ion conduction specificities related to their variable pore sequences, and divergent ligand-binding domains function in odor recognition and cilia localization. Our results provide insights into the conserved and distinct architecture of these olfactory and synaptic ion channels and offer perspectives into use of IRs as genetically encoded chemical sensors. PMID:21220098

  20. Acute Modulation of Cortical Glutamate and GABA Content by Physical Activity.

    PubMed

    Maddock, Richard J; Casazza, Gretchen A; Fernandez, Dione H; Maddock, Michael I

    2016-02-24

    Converging evidence demonstrates that physical activity evokes a brain state characterized by distinctive changes in brain metabolism and cortical function. Human studies have shown that physical activity leads to a generalized increase in electroencephalography power across regions and frequencies, and a global increase in brain nonoxidative metabolism of carbohydrate substrates. This nonoxidative consumption of carbohydrate has been hypothesized to include increased de novo synthesis of amino acid neurotransmitters, especially glutamate and GABA. Here, we conducted a series of proton magnetic resonance spectroscopy studies in human volunteers before and after vigorous exercise (≥80% of predicted maximal heart rate). Results showed that the resonance signals of both glutamate and GABA increased significantly in the visual cortex following exercise. We further demonstrated a similar increase in glutamate following exercise in an executive region, the anterior cingulate cortex. The increase in glutamate was similar when using echo times of 30 and 144 ms, indicating that exercise-related T2 relaxation effects across this range of relaxation times did not account for the findings. In addition, we found preliminary evidence that more physical activity during the preceding week predicts higher resting glutamate levels. Overall, the results are consistent with an exercise-induced expansion of the cortical pools of glutamate and GABA, and add to a growing understanding of the distinctive brain state associated with physical activity. A more complete understanding of this brain state may reveal important insights into mechanisms underlying the beneficial effects of physical exercise in neuropsychiatric disorders, neurorehabilitation, aging, and cognition.

  1. Increased extracellular levels of glutamate in the hippocampus of chronically epileptic rats.

    PubMed

    Soukupova, M; Binaschi, A; Falcicchia, C; Palma, E; Roncon, P; Zucchini, S; Simonato, M

    2015-08-20

    An increase in the release of excitatory amino acids has consistently been observed in the hippocampus during seizures, both in humans and animals. However, very little or nothing is known about the extracellular levels of glutamate and aspartate during epileptogenesis and in the interictal chronic period of established epilepsy. The aim of this study was to systematically evaluate the relationship between seizure activity and changes in hippocampal glutamate and aspartate extracellular levels under basal and high K(+)-evoked conditions, at various time-points in the natural history of experimental temporal lobe epilepsy, using in vivo microdialysis. Hippocampal extracellular glutamate and aspartate levels were evaluated: 24h after pilocarpine-induced status epilepticus (SE); during the latency period preceding spontaneous seizures; immediately after the first spontaneous seizure; in the chronic (epileptic) period. We found that (i) basal (spontaneous) glutamate outflow is increased in the interictal phases of the chronic period, whereas basal aspartate outflow remains stable for the entire course of the disease; (ii) high K(+) perfusion increased glutamate and aspartate outflow in both control and pilocarpine-treated animals, and the overflow of glutamate was clearly increased in the chronic group. Our data suggest that the glutamatergic signaling is preserved and even potentiated in the hippocampus of epileptic rats, and thus may favor the occurrence of spontaneous recurrent seizures. Together with an impairment of GABA signaling (Soukupova et al., 2014), these data suggest that a shift toward excitation occurs in the excitation/inhibition balance in the chronic epileptic state. PMID:26073699

  2. [Vestibular evoked potentials in people].

    PubMed

    Fraczkowski, K; Pośpiech, L; Orendorz-Fraczkowska, K

    1997-01-01

    In the article had been presented the structure and functioning of a prototype system for stimulation and registration vestibular evoked potentials, and the first recording of evoked vestibular potentials (VsEPs) in human beings. This system consist of a original stimulator accelerated for the stimulation of vestibular organ, modul registratory VsEPs as well as string elements and synchronizing stimulation with recording. IMB PC 486 is quickly process of investigation with help of standard interface and a original computer programme. Vestibular organ had been evoked by 200 to 500 cyclicly repealed angular decelerations of 4000 degrees/s2. During investigation white noise was used for masking to avoid the evoking of auditory potentials. Seven of the examined healthy persons (including one deaf person whose vestibular organ was not damaged) had registered a response consisting of several waves with vertex positive peaks. The first two waves P1 and P2 with the mean value 2.02 ms and 5.6 ms are most often during in the 10 ms. The registered deaf persons response does not differ from the record of healthy persons. PMID:9518319

  3. Single-file diffusion and neurotransmitter transporters: Hodgkin and Keynes model revisited.

    PubMed

    DeFelice, L J; Adams, S V; Ypey, D L

    2001-01-01

    Norepinephrine transporters (NETs) use the Na gradient to remove norepinephrine (NE) from the synaptic cleft of adrenergic neurons following NE release from the presynaptic terminal. By coupling NE to the inwardly directed Na gradient, it is possible to concentrate NE inside cells. This mechanism, which is referred to as co-transport or secondary transport (Läuger, 1991, Electrogenic Ion Pumps, Sinauer Associates) is apparently universal: Na coupled transport applies to serotonin transporters (SERTs), dopamine transporters (DATs), glutamate transporters, and many others, including transporters for osmolites, metabolites and substrates such as sugar. Recently we have shown that NETs and SERTs transport norepinephrine or serotonin as if Na and the transmitter permeated through an ion channel together 'Galli et al., 1998, PNAS 95, 13260-13265; Petersen and DeFelice, 1999, Nature Neurosci. 2, 605-610'. These data are paradoxical because it has been difficult to envisage how NE, for example, would couple to Na if these ions move passively through an open pore. An 'alternating access' model is usually evoked to explain coupling: in such models NE and Na bind to NET, which then undergoes a conformational change to release NE and Na on the inside. The empty transporter then turns outward to complete the cycle. Alternating-access models never afford access to an open channel. Rather, substrates and co-transported ions are occluded in the transporter and carried across the membrane. The coupling mechanism we propose is fundamentally different than the coupling mechanism evoked in the alternating access model. To explain coupling in co-transporters, we use a mechanism first evoked by 'Hodgkin and Keynes (1955) J. Physiol. 128, 61-88' to explain ion interactions in K-selective channels. In the Hodgkin and Keynes model, K ions move single-file through a long narrow pore. Their model accounted for the inward/outward flux ratio if they assumed that two K ions queue within the

  4. Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors

    NASA Astrophysics Data System (ADS)

    Okumoto, Sakiko; Looger, Loren L.; Micheva, Kristina D.; Reimer, Richard J.; Smith, Stephen J.; Frommer, Wolf B.

    2005-06-01

    Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed. The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate/aspartate binding protein ybeJ from Escherichia coli fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery. aspartate | hippocampal neuron | neurotransmitter | secretion | transport

  5. Effects of a high molecular weight toxin from Physalia physalis on glutamate responses.

    PubMed

    Mas, R; Menéndez, R; Garateix, A; Garcia, M; Chávez, M

    1989-01-01

    The effects of a high molecular weight toxin from Physalia physalis (P3) were investigated on glutamate evoked potentials in snail (Zachrysia guanesis) neurons and in crayfish (Cambarus clarkii) neuromuscular junction. The glutamate evoked potentials of snail neurons were reversibly blocked by P3 in a dose-dependent manner (2-200 microM). A reversible blocking action was also found for P3 on excitatory junctional potentials and on glutamate potentials of crayfish at a concentration range of 6 nM-60 microM. Experiments carried out with independent stimulation of the excitatory and inhibitory nerves showed that the effect of P3 (60 nM-10 microM) was exerted predominantly on excitatory junctional potentials. However, at higher doses (greater than 10 microM) a slight reduction of the inhibitory potentials was also observed. These results suggest that P3 reversibly blocks glutamate receptors. Thus, it could be a promising tool for further studies on glutamatergic transmission. PMID:2576111

  6. Methylphenidate Increases Glutamate Uptake in Bergmann Glial Cells.

    PubMed

    Guillem, Alain M; Martínez-Lozada, Zila; Hernández-Kelly, Luisa C; López-Bayghen, Esther; López-Bayghen, Bruno; Calleros, Oscar A; Campuzano, Marco R; Ortega, Arturo

    2015-11-01

    Glutamate, the main excitatory transmitter in the vertebrate brain, exerts its actions through the activation of specific membrane receptors present in neurons and glial cells. Over-stimulation of glutamate receptors results in neuronal death, phenomena known as excitotoxicity. A family of glutamate uptake systems, mainly expressed in glial cells, removes the amino acid from the synaptic cleft preventing an excessive glutamatergic stimulation and thus neuronal damage. Autism spectrum disorders comprise a group of syndromes characterized by impaired social interactions and anxiety. One or the most common drugs prescribed to treat these disorders is Methylphenidate, known to increase dopamine extracellular levels, although it is not clear if its sedative effects are related to a plausible regulation of the glutamatergic tone via the regulation of the glial glutamate uptake systems. To gain insight into this possibility, we used the well-established model system of cultured chick cerebellum Bergmann glia cells. A time and dose-dependent increase in the activity and protein levels of glutamate transporters was detected upon Methylphenidate exposure. Interestingly, this increase is the result of an augmentation of both the synthesis as well as the insertion of these protein complexes in the plasma membrane. These results favour the notion that glial cells are Methylphenidate targets, and that by these means could regulate dopamine turnover.

  7. Acacetin Inhibits Glutamate Release and Prevents Kainic Acid-Induced Neurotoxicity in Rats

    PubMed Central

    Lin, Tzu-Yu; Huang, Wei-Jan; Wu, Chia-Chan; Lu, Cheng-Wei; Wang, Su-Jane

    2014-01-01

    An excessive release of glutamate is considered to be a molecular mechanism associated with several neurological diseases that causes neuronal damage. Therefore, searching for compounds that reduce glutamate neurotoxicity is necessary. In this study, the possibility that the natural flavone acacetin derived from the traditional Chinese medicine Clerodendrum inerme (L.) Gaertn is a neuroprotective agent was investigated. The effect of acacetin on endogenous glutamate release in rat hippocampal nerve terminals (synaptosomes) was also investigated. The results indicated that acacetin inhibited depolarization-evoked glutamate release and cytosolic free Ca2+ concentration ([Ca2+]C) in the hippocampal nerve terminals. However, acacetin did not alter synaptosomal membrane potential. Furthermore, the inhibitory effect of acacetin on evoked glutamate release was prevented by the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker known as ω-conotoxin MVIIC. In a kainic acid (KA) rat model, an animal model used for excitotoxic neurodegeneration experiments, acacetin (10 or 50 mg/kg) was administrated intraperitoneally to the rats 30 min before the KA (15 mg/kg) intraperitoneal injection, and subsequently induced the attenuation of KA-induced neuronal cell death and microglia activation in the CA3 region of the hippocampus. The present study demonstrates that the natural compound, acacetin, inhibits glutamate release from hippocampal synaptosomes by attenuating voltage-dependent Ca2+ entry and effectively prevents KA-induced in vivo excitotoxicity. Collectively, these data suggest that acacetin has the therapeutic potential for treating neurological diseases associated with excitotoxicity. PMID:24520409

  8. Glutamate-dependent transcriptional regulation of GLAST/EAAT1: a role for YY1.

    PubMed

    Rosas, Sandra; Vargas, Miguel A; López-Bayghen, Esther; Ortega, Arturo

    2007-05-01

    Glutamate is the major excitatory transmitter in the vertebrate brain and its extracellular levels are tightly regulated to prevent excitotoxic effects. The Na(+)-dependent glutamate/aspartate transporter GLAST/EAAT1 is regulated in the short and in the long term by glutamate. A receptors-independent change in its membrane translocation rate, accounts for an acute modulation in GLAST/EAAT1 transport. In contrast, activation of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate subtype of glutamate receptors represses the transcription of the chick glast gene. A glutamate responsive element has been mapped to the promoter region of this gene containing a bonafide binding site for the transcription factor Ying-Yang 1. Using cultured chick cerebellar Bergmann glia cells, glutamate elicited a time and dose-dependent increase in Ying-Yang 1 DNA binding consistent with the negative response generated in a reporter gene construct controlled for Ying-Yang 1. Over-expression of this transcription factor leads to a substantial reduction in GLAST/EAAT1 transporter uptake and an important decrease in mRNA levels, all associated with the transcriptional repression of the chick glast promoter activity. These results provide evidence for an involvement of Ying-Yang 1 in the transcriptional response to glutamate in glial cells and favor the notion of a relevant role of this factor in GLAST/EAAT1 transcriptional control.

  9. Antagonistic interaction between adenosine A2A receptors and Na+/K+-ATPase-α2 controlling glutamate uptake in astrocytes.

    PubMed

    Matos, Marco; Augusto, Elisabete; Agostinho, Paula; Cunha, Rodrigo A; Chen, Jiang-Fan

    2013-11-20

    Astrocytic glutamate transporter-1 (GLT-I) is critical to control the bulk of glutamate uptake and, thus, to regulate synaptic plasticity and excitotoxicity. GLT-I glutamate uptake is driven by the sodium gradient implemented by Na(+)/K(+)-ATPases (NKAs) and the α2 subunit of NKA (NKA-α2) is actually linked to GLT-I to regulate astrocytic glutamate transport. We recently found that adenosine A2A receptors (A2ARs), which control synaptic plasticity and neurodegeneration, regulate glutamate uptake through unknown mechanisms. Here we report that A2AR activation decreases NKA activity selectively in astrocytes to inhibit glutamate uptake. Furthermore, we found a physical association of A2ARs with NKA-α2s in astrocytes, as gauged by coimmunoprecipitation and in situ proximity ligation assays, in the cerebral cortex and striatum, two brain regions where A2ARs inhibit the astrocytic glutamate uptake. Moreover, the selective deletion of A2ARs in astrocytes (using Gfa2-A2AR-KO mice) leads to a concurrent increase of both astrocytic glutamate uptake and NKA-α2 levels and activity in the striatum and cortex. This coupling of astrocytic A2ARs to the regulation of glutamate transport through modulation of NKA-α2 activity provides a novel mechanism linking neuronal activity to ion homeostasis controlling glutamatergic activity, all of which are processes intricately associated with the etiology of several brain diseases.

  10. Evoked response to taste stimulations.

    PubMed

    Wada, Masashi

    2005-01-01

    For the recording of gustatory evoked responses, the tip of a stimulator is pressed vertically on one side of the tongue until the trigger pulses are generated by a switch attached to the bottom of the stimulator. According to our results, no detectable response was observed in the absence of taste. The positive waves were distinguishable by using the technique of superimposition before averaging, and the positive wave was made clearer by averaging.

  11. An essential role of acetylcholine-glutamate synergy at habenular synapses in nicotine dependence.

    PubMed

    Frahm, Silke; Antolin-Fontes, Beatriz; Görlich, Andreas; Zander, Johannes-Friedrich; Ahnert-Hilger, Gudrun; Ibañez-Tallon, Ines

    2015-12-01

    A great deal of interest has been focused recently on the habenula and its critical role in aversion, negative-reward and drug dependence. Using a conditional mouse model of the ACh-synthesizing enzyme choline acetyltransferase (Chat), we report that local elimination of acetylcholine (ACh) in medial habenula (MHb) neurons alters glutamate corelease and presynaptic facilitation. Electron microscopy and immuno-isolation analyses revealed colocalization of ACh and glutamate vesicular transporters in synaptic vesicles (SVs) in the central IPN. Glutamate reuptake in SVs prepared from the IPN was increased by ACh, indicating vesicular synergy. Mice lacking CHAT in habenular neurons were insensitive to nicotine-conditioned reward and withdrawal. These data demonstrate that ACh controls the quantal size and release frequency of glutamate at habenular synapses, and suggest that the synergistic functions of ACh and glutamate may be generally important for modulation of cholinergic circuit function and behavior.

  12. The minimal inhibitory synaptic currents evoked in neonatal rat motoneurones.

    PubMed Central

    Takahashi, T

    1992-01-01

    1. Tight-seal whole-cell recordings were made from lumbar motoneurones visually identified in thin slices of neonatal rat spinal cord. The inhibitory postsynaptic currents (IPSCs) were evoked by extracellular stimulation of a neighbouring internuncial neurone in the presence of glutamate receptor antagonists. 2. Glycinergic IPSCs were recorded in the presence of bicuculline. The IPSCs appeared in an all-or-none manner as the graded stimulus intensity exceeded a certain threshold. Their latencies showed a unimodal distribution with a mean of 0.81 ms at 37 degrees C. Thus, the observed IPSCs are suggested to be monosynaptically evoked unitary IPSCs. The mean conductance of unitary IPSCs was 2.9 +/- 1.2 nS (+/- S.D.). 3. When the external Ca2+ concentration ([Ca2+]o) was reduced, the number of failures in response to stimulation increased, thereby reducing the mean amplitude of IPSCs. The mean amplitude of IPSCs was linearly related to the [Ca2+]o (0.35-1.4 mM) with a mean slope of 3.1 +/- 0.67 on double logarithmic co-ordinates. 4. The amplitude of individual IPSCs decreased with decrease in [Ca2+]o. However, below 0.7 mM [Ca2+]o, the mean amplitude of IPSCs (excluding failures) reached a steady minimum level. The mean conductance of these IPSCs measured in 0.5 mM [Ca2+]o was 657 +/- 281 pS. 5. The minimal IPSCs had a coefficient of variation of 0.50 +/- 0.13. No clear correlation was observed between the rise time and the amplitude of minimal IPSCs evoked in individual motoneurones, indicating that the amplitude variability is not due to the different synaptic locations. 6. Spontaneous miniature IPSCs were recorded from motoneurones in the presence of tetrodotoxin. The miniature IPSCs had a mean conductance of 739 +/- 278 pS, being comparable to the minimal evoked IPSCs. 7. Under various internal and external Cl- concentration, the reversal potential of the IPSCs (EIPSC) approximately coincided with the Cl- equilibrium potential. A 730-fold change in the potassium

  13. Glutamate receptors at atomic resolution

    SciTech Connect

    Mayer, Mark L.

    2010-12-03

    At synapses throughout the brain and spinal cord, the amino-acid glutamate is the major excitatory neurotransmitter. During evolution, a family of glutamate-receptor ion channels seems to have been assembled from a kit consisting of discrete ligand-binding, ion-channel, modulatory and cytoplasmic domains. Crystallographic studies that exploit this unique architecture have greatly aided structural analysis of the ligand-binding core, but the results also pose a formidable challenge, namely that of resolving the allosteric mechanisms by which individual domains communicate and function in an intact receptor.

  14. Glycine effects on glutamate-receptor elicited acetylcholinesterase release from slices and synaptosomes of the spinal ventral horn.

    PubMed

    Rodríguez-Ithurralde, D; Olivera, S; La Paz, A; Vincent, O; Rondeau, A

    1996-08-01

    To study the mechanisms by which glutamate-elicited acetylcholinesterase release (GEAR) might play a part in the pathogenesis of excitotoxically triggered motor neurone disease, and to investigate the interaction of GEAR with spinal glycinergic mechanisms, we measured acetylcholinesterase (AChE) and cholinergic markers, after stimulating ventral horn slices and synaptosomes from the mouse spinal cord, with both glutamate- and glycine-receptor agonists. Glutamate (GLU), kainate and AMPA, as well as glycine (GLY) evoked dose-related, calcium-dependent liberation of soluble forms of AChE from both slices and synaptosomes. GLY-evoked AChE release showed remarkable age-related postnatal changes. In the immature slice of the ventral horn. GLY potentiated the GEAR response in the presence of strychnine, suggesting N-methyl-D-aspartate (NMDA) receptor involvement, and was also able to evoke a strychnine-sensitive AChE release in the absence of exogenous GLU. After the 28th postnatal day, nearly all the AChE secreted was released either after the activation of non-NMDA glutamate receptors or by strychnine-sensitive GLY-evoked AChE release mechanisms. Both GEAR and GLY-evoked AChE release might impair the negative feedback loop which modulates the overactivation of motor neurones, and cause prolonged extracellular rises of soluble AChE. These effects might augment the vulnerability of motor neurones to excitotoxic stress, promote fiber outgrowth, and eventually accelerate the metabolic exhaustion of lower motor neurones. It is possible that the mechanisms described are operative at the spinal cord of ALS/MND patients.

  15. Excitatory amino acid transporters tonically restrain nTS synaptic and neuronal activity to modulate cardiorespiratory function.

    PubMed

    Matott, Michael P; Ruyle, Brian C; Hasser, Eileen M; Kline, David D

    2016-03-01

    The nucleus tractus solitarii (nTS) is the initial central termination site for visceral afferents and is important for modulation and integration of multiple reflexes including cardiorespiratory reflexes. Glutamate is the primary excitatory neurotransmitter in the nTS and is removed from the extracellular milieu by excitatory amino acid transporters (EAATs). The goal of this study was to elucidate the role of EAATs in the nTS on basal synaptic and neuronal function and cardiorespiratory regulation. The majority of glutamate clearance in the central nervous system is believed to be mediated by astrocytic EAAT 1 and 2. We confirmed the presence of EAAT 1 and 2 within the nTS and their colocalization with astrocytic markers. EAAT blockade withdl-threo-β-benzyloxyaspartic acid (TBOA) produced a concentration-related depolarization, increased spontaneous excitatory postsynaptic current (EPSC) frequency, and enhanced action potential discharge in nTS neurons. Solitary tract-evoked EPSCs were significantly reduced by EAAT blockade. Microinjection of TBOA into the nTS of anesthetized rats induced apneic, sympathoinhibitory, depressor, and bradycardic responses. These effects mimicked the response to microinjection of exogenous glutamate, and glutamate responses were enhanced by EAAT blockade. Together these data indicate that EAATs tonically restrain nTS excitability to modulate cardiorespiratory function.

  16. Ferrous-iron-dependent uptake of L-glutamate by a mesophilic, mixotrophic iron-oxidizing bacterium strain OKM-9.

    PubMed

    Inoue, Takao; Kamimura, Kazuo; Sugio, Tsuyoshi

    2002-10-01

    Strain OKM-9 is a mesophilic, mixotrophic iron-oxidizing bacterium that absolutely requires ferrous iron as its energy source and L-amino acids (including L-glutamate) as carbon sources for growth. The properties of the L-glutamate transport system were studied with OKM-9 resting cells, plasma membranes, and actively reconstituted proteoliposomes. L-Glutamate uptake into resting cells was totally dependent on ferrous iron that was added to the reaction mixture. Potassium cyanide, an iron oxidase inhibitor, completely inhibited the activity at 1 mM. The optimum pH for Fe2+-dependent uptake activity of L-glutamate was 3.5-4.0. Uptake activity was dependent on the concentration of the L-glutamate. The Km and Vmax for L-glutamate were 0.4 mM and 11.3 nmol x min(-1) x mg(-1), respectively. L-Aspartate, D-aspartate, D-glutamate, and L-cysteine strongly inhibited L-glutamate uptake. L-Aspartate competitively inhibited the activity, and the apparent Ki for this amino acid was 75.9 microM. 2,4-Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, gramicidin D, valinomycin, and monensin did not inhibit Fe2+-dependent L-glutamate uptake. The OKM-9 plasma membranes had approximately 40% of the iron-oxidizing activity of the resting cells and approximately 85% of the Fe2+-dependent uptake activity. The glutamate transport system was solubilized from the membranes with 1% n-octyl-beta-D-glucopyranoside and reconstituted into a lecithin liposome. The L-glutamate transport activity of the reconstituted proteoliposomes was 8-fold than that of the resting cells. The Fe2+-dependent L-glutamate uptake observed here seems to explain the mixotrophic nature of this strain, which absolutely requires Fe2+ oxidation when using amino acids as carbon sources. PMID:12450111

  17. Second-by-second analysis of alpha 7 nicotine receptor regulation of glutamate release in the prefrontal cortex of awake rats.

    PubMed

    Konradsson-Geuken, Asa; Gash, Clelland R; Alexander, Kathleen; Pomerleau, Francois; Huettl, Peter; Gerhardt, Greg A; Bruno, John P

    2009-12-01

    These experiments utilized an enzyme-based microelectrode selective for the second-by-second detection of extracellular glutamate to reveal the alpha 7-based nicotinic modulation of glutamate release in the prefrontal cortex (PFC) of freely moving rats. Rats received intracortical infusions of the nonselective nicotinic agonist nicotine (12.0 mM, 1.0 microg/0.4 microl) or the selective alpha 7 agonist choline (2.0 mM/0.4 microl). The selectivity of drug-induced glutamate release was assessed in subgroups of animals pretreated with the alpha 7 antagonist, alpha-bungarotoxin (alpha-BGT, 10 microM), or kynurenine (10 microM) the precursor of the astrocyte-derived, negative allosteric alpha 7 modulator kynurenic acid. Local administration of nicotine increased glutamate signals (maximum amplitude = 4.3 +/- 0.6 microM) that were cleared to baseline levels in 493 +/- 80 seconds. Pretreatment with alpha-BGT or kynurenine attenuated nicotine-induced glutamate by 61% and 60%, respectively. Local administration of choline also increased glutamate signals (maximum amplitude = 6.3 +/- 0.9 microM). In contrast to nicotine-evoked glutamate release, choline-evoked signals were cleared more quickly (28 +/- 6 seconds) and pretreatment with alpha-BGT or kynurenine completely blocked the stimulated glutamate release. Using a method that reveals the temporal dynamics of in vivo glutamate release and clearance, these data indicate a nicotinic modulation of cortical glutamate release that is both alpha 7- and non-alpha 7-mediated. Furthermore, these data may also provide a mechanism underlying the recent focus on alpha 7 full and partial agonists as therapeutic agents in the treatment of cortically mediated cognitive deficits in schizophrenia.

  18. Regional regulation of glutamate signaling during cuprizone-induced demyelination in the brain.

    PubMed

    Azami Tameh, Abolfazl; Clarner, Tim; Beyer, Cordian; Atlasi, Mohammad Ali; Hassanzadeh, Gholamreza; Naderian, Homayoun

    2013-10-01

    Glutamate excitotoxicity is associated with a wide range of neurodegenerative disorders and also seems to be involved in the pathology of demyelinating disorders such as multiple sclerosis (MS). Cuprizone-induced toxic demyelination shows clear characteristics of MS such as demyelination and axonal damage without the involvement of the innate immune system. In this study, we have evaluated glutamate signaling during cuprizone-induced demyelination in the white and gray matter of mouse brain by studying the expression of ionotropic and metabotropic glutamate-receptors and -transporters by Affymetrix gene array analysis, followed by real-time PCR and western blot analysis. Cellular localization of glutamate transporters was investigated by fluorescence double-labeling experiments. Comparing white and gray matter areas, the expression of glutamate receptors was region-specific. Among NMDA receptor subunits, NR2A was up-regulated in the demyelinated corpus callosum (CC), whereas the metabotropic glutamate receptor mGluR2 was down-regulated in demyelinated gray matter. Glutamate-aspartate transporter (GLAST) co-localizing with GFAP(+) astrocytes was increased in both demyelinated CC and telencephalic cortex, whereas Slc1a4 transporter was up-regulated only in CC. Our data indicate that cuprizone treatment affects glutamate-receptors and -transporters differently in gray and white matter brain areas revealing particularly regulation of GLAST and Slc1a4 compared with other genes. This might have an important influence on brain-region selective sensitivity to neurotoxic compounds and the progression of demyelination as has been reported for MS and other demyelinating neurological diseases. PMID:23711509

  19. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  20. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  1. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and... GENERALLY RECOGNIZED AS SAFE Multiple Purpose GRAS Food Substances § 182.1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation. This substance is generally recognized...

  2. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  3. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  4. 21 CFR 582.1500 - Monoammonium glutamate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Monoammonium glutamate. 582.1500 Section 582.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1500 Monoammonium glutamate. (a) Product. Monoammonium glutamate. (b) Conditions of...

  5. 21 CFR 182.1500 - Monoammonium glutamate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Monoammonium glutamate. 182.1500 Section 182.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD... Substances § 182.1500 Monoammonium glutamate. (a) Product. Monoammonium glutamate. (b) Conditions of...

  6. 21 CFR 582.1500 - Monoammonium glutamate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Monoammonium glutamate. 582.1500 Section 582.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1500 Monoammonium glutamate. (a) Product. Monoammonium glutamate. (b) Conditions of...

  7. 21 CFR 182.1516 - Monopotassium glutamate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Monopotassium glutamate. 182.1516 Section 182.1516 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD... Substances § 182.1516 Monopotassium glutamate. (a) Product. Monopotassium glutamate. (b) Conditions of...

  8. 21 CFR 582.1500 - Monoammonium glutamate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Monoammonium glutamate. 582.1500 Section 582.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1500 Monoammonium glutamate. (a) Product. Monoammonium glutamate. (b) Conditions of...

  9. 21 CFR 582.1516 - Monopotassium glutamate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Monopotassium glutamate. 582.1516 Section 582.1516 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1516 Monopotassium glutamate. (a) Product. Monopotassium glutamate. (b) Conditions of...

  10. 21 CFR 182.1500 - Monoammonium glutamate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Monoammonium glutamate. 182.1500 Section 182.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD... Substances § 182.1500 Monoammonium glutamate. (a) Product. Monoammonium glutamate. (b) Conditions of...

  11. 21 CFR 182.1516 - Monopotassium glutamate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Monopotassium glutamate. 182.1516 Section 182.1516 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD... Substances § 182.1516 Monopotassium glutamate. (a) Product. Monopotassium glutamate. (b) Conditions of...

  12. 21 CFR 582.1516 - Monopotassium glutamate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Monopotassium glutamate. 582.1516 Section 582.1516 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1516 Monopotassium glutamate. (a) Product. Monopotassium glutamate. (b) Conditions of...

  13. 21 CFR 182.1500 - Monoammonium glutamate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Monoammonium glutamate. 182.1500 Section 182.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD... Substances § 182.1500 Monoammonium glutamate. (a) Product. Monoammonium glutamate. (b) Conditions of...

  14. 21 CFR 582.1516 - Monopotassium glutamate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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  15. 21 CFR 582.1500 - Monoammonium glutamate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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  16. 21 CFR 182.1516 - Monopotassium glutamate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Monopotassium glutamate. 182.1516 Section 182.1516 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD... Substances § 182.1516 Monopotassium glutamate. (a) Product. Monopotassium glutamate. (b) Conditions of...

  17. 21 CFR 582.1516 - Monopotassium glutamate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Monopotassium glutamate. 582.1516 Section 582.1516 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1516 Monopotassium glutamate. (a) Product. Monopotassium glutamate. (b) Conditions of...

  18. 21 CFR 182.1500 - Monoammonium glutamate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Monoammonium glutamate. 182.1500 Section 182.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... glutamate. (a) Product. Monoammonium glutamate. (b) Conditions of use. This substance is...

  19. 21 CFR 182.1500 - Monoammonium glutamate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Monoammonium glutamate. 182.1500 Section 182.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD... Substances § 182.1500 Monoammonium glutamate. (a) Product. Monoammonium glutamate. (b) Conditions of...

  20. 21 CFR 582.1516 - Monopotassium glutamate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Monopotassium glutamate. 582.1516 Section 582.1516 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1516 Monopotassium glutamate. (a) Product. Monopotassium glutamate. (b) Conditions of...

  1. 21 CFR 182.1516 - Monopotassium glutamate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Monopotassium glutamate. 182.1516 Section 182.1516 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... glutamate. (a) Product. Monopotassium glutamate. (b) Conditions of use. This substance is...

  2. 21 CFR 182.1516 - Monopotassium glutamate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Monopotassium glutamate. 182.1516 Section 182.1516 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD... Substances § 182.1516 Monopotassium glutamate. (a) Product. Monopotassium glutamate. (b) Conditions of...

  3. 21 CFR 582.1500 - Monoammonium glutamate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Monoammonium glutamate. 582.1500 Section 582.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1500 Monoammonium glutamate. (a) Product. Monoammonium glutamate. (b) Conditions of...

  4. Brain neuroprotection by scavenging blood glutamate.

    PubMed

    Zlotnik, Alexander; Gurevich, Boris; Tkachov, Sergei; Maoz, Ilana; Shapira, Yoram; Teichberg, Vivian I

    2007-01-01

    Excess glutamate in brain fluids characterizes acute brain insults such as traumatic brain injury and stroke. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As blood glutamate scavenging has been demonstrated to increase the efflux of excess glutamate from brain into blood, we tested the prediction that oxaloacetate-mediated blood glutamate scavenging causes neuroprotection in a pathological situation such as closed head injury (CHI), in which there is a well established deleterious increase of glutamate in brain fluids. We observed highly significant improvements of the neurological status of rats submitted to CHI following an intravenous treatment with 1 mmol oxaloacetate/100 g rat weight which decreases blood glutamate levels by 40%. No detectable therapeutic effect was obtained when rats were treated IV with 1 mmol oxaloacetate together with 1 mmol glutamate/100 g rat. The treatment with 0.005 mmol/100 g rat oxaloacetate was no more effective than saline but when it was combined with the intravenous administration of 0.14 nmol/100 g of recombinant glutamate-oxaloacetate transaminase, recovery was almost complete. Oxaloacetate provided neuroprotection when administered before CHI or at 60 min post CHI but not at 120 min post CHI. Since neurological recovery from CHI was highly correlated with the decrease of blood glutamate levels (r=0.89, P=0.001), we conclude that blood glutamate scavenging affords brain neuroprotection Blood glutamate scavenging may open now new therapeutic options.

  5. Traumatic Brain Injury Increases Cortical Glutamate Network Activity by Compromising GABAergic Control

    PubMed Central

    Cantu, David; Walker, Kendall; Andresen, Lauren; Taylor-Weiner, Amaro; Hampton, David; Tesco, Giuseppina; Dulla, Chris G.

    2015-01-01

    Traumatic brain injury (TBI) is a major risk factor for developing pharmaco-resistant epilepsy. Although disruptions in brain circuitry are associated with TBI, the precise mechanisms by which brain injury leads to epileptiform network activity is unknown. Using controlled cortical impact (CCI) as a model of TBI, we examined how cortical excitability and glutamatergic signaling was altered following injury. We optically mapped cortical glutamate signaling using FRET-based glutamate biosensors, while simultaneously recording cortical field potentials in acute brain slices 2–4 weeks following CCI. Cortical electrical stimulation evoked polyphasic, epileptiform field potentials and disrupted the input–output relationship in deep layers of CCI-injured cortex. High-speed glutamate biosensor imaging showed that glutamate signaling was significantly increased in the injured cortex. Elevated glutamate responses correlated with epileptiform activity, were highest directly adjacent to the injury, and spread via deep cortical layers. Immunoreactivity for markers of GABAergic interneurons were significantly decreased throughout CCI cortex. Lastly, spontaneous inhibitory postsynaptic current frequency decreased and spontaneous excitatory postsynaptic current increased after CCI injury. Our results suggest that specific cortical neuronal microcircuits may initiate and facilitate the spread of epileptiform activity following TBI. Increased glutamatergic signaling due to loss of GABAergic control may provide a mechanism by which TBI can give rise to post-traumatic epilepsy. PMID:24610117

  6. Voltage-dependent drug blockade of L-glutamate activated channels of the crayfish.

    PubMed Central

    Dekin, M S; Edwards, C

    1983-01-01

    The actions of d-tubocurarine (d-TC) and local anaesthetics on the L-glutamate activated channel at the voltage-clamped crayfish neuromuscular junction were studied. The effect of d-TC and local anaesthetics on the dose-response relationship between ionophoretically applied L-glutamate and synaptic current suggested that both acted as non-competitive inhibitors. The amount of inhibition was voltage dependent, and increased as the membrane potential was hyperpolarized. This voltage-dependent block was also manifest in a flattening of the I-V relationship between L-glutamate induced current and membrane potential in the presence of d-TC. However, the reversal potential for the L-glutamate activated channel was not affected; it was about +7 mV in both the presence and absence of d-TC. The neurally evoked excitatory post-synaptic current (e.p.s.c.) was depressed in the presence of these drugs and this effect was also voltage dependent. The time course of the e.p.s.c. was affected, but less so than expected if the L-glutamate activated channel were identical to the channel opened by acetylcholine at the vertebrate neuromuscular junction. Possible reasons for this discrepancy are discussed. PMID:6312026

  7. Modes of glutamate receptor gating

    PubMed Central

    Popescu, Gabriela K

    2012-01-01

    Abstract The time course of excitatory synaptic currents, the major means of fast communication between neurons of the central nervous system, is encoded in the dynamic behaviour of post-synaptic glutamate-activated channels. First-pass attempts to explain the glutamate-elicited currents with mathematical models produced reaction mechanisms that included only the most basic functionally defined states: resting vs. liganded, closed vs. open, responsive vs. desensitized. In contrast, single-molecule observations afforded by the patch-clamp technique revealed an unanticipated kinetic multiplicity of transitions: from microseconds-lasting flickers to minutes-long modes. How these kinetically defined events impact the shape of the synaptic response, how they relate to rearrangements in receptor structure, and whether and how they are physiologically controlled represent currently active research directions. Modal gating, which refers to the slowest, least frequently observed ion-channel transitions, has been demonstrated for representatives of all ion channel families. However, reaction schemes have been largely confined to the short- and medium-range time scales. For glutamate receptors as well, modal gating has only recently come under rigorous scrutiny. This article reviews the evidence for modal gating of glutamate receptors and the still developing hypotheses about the mechanism(s) by which modal shifts occur and the ways in which they may impact the time course of synaptic transmission. PMID:22106181

  8. Vanilloids selectively sensitize thermal glutamate release from TRPV1 expressing solitary tract afferents.

    PubMed

    Hofmann, Mackenzie E; Andresen, Michael C

    2016-02-01

    Vanilloids, high temperature, and low pH activate the transient receptor potential vanilloid type 1 (TRPV1) receptor. In spinal dorsal root ganglia, co-activation of one of these gating sites on TRPV1 sensitized receptor gating by other modes. Here in rat brainstem slices, we examined glutamate synaptic transmission in nucleus of the solitary tract (NTS) neurons where most cranial primary afferents express TRPV1, but TRPV1 sensitization is unknown. Electrical shocks to the solitary tract (ST) evoked EPSCs (ST-EPSCs). Activation of TRPV1 with capsaicin (100 nM) increased spontaneous EPSCs (sEPSCs) but inhibited ST-EPSCs. High concentrations of the ultra-potent vanilloid resiniferatoxin (RTX, 1 nM) similarly increased sEPSC rates but blocked ST-EPSCs. Lowering the RTX concentration to 150 pM modestly increased the frequency of the sEPSCs without causing failures in the evoked ST-EPSCs. The sEPSC rate increased with raising bath temperature to 36 °C. Such thermal responses were larger in 150 pM RTX, while the ST-EPSCs remained unaffected. Vanilloid sensitization of thermal responses persisted in TTX but was blocked by the TRPV1 antagonist capsazepine. Our results demonstrate that multimodal activation of TRPV1 facilitates sEPSC responses in more than the arithmetic sum of the two activators, i.e. co-activation sensitizes TRPV1 control of spontaneous glutamate release. Since action potential evoked glutamate release is unaltered, the work provides evidence for cooperativity in gating TRPV1 plus a remarkable separation of calcium mechanisms governing the independent vesicle pools responsible for spontaneous and evoked release at primary afferents in the NTS.

  9. Sensory transduction of weak electromagnetic fields: role of glutamate neurotransmission mediated by NMDA receptors.

    PubMed

    Frilot, C; Carrubba, S; Marino, A A

    2014-01-31

    Subliminal electromagnetic fields (EMFs) triggered nonlinear evoked potentials in awake but not anesthetized animals, and increased glucose metabolism in the hindbrain. Field detection occurred somewhere in the head and possibly was an unrecognized function of sensory neurons in facial skin, which synapse in the trigeminal nucleus and project to the thalamus via glutamate-dependent pathways. If so, anesthetic agents that antagonize glutamate neurotransmission would be expected to degrade EMF-evoked potentials (EEPs) to a greater extent than agents having different pharmacological effects. We tested the hypothesis using ketamine which blocks N-methyl-d-aspartate (NMDA) receptors (NMDARs), and xylazine which is an α₂-adrenoreceptor agonist. Electroencephalograms (EEGs) of rats were examined using recurrence analysis to observe EEPs in the presence and absence of ketamine and/or xylazine anesthesia. Auditory evoked potentials (AEPs) served as positive controls. The frequency of observation of evoked potentials in a given condition (wake or anesthesia) was compared with that due to chance using the Fisher's exact test. EEPs were observed in awake rats but not while they were under anesthesia produced using a cocktail of xylazine and ketamine. In another experiment each rat was measured while awake and while under anesthesia produced using either xylazine or ketamine. EEPs and AEPs were detected during wake and under xylazine (P<0.05 in each of the four measurements). In contrast, neither EEPs nor AEPs were observed when anesthesia was produced partly or wholly using ketamine. The duration and latency of the EEPs was unaltered by xylazine anesthesia. The afferent signal triggered by the transduction of weak EMFs was likely mediated by NMDAR-mediated glutamate neurotransmission.

  10. Sensory transduction of weak electromagnetic fields: role of glutamate neurotransmission mediated by NMDA receptors.

    PubMed

    Frilot, C; Carrubba, S; Marino, A A

    2014-01-31

    Subliminal electromagnetic fields (EMFs) triggered nonlinear evoked potentials in awake but not anesthetized animals, and increased glucose metabolism in the hindbrain. Field detection occurred somewhere in the head and possibly was an unrecognized function of sensory neurons in facial skin, which synapse in the trigeminal nucleus and project to the thalamus via glutamate-dependent pathways. If so, anesthetic agents that antagonize glutamate neurotransmission would be expected to degrade EMF-evoked potentials (EEPs) to a greater extent than agents having different pharmacological effects. We tested the hypothesis using ketamine which blocks N-methyl-d-aspartate (NMDA) receptors (NMDARs), and xylazine which is an α₂-adrenoreceptor agonist. Electroencephalograms (EEGs) of rats were examined using recurrence analysis to observe EEPs in the presence and absence of ketamine and/or xylazine anesthesia. Auditory evoked potentials (AEPs) served as positive controls. The frequency of observation of evoked potentials in a given condition (wake or anesthesia) was compared with that due to chance using the Fisher's exact test. EEPs were observed in awake rats but not while they were under anesthesia produced using a cocktail of xylazine and ketamine. In another experiment each rat was measured while awake and while under anesthesia produced using either xylazine or ketamine. EEPs and AEPs were detected during wake and under xylazine (P<0.05 in each of the four measurements). In contrast, neither EEPs nor AEPs were observed when anesthesia was produced partly or wholly using ketamine. The duration and latency of the EEPs was unaltered by xylazine anesthesia. The afferent signal triggered by the transduction of weak EMFs was likely mediated by NMDAR-mediated glutamate neurotransmission. PMID:24239718

  11. Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

    PubMed

    Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

    2014-08-01

    Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes.

  12. Excitatory amino acid glutamate: role in peripheral nociceptive transduction and inflammation in experimental and clinical osteoarthritis.

    PubMed

    Wen, Z-H; Chang, Y-C; Jean, Y-H

    2015-11-01

    Although a large proportion of patients with osteoarthritis (OA) show inflammation in their affected joints, the pathological role of inflammation in the development and progression of OA has yet to be clarified. Glutamate is considered an excitatory amino acid (EAA) neurotransmitter in the mammalian central nervous system (CNS). There are cellular membrane glutamate receptors and transporters for signal input modulation and termination as well as vesicular glutamate transporters (VGLUTs) for signal output through exocytotic release. Glutamate been shown to mediate intercellular communications in bone cells in a manner similar to synaptic transmission within the CNS. Glutamate-mediated events may also contribute to the pathogenesis and ongoing processes of peripheral nociceptive transduction and inflammation of experimental arthritis models as well as human arthritic conditions. This review will discuss the differential roles of glutamate signaling and blockade in peripheral neuronal and non-neuronal joint tissues, including bone remodeling systems and their potentials to impact OA-related inflammation and progression. This will serve to identify several potential targets to direct novel therapies for OA. Future studies will further elucidate the role of glutamate in the development and progression of OA, as well as its association with the clinical features of the disease.

  13. Among the twenty classical L-amino acids, only glutamate directly activates metabotropic glutamate receptors.

    PubMed

    Frauli, Mélanie; Neuville, Pascal; Vol, Claire; Pin, Jean-Philippe; Prézeau, Laurent

    2006-02-01

    Under pathophysiological conditions, cellular amino acids can be profusely released from cells into the cerebral interstitial space. Because several class-C G protein coupled receptors (GPCRs) display a broad natural ligand spectrum, being sensitive to more than one endogenous ligand, we wondered whether the related metabotropic glutamate (mGlu) receptors could be modulated by various types of L-amino acids, allowing them to sense large increase in extracellular amino acid concentration. Here, the agonist, antagonist and allosteric effects of the twenty classical L-amino acids were evaluated on the eight mGlu receptor subtypes. We show that, in addition to glutamate (Glu), cysteine, aspartate and asparagine also lead to the activation of mGlu3, 4 and 5. Interestingly, our data demonstrate that the effect of these three amino acids did not result from a direct activation of the receptors, but from an indirect action involving Glu-transporters/exchangers. These data first demonstrate that mGlu receptors, unlike other class-C GPCRs, display an extremely high selectivity towards one ligand. Moreover, our results also show that Glu transport systems allow mGlu receptors to sense large increase in the extracellular concentration of some amino acids. Such a system will certainly lead to a large increase in some mGlu receptor activity under pathological conditions, such as seizure, ischemia or other brain injuries. PMID:16310227

  14. Fluorescence imaging of glutamate release in neurons

    SciTech Connect

    Wang, Ziqiang; Yeung, Edward S.

    1999-12-01

    A noninvasive detection scheme based on glutamate dehydrogenase (GDH) enzymatic assay combined with microscopy was developed to measure the glutamate release in cultured cells from the central nervous system (CNS). The enzyme reaction is very specific and sensitive. The detection limit with charge-coupled device (CCD) imaging is down to {mu}M levels of glutamate with reasonable response time ({approx}30 s). The standard glutamate test shows a linear response over 3 orders of magnitude, from {mu}M to 0.1 mM range. The in vitro monitoring of glutamate release from cultured neuron cells demonstrated excellent spatial and temporal resolution. (c) 1999 Society for Applied Spectroscopy.

  15. Intradermal glutamate and capsaicin injections: intra- and interindividual variability of provoked hyperalgesia and allodynia.

    PubMed

    Nilsson, Matias; Lassen, Dorte; Andresen, Trine; Nielsen, Anders K; Arendt-Nielsen, Lars; Drewes, Asbjørn M

    2014-06-01

    Intradermal injections of glutamate and capsaicin are attractive to use in human experimental pain models because hyperalgesia and allodynia mimic isolated aspects of clinical pain disorders. The aim of the present study was to investigate the reproducibility of these models. Twenty healthy male volunteers (mean age 24 years; range 18-38 years) received intradermal injections of glutamate and capsaicin in the volar forearm. Magnitudes of secondary pinprick hyperalgesia and brush-evoked allodynia were investigated using von Frey filaments (gauges 10, 15, 60 and 100 g) and brush strokes. Areas of secondary hyperalgesia and allodynia were quantified immediately after injection and after 15, 30 and 60 min. Two identical experiments separated by at least 7 days were performed. Reproducibility across and within volunteers (inter- and intra-individual variation, respectively) was assessed using intraclass correlation coefficient (ICC) and coefficient of variation (CV). Secondary pinprick hyperalgesia was observed as a marked increase in the visual analogue scale (VAS) response to von Frey gauges 60 and 100 g (P < 0.001) after glutamate injection. For capsaicin, secondary pinprick hyperalgesia was detected with all von Frey gauges (P < 0.001). Glutamate evoked reproducible VAS response to all von Frey gauges (ICC > 0.60) and brush strokes (ICC > 0.83). Capsaicin injection was reproducible for secondary hyperalgesia (ICC > 0.70) and allodynia (ICC > 0.71). Intra-individual variability was generally lower for the VAS response to von Frey and brush compared with areas of secondary hyperalgesia and allodynia. In conclusion, glutamate and capsaicin yield reproducible hyperalgesic and allodynic responses, and the present model is well suited for basic research, as well as for assessing the modulation of central phenomena.

  16. Achieving Presence through Evoked Reality

    PubMed Central

    Pillai, Jayesh S.; Schmidt, Colin; Richir, Simon

    2013-01-01

    The sense of “Presence” (evolving from “telepresence”) has always been associated with virtual reality research and is still an exceptionally mystifying constituent. Now the study of presence clearly spans over various disciplines associated with cognition. This paper attempts to put forth a concept that argues that it’s an experience of an “Evoked Reality (ER)” (illusion of reality) that triggers an “Evoked Presence (EP)” (sense of presence) in our minds. A Three Pole Reality Model is proposed to explain this phenomenon. The poles range from Dream Reality to Simulated Reality with Primary (Physical) Reality at the center. To demonstrate the relationship between ER and EP, a Reality-Presence Map is developed. We believe that this concept of ER and the proposed model may have significant applications in the study of presence, and in exploring the possibilities of not just virtual reality but also what we call “reality.” PMID:23550234

  17. Evoked Electromyographically Controlled Electrical Stimulation

    PubMed Central

    Hayashibe, Mitsuhiro

    2016-01-01

    Time-variant muscle responses under electrical stimulation (ES) are often problematic for all the applications of neuroprosthetic muscle control. This situation limits the range of ES usage in relevant areas, mainly due to muscle fatigue and also to changes in stimulation electrode contact conditions, especially in transcutaneous ES. Surface electrodes are still the most widely used in noninvasive applications. Electrical field variations caused by changes in the stimulation contact condition markedly affect the resulting total muscle activation levels. Fatigue phenomena under functional electrical stimulation (FES) are also well known source of time-varying characteristics coming from muscle response under ES. Therefore, it is essential to monitor the actual muscle state and assess the expected muscle response by ES so as to improve the current ES system in favor of adaptive muscle-response-aware FES control. To deal with this issue, we have been studying a novel control technique using evoked electromyography (eEMG) signals to compensate for these muscle time-variances under ES for stable neuroprosthetic muscle control. In this perspective article, I overview the background of this topic and highlight important points to be aware of when using ES to induce the desired muscle activation regardless of the time-variance. I also demonstrate how to deal with the common critical problem of ES to move toward robust neuroprosthetic muscle control with the Evoked Electromyographically Controlled Electrical Stimulation paradigm. PMID:27471448

  18. Rat epileptic seizures evoked by BmK {alpha}IV and its possible mechanisms involved in sodium channels

    SciTech Connect

    Chai Zhifang; Bai Zhantao; Zhang Xuying; Liu Tong; Pang Xueyan; Ji Yonghua . E-mail: yhji@server.shcnc.ac.cn

    2007-05-01

    This study showed that rat unilateral intracerebroventricular injection of BmK {alpha}IV, a sodium channel modulator derived from scorpion Buthus martensi Karsch, induced clusters of spikes, epileptic discharges and convulsion-related behavioral changes. BmK {alpha}IV potently promoted the release of endogenous glutamate from rat cerebrocortical synaptosomes. In vitro examination of the effect of BmK {alpha}IV on intrasynaptosomal free calcium concentration [Ca{sup 2+}]{sub i} and sodium concentration [Na{sup +}]{sub i} revealed that BmK {alpha}IV-evoked glutamate release from synaptosomes was associated with an increase in Ca{sup 2+} and Na{sup +} influx. Moreover, BmK {alpha}IV-mediated glutamate release and ion influx was completely blocked by tetrodotoxin, a blocker of sodium channel. Together, these results suggest that the induction of BmK {alpha}IV-evoked epileptic seizures may be involved in the modulation of BmK {alpha}IV on tetrodotoxin-sensitive sodium channels located on the nerve terminal, which subsequently enhances the Ca{sup 2+} influx to cause an increase of glutamate release. These findings may provide some insight regarding the mechanism of neuronal action of BmK {alpha}IV in the central nervous system for understanding epileptogenesis involved in sodium channels.

  19. Disruptions in the Regulation of Extracellular Glutamate by Neurons and Glia in the Rat Striatum Two Days after Diffuse Brain Injury

    PubMed Central

    Hinzman, Jason M.; Thomas, Theresa Currier; Quintero, Jorge E.; Gerhardt, Greg A.

    2012-01-01

    Abstract Disrupted regulation of extracellular glutamate in the central nervous system contributes to and can exacerbate the acute pathophysiology of traumatic brain injury (TBI). Previously, we reported increased extracellular glutamate in the striatum of anesthetized rats 2 days after diffuse brain injury. To determine the mechanism(s) responsible for increased extracellular glutamate, we used enzyme-based microelectrode arrays (MEAs) coupled with specific pharmacological agents targeted at in vivo neuronal and glial regulation of extracellular glutamate. After TBI, extracellular glutamate was significantly increased in the striatum by (∼90%) averaging 4.1±0.6 μM compared with sham 2.2±0.4 μM. Calcium-dependent neuronal glutamate release, investigated by local application of an N-type calcium channel blocker, was no longer a significant source of extracellular glutamate after TBI, compared with sham. In brain-injured animals, inhibition of glutamate uptake with local application of an excitatory amino acid transporter inhibitor produced significantly greater increase in glutamate spillover (∼ 65%) from the synapses compared with sham. Furthermore, glutamate clearance measured by locally applying glutamate into the extracellular space revealed significant reductions in glutamate clearance parameters in brain-injured animals compared with sham. Taken together, these data indicate that disruptions in calcium-mediated glutamate release and glial regulation of extracellular glutamate contribute to increased extracellular glutamate in the striatum 2 days after diffuse brain injury. Overall, these data suggest that therapeutic strategies used to regulate glutamate release and uptake may improve excitatory circuit function and, possibly, outcomes following TBI. PMID:22233432

  20. Abnormal glutamate release in aged BTBR mouse model of autism.

    PubMed

    Wei, Hongen; Ding, Caiyun; Jin, Guorong; Yin, Haizhen; Liu, Jianrong; Hu, Fengyun

    2015-01-01

    Autism is a neurodevelopmental disorder characterized by abnormal reciprocal social interactions, communication deficits, and repetitive behaviors with restricted interests. Most of the available research on autism is focused on children and young adults and little is known about the pathological alternation of autism in older adults. In order to investigate the neurobiological alternation of autism in old age stage, we compared the morphology and synaptic function of excitatory synapses between the BTBR mice with low level sociability and B6 mice with high level sociability. The results revealed that the number of excitatory synapse colocalized with pre- and post-synaptic marker was not different between aged BTBR and B6 mice. The aged BTBR mice had a normal structure of dendritic spine and the expression of Shank3 protein in the brain as well as that in B6 mice. The baseline and KCl-evoked glutamate release from the cortical synaptoneurosome in aged BTBR mice was lower than that in aged B6 mice. Overall, the data indicate that there is a link between disturbances of the glutamate transmission and autism. These findings provide new evidences for the hypothesis of excitation/inhibition imbalance in autism. Further work is required to determine the cause of this putative abnormality.

  1. Glutamate down-regulates GLAST expression through AMPA receptors in Bergmann glial cells.

    PubMed

    López-Bayghen, Esther; Espinoza-Rojo, Mónica; Ortega, Arturo

    2003-07-01

    The Na(+)-dependent glutamate/aspartate transporter GLAST plays a major role in the removal of glutamate from the synaptic cleft. Short-, as well as long-term changes in transporter activity are triggered by glutamate. An important locus of regulation is the density of transporter molecules at the plasma membrane. A substrate-dependent change in the translocation rate accounts for the short-term effect, whereas the mechanisms of long-term modulation are less understood. Using cultured chick cerebellar Bergmann glial cells, we report here that glutamate receptors mediate a substantial reduction in GLAST mRNA levels, suggesting a transcriptional level of regulation. Moreover, when the 5' proximal region of the GLAST gene was cloned and transfected into Bergmann glia cells, a decrease in promoter activity was induced by glutamate exposure. The use of specific pharmacological tools established the involvement of Ca(2+)-permeable alpha-amino 3-hydroxy-5-methyl-4-isoaxazolepropionate (AMPA) receptors via protein kinase C and c-Jun. These results demonstrate that GLAST is under transcriptional control through glutamate receptors activation, and further supports the participation of Bergmann glia cells in the modulation of glutamatergic transmission.

  2. Ligands for Ionotropic Glutamate Receptors

    NASA Astrophysics Data System (ADS)

    Swanson, Geoffrey T.; Sakai, Ryuichi

    Marine-derived small molecules and peptides have played a central role in elaborating pharmacological specificities and neuronal functions of mammalian ionotropic glutamate receptors (iGluRs), the primary mediators of excitatory syn-aptic transmission in the central nervous system (CNS). As well, the pathological sequelae elicited by one class of compounds (the kainoids) constitute a widely-used animal model for human mesial temporal lobe epilepsy (mTLE). New and existing molecules could prove useful as lead compounds for the development of therapeutics for neuropathologies that have aberrant glutamatergic signaling as a central component. In this chapter we discuss natural source origins and pharmacological activities of those marine compounds that target ionotropic glutamate receptors.

  3. Ligands for Ionotropic Glutamate Receptors

    PubMed Central

    Swanson, Geoffrey T.; Sakai, Ryuichi

    2010-01-01

    Marine-derived small molecules and peptides have played a central role in elaborating pharmacological specificities and neuronal functions of mammalian ionotropic glutamate receptors (iGluRs), the primary mediators of excitatory synaptic transmission in the central nervous system (CNS). As well, the pathological sequelae elicited by one class of compounds (the kainoids) constitute a widely-used animal model for human mesial temporal lobe epilepsy (mTLE). New and existing molecules could prove useful as lead compounds for the development of therapeutics for neuropathologies that have aberrant glutamatergic signaling as a central component. In this chapter we discuss natural source origins and pharmacological activities of those marine compounds that target ionotropic glutamate receptors. PMID:19184587

  4. Manipulation of isolated brain nerve terminals by an external magnetic field using D-mannose-coated γ-Fe2O3 nano-sized particles and assessment of their effects on glutamate transport

    PubMed Central

    Krisanova, Natalia; Borуsov, Arsenii; Sivko, Roman; Ostapchenko, Ludmila; Babic, Michal; Horak, Daniel

    2014-01-01

    Summary The manipulation of brain nerve terminals by an external magnetic field promises breakthroughs in nano-neurotechnology. D-Mannose-coated superparamagnetic nanoparticles were synthesized by coprecipitation of Fe(II) and Fe(III) salts followed by oxidation with sodium hypochlorite and addition of D-mannose. Effects of D-mannose-coated superparamagnetic maghemite (γ-Fe2O3) nanoparticles on key characteristics of the glutamatergic neurotransmission were analysed. Using radiolabeled L-[14C]glutamate, it was shown that D-mannose-coated γ-Fe2O3 nanoparticles did not affect high-affinity Na+-dependent uptake, tonic release and the extracellular level of L-[14C]glutamate in isolated rat brain nerve terminals (synaptosomes). Also, the membrane potential of synaptosomes and acidification of synaptic vesicles was not changed as a result of the application of D-mannose-coated γ-Fe2O3 nanoparticles. This was demonstrated with the potential-sensitive fluorescent dye rhodamine 6G and the pH-sensitive dye acridine orange. The study also focused on the analysis of the potential use of these nanoparticles for manipulation of nerve terminals by an external magnetic field. It was shown that more than 84.3 ± 5.0% of L-[14C]glutamate-loaded synaptosomes (1 mg of protein/mL) incubated for 5 min with D-mannose-coated γ-Fe2O3 nanoparticles (250 µg/mL) moved to an area, in which the magnet (250 mT, gradient 5.5 Т/m) was applied compared to 33.5 ± 3.0% of the control and 48.6 ± 3.0% of samples that were treated with uncoated nanoparticles. Therefore, isolated brain nerve terminals can be easily manipulated by an external magnetic field using D-mannose-coated γ-Fe2O3 nanoparticles, while the key characteristics of glutamatergic neurotransmission are not affected. In other words, functionally active synaptosomes labeled with D-mannose-coated γ-Fe2O3 nanoparticles were obtained. PMID:24991515

  5. The human auditory evoked response

    NASA Technical Reports Server (NTRS)

    Galambos, R.

    1974-01-01

    Figures are presented of computer-averaged auditory evoked responses (AERs) that point to the existence of a completely endogenous brain event. A series of regular clicks or tones was administered to the ear, and 'odd-balls' of different intensity or frequency respectively were included. Subjects were asked either to ignore the sounds (to read or do something else) or to attend to the stimuli. When they listened and counted the odd-balls, a P3 wave occurred at 300msec after stimulus. When the odd-balls consisted of omitted clicks or tone bursts, a similar response was observed. This could not have come from auditory nerve, but only from cortex. It is evidence of recognition, a conscious process.

  6. Glutamate racemization and catabolism in Fusobacterium varium.

    PubMed

    Ramezani, Mohammad; Resmer, Kelly L; White, Robert L

    2011-07-01

    The pathways of glutamate catabolism in the anaerobic bacterium Fusobacterium varium, grown on complex, undefined medium and chemically defined, minimal medium, were investigated using specifically labelled (13)C-glutamate. The metabolic end-products acetate and butyrate were isolated from culture fluids and derivatized for analysis by nuclear magnetic resonance and mass spectrometry. On complex medium, labels from L-[1-(13)C]glutamate and L-[4-(13)C]glutamate were incorporated into C1 of acetate and equally into C1/C3 of butyrate, while label derived from L-[5-(13)C]glutamate was not incorporated. The isotopic incorporation results and the detection of glutamate mutase and 3-methylaspartate ammonia lyase in cell extracts are most consistent with the methylaspartate pathway, the best known route of glutamate catabolism in Clostridium species. When F. varium was grown on defined medium, label from L-[4-(13)C]glutamate was incorporated mainly into C4 of butyrate, demonstrating a major role for the hydroxyglutarate pathway. Upon addition of coenzyme B(12) or cobalt ion to the defined medium in replicate experiments, isotope was located equally at C1/C3 of butyrate in accord with the methylaspartate pathway. Racemization of D-glutamate and subsequent degradation of L-glutamate via the methylaspartate pathway are supported by incorporation of label into C2 of acetate and equally into C2/C4 of butyrate from D-[3-(13)C]glutamate and the detection of a cofactor-independent glutamate racemase in cell extracts. Together the results demonstrate a major role for the methylaspartate pathway of glutamate catabolism in F. varium and substantial participation of the hydroxyglutarate pathway when coenzyme B(12) is not available.

  7. Current-evoked transcellular K+ flux in frog retina.

    PubMed

    Karwoski, C J; Coles, J A; Lu, H K; Huang, B

    1989-05-01

    1. Changes in extracellular K+ concentration (delta[K+]o) evoked by electrical current were measured with K+-selective microelectrodes (K-ISMs) in the retina of the frog eyecup. 2. In the superfusate at 20 microns above the inner limiting membrane (ILM), current-evoked delta[K+] was a function of current polarity and strength; its amplitude decreased as the K-ISM was moved higher above the ILM. Responses were similar whether measured with K-ISMs containing the Corning exchanger or a valinomycin-based liquid membrane. No current-evoked delta[Ca2+] could be detected with Ca-selective microelectrodes (Ca-ISMs). 3. Within the retina, a complex spatiotemporal profile of current-evoked delta[K+]o was observed. Strophanthidin abolished responses in the proximal retina, but had little effect on the response in the superfusate. A blocker of K+ channels (Ba2+) depressed responses in the superfusate, but not in the proximal retina. 4. Quantitative analysis of these responses indicates a transport number for K+ of 0.18 at onset of current, and that decreases over a few seconds. In contrast, a transport number of approximately 0.01 is predicted from the expected ionic concentrations within extracellular space. 5. These findings are compatible with the delta[K+] above the ILM being due to transcellular movement of K+ through Müller cells. The results suggest that K+ spatial buffering may be particularly potent in the retina. Furthermore, determinations of tissue characteristics by passage of electrical current must take into account that at least 17% of the current does not travel through extracellular space.

  8. Real-time imaging of glutamate clearance reveals normal striatal uptake in Huntington disease mouse models

    PubMed Central

    Parsons, Matthew P.; Vanni, Matthieu P.; Woodard, Cameron L.; Kang, Rujun; Murphy, Timothy H.; Raymond, Lynn A.

    2016-01-01

    It has become well accepted that Huntington disease (HD) is associated with impaired glutamate uptake, resulting in a prolonged time-course of extracellular glutamate that contributes to excitotoxicity. However, the data supporting this view come largely from work in synaptosomes, which may overrepresent nerve-terminal uptake over astrocytic uptake. Here, we quantify real-time glutamate dynamics in HD mouse models by high-speed imaging of an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) and electrophysiological recordings of synaptically activated transporter currents in astrocytes. These techniques reveal a disconnect between the results obtained in synaptosomes and those in situ. Exogenous glutamate uptake is impaired in synaptosomes, whereas real-time measures of glutamate clearance in the HD striatum are normal or even accelerated, particularly in the aggressive R6/2 model. Our results highlight the importance of quantifying glutamate dynamics under endogenous release conditions, and suggest that the widely cited uptake impairment in HD does not contribute to pathogenesis. PMID:27052848

  9. Real-time imaging of glutamate clearance reveals normal striatal uptake in Huntington disease mouse models.

    PubMed

    Parsons, Matthew P; Vanni, Matthieu P; Woodard, Cameron L; Kang, Rujun; Murphy, Timothy H; Raymond, Lynn A

    2016-01-01

    It has become well accepted that Huntington disease (HD) is associated with impaired glutamate uptake, resulting in a prolonged time-course of extracellular glutamate that contributes to excitotoxicity. However, the data supporting this view come largely from work in synaptosomes, which may overrepresent nerve-terminal uptake over astrocytic uptake. Here, we quantify real-time glutamate dynamics in HD mouse models by high-speed imaging of an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) and electrophysiological recordings of synaptically activated transporter currents in astrocytes. These techniques reveal a disconnect between the results obtained in synaptosomes and those in situ. Exogenous glutamate uptake is impaired in synaptosomes, whereas real-time measures of glutamate clearance in the HD striatum are normal or even accelerated, particularly in the aggressive R6/2 model. Our results highlight the importance of quantifying glutamate dynamics under endogenous release conditions, and suggest that the widely cited uptake impairment in HD does not contribute to pathogenesis. PMID:27052848

  10. High t-PA release by neonate brain microvascular endothelial cells under glutamate exposure affects neuronal fate.

    PubMed

    Henry, Vincent Jean; Lecointre, Maryline; Laudenbach, Vincent; Ali, Carine; Macrez, Richard; Jullienne, Amandine; Berezowski, Vincent; Carmeliet, Peter; Vivien, Denis; Marret, Stéphane; Gonzalez, Bruno José; Leroux, Philippe

    2013-02-01

    Glutamate excitotoxicity is a consolidated hypothesis in neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor mediated effects. In brain microvascular endothelial cell (nBMEC) cultures from neonates, t-PA content and release upon glutamate are higher than in adult (aBMECs) cultures. Owing to the variety of t-PA substrates and receptor targets, the study was aimed at determining the putative roles of endothelial t-PA in the neonatal brain parenchyma under glutamate challenge. Basal t-PA release was 4.4 fold higher in nBMECs vs aBMECs and glutamate was 20 fold more potent to allow Evans blue vascular permeability in neonate microvessels indicating that, under noxious glutamate (50 μM) exposure, high amounts of endothelial t-PA stores may be mobilized and may access the nervous parenchyma. Culture media from nBMECS or aBMECs challenged by excitotoxic glutamate were applied to neuron cultures at DIV 11. While media from adult cells did not evoke more LDH release in neuronal cultures that under glutamate alone, media from nBMECs enhanced 2.2 fold LDH release. This effect was not observed with media from t-PA(-/-) nBMECs and was inhibited by hr-PAI-1. In Cortical slices from 10 day-old mice, hrt-PA associated with glutamate evoked neuronal necrosis in deeper (more mature) layers, an effect reversed by NMDA receptor GluN1 amino-terminal domain antibody capable of inhibiting t-PA potentiation of the receptor. In superficial layers (less mature), hrt-PA alone inhibited apoptosis, an effect reversed by the EGF receptor antagonist AG1478. Applied to immature neurons in culture (DIV5), media from nBMEC rescued 85.1% of neurons from cell death induced by serum deprivation. In cortical slices, the anti-apoptotic effect of t-PA fitted with age dependent localization of less mature neurons. These data suggest that in the immature brain, propensity of vessels to release high amounts of t-PA may not only

  11. Glutamate and GABA in Vestibulo-Sympathetic Pathway Neurons

    PubMed Central

    Holstein, Gay R.; Friedrich, Victor L. Jr.; Martinelli, Giorgio P.

    2016-01-01

    The vestibulo-sympathetic reflex (VSR) actively modulates blood pressure during changes in posture. This reflex allows humans to stand up and quadrupeds to rear or climb without a precipitous decline in cerebral perfusion. The VSR pathway conveys signals from the vestibular end organs to the caudal vestibular nuclei. These cells, in turn, project to pre-sympathetic neurons in the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). The present study assessed glutamate- and GABA-related immunofluorescence associated with central vestibular neurons of the VSR pathway in rats. Retrograde FluoroGold tract tracing was used to label vestibular neurons with projections to RVLM or CVLM, and sinusoidal galvanic vestibular stimulation (GVS) was employed to activate these pathways. Central vestibular neurons of the VSR were identified by co-localization of FluoroGold and cFos protein, which accumulates in some vestibular neurons following galvanic stimulation. Triple-label immunofluorescence was used to co-localize glutamate- or GABA- labeling in the identified VSR pathway neurons. Most activated projection neurons displayed intense glutamate immunofluorescence, suggestive of glutamatergic neurotransmission. To support this, anterograde tracer was injected into the caudal vestibular nuclei. Vestibular axons and terminals in RVLM and CVLM co-localized the anterograde tracer and vesicular glutamate transporter-2 signals. Other retrogradely-labeled cFos-positive neurons displayed intense GABA immunofluorescence. VSR pathway neurons of both phenotypes were present in the caudal medial and spinal vestibular nuclei, and projected to both RVLM and CVLM. As a group, however, triple-labeled vestibular cells with intense glutamate immunofluorescence were located more rostrally in the vestibular nuclei than the GABAergic neurons. Only the GABAergic VSR pathway neurons showed a target preference, projecting predominantly to CVLM. These data provide the first

  12. Glutamate and GABA in Vestibulo-Sympathetic Pathway Neurons.

    PubMed

    Holstein, Gay R; Friedrich, Victor L; Martinelli, Giorgio P

    2016-01-01

    The vestibulo-sympathetic reflex (VSR) actively modulates blood pressure during changes in posture. This reflex allows humans to stand up and quadrupeds to rear or climb without a precipitous decline in cerebral perfusion. The VSR pathway conveys signals from the vestibular end organs to the caudal vestibular nuclei. These cells, in turn, project to pre-sympathetic neurons in the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). The present study assessed glutamate- and GABA-related immunofluorescence associated with central vestibular neurons of the VSR pathway in rats. Retrograde FluoroGold tract tracing was used to label vestibular neurons with projections to RVLM or CVLM, and sinusoidal galvanic vestibular stimulation (GVS) was employed to activate these pathways. Central vestibular neurons of the VSR were identified by co-localization of FluoroGold and cFos protein, which accumulates in some vestibular neurons following galvanic stimulation. Triple-label immunofluorescence was used to co-localize glutamate- or GABA- labeling in the identified VSR pathway neurons. Most activated projection neurons displayed intense glutamate immunofluorescence, suggestive of glutamatergic neurotransmission. To support this, anterograde tracer was injected into the caudal vestibular nuclei. Vestibular axons and terminals in RVLM and CVLM co-localized the anterograde tracer and vesicular glutamate transporter-2 signals. Other retrogradely-labeled cFos-positive neurons displayed intense GABA immunofluorescence. VSR pathway neurons of both phenotypes were present in the caudal medial and spinal vestibular nuclei, and projected to both RVLM and CVLM. As a group, however, triple-labeled vestibular cells with intense glutamate immunofluorescence were located more rostrally in the vestibular nuclei than the GABAergic neurons. Only the GABAergic VSR pathway neurons showed a target preference, projecting predominantly to CVLM. These data provide the first

  13. Glutamate neurons within the midbrain dopamine regions.

    PubMed

    Morales, M; Root, D H

    2014-12-12

    Midbrain dopamine systems play important roles in Parkinson's disease, schizophrenia, addiction, and depression. The participation of midbrain dopamine systems in diverse clinical contexts suggests these systems are highly complex. Midbrain dopamine regions contain at least three neuronal phenotypes: dopaminergic, GABAergic, and glutamatergic. Here, we review the locations, subtypes, and functions of glutamatergic neurons within midbrain dopamine regions. Vesicular glutamate transporter 2 (VGluT2) mRNA-expressing neurons are observed within each midbrain dopamine system. Within rat retrorubral field (RRF), large populations of VGluT2 neurons are observed throughout its anteroposterior extent. Within rat substantia nigra pars compacta (SNC), VGluT2 neurons are observed centrally and caudally, and are most dense within the laterodorsal subdivision. RRF and SNC rat VGluT2 neurons lack tyrosine hydroxylase (TH), making them an entirely distinct population of neurons from dopaminergic neurons. The rat ventral tegmental area (VTA) contains the most heterogeneous populations of VGluT2 neurons. VGluT2 neurons are found in each VTA subnucleus but are most dense within the anterior midline subnuclei. Some subpopulations of rat VGluT2 neurons co-express TH or glutamic acid decarboxylase (GAD), but most of the VGluT2 neurons lack TH or GAD. Different subsets of rat VGluT2-TH neurons exist based on the presence or absence of vesicular monoamine transporter 2, dopamine transporter, or D2 dopamine receptor. Thus, the capacity by which VGluT2-TH neurons may release dopamine will differ based on their capacity to accumulate vesicular dopamine, uptake extracellular dopamine, or be autoregulated by dopamine. Rat VTA VGluT2 neurons exhibit intrinsic VTA projections and extrinsic projections to the accumbens and to the prefrontal cortex. Mouse VTA VGluT2 neurons project to accumbens shell, prefrontal cortex, ventral pallidum, amygdala, and lateral habenula. Given their molecular

  14. Calcium influx via ionotropic glutamate receptors causes long lasting inhibition of metabotropic glutamate receptor-coupled phosphoinositide hydrolysis.

    PubMed

    Facchinetti, F; Hack, N J; Balázs, R

    1998-09-01

    Functional interaction between ionotropic and metabotropic glutamate receptors (iGluR and mGluR respectively) was studied in cerebellar granule cell cultures using quisqualate (QA), the most potent agonist of phosphoinositide hydrolysis coupled mGluR, and N-methyl-D-aspartate (NMDA) or kainate (KA) that activate different classes of iGluR. Two h exposure to NMDA or KA resulted in a marked reduction (about 75%) of QA-evoked PI hydrolysis. The efficacy of the two agonists was about the same, but the potencies were different (IC50 for NMDA about 35 microM and for KA about 70 microM). NMDA-induced depression of QA-stimulated PI hydrolysis was relatively long lasting but reversible. Recovery required protein synthesis. In nominally Ca2+-free medium both NMDA and KA failed to attenuate QA-stimulated PI hydrolysis. The effect of NMDA was prevented by the NMDA receptor antagonist MK801, but not by the wide spectrum protein kinase inhibitor staurosporin nor by the nitric oxide synthase inhibitor N omega-nitro-L-arginine. Cycloheximide and concanavalin A were also ineffective. The effect of KA was prevented by the selective non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX). Voltage sensitive Ca2+ channel antagonists together with MK801 did not counteract the inhibition by KA of the QA response. Both NMDA and KA attenuated PI hydrolysis evoked by the muscarinic receptor agonist carbachol (about 30%), indicating that the activation of iGluRs exerts a relatively general inhibitory effect on the function of different PLC-coupled metabotropic receptors. Consistent with this observation is that treatments either with KA and NMDA induced an inhibition (about 30%) of NaF-stimulated PI hydrolysis which occurs through the direct activation of G proteins. Our observations show that ionotropic glutamate receptor stimulation induces a long lasting suppression of QA-evoked PI breakdown through a Ca2+ dependent mechanism which seems to involve

  15. Dentate gyrus–CA3 glutamate release/NMDA transmission mediates behavioral despair and antidepressant-like responses to leptin

    PubMed Central

    Wang, Xuezhen; Zhang, Di; Lu, Xin-Yun

    2014-01-01

    Compelling evidence supports the important role of the glutamatergic system in the pathophysiology of major depression and also as a target for rapid-acting antidepressants. However, the functional role of glutamate release/transmission in behavioral processes related to depression and antidepressant efficacy remains to be elucidated. In this study, glutamate release and behavioral responses to tail suspension, a procedure commonly used for inducing behavioral despair, were simultaneously monitored in real time. The onset of tail suspension stress evoked a rapid increase in glutamate release in hippocampal field CA3, which declined gradually after its offset. Blockade of NMDA receptors by intra-CA3 infusion of MK-801, a non-competitive NMDA receptor antagonist, reversed behavioral despair. The CA3 was innervated by granule neurons expressing the leptin receptor (LepRb) in the dentate gyrus (DG), representing a subpopulation of granule neurons that were devoid of stress-induced activation. Leptin treatment dampened tail suspension-evoked glutamate release in CA3. On the other hand, intra-CA3 infusion of NMDA blocked the antidepressant-like effect of leptin in reversing behavioral despair in both the tail suspension and forced swim tests, which involved activation of Akt signaling in DG. Together, these results suggest that the DG-CA3 glutamatergic pathway is critical for mediating behavioral despair and antidepressant-like responses to leptin. PMID:25092243

  16. VGLUTs and Glutamate Synthesis—Focus on DRG Neurons and Pain

    PubMed Central

    Malet, Mariana; Brumovsky, Pablo R.

    2015-01-01

    The amino acid glutamate is the principal excitatory transmitter in the nervous system, including in sensory neurons that convey pain sensation from the periphery to the brain. It is now well established that a family of membrane proteins, termed vesicular glutamate transporters (VGLUTs), serve a critical function in these neurons: they incorporate glutamate into synaptic vesicles. VGLUTs have a central role both under normal neurotransmission and pathological conditions, such as neuropathic or inflammatory pain. In the present short review, we will address VGLUTs in the context of primary afferent neurons. We will focus on the role of VGLUTs in pain triggered by noxious stimuli, peripheral nerve injury, and tissue inflammation, as mostly explored in transgenic mice. The possible interplay between glutamate biosynthesis and VGLUT-dependent packaging in synaptic vesicles, and its potential impact in various pain states will be presented. PMID:26633536

  17. VGLUTs and Glutamate Synthesis-Focus on DRG Neurons and Pain.

    PubMed

    Malet, Mariana; Brumovsky, Pablo R

    2015-12-02

    The amino acid glutamate is the principal excitatory transmitter in the nervous system, including in sensory neurons that convey pain sensation from the periphery to the brain. It is now well established that a family of membrane proteins, termed vesicular glutamate transporters (VGLUTs), serve a critical function in these neurons: they incorporate glutamate into synaptic vesicles. VGLUTs have a central role both under normal neurotransmission and pathological conditions, such as neuropathic or inflammatory pain. In the present short review, we will address VGLUTs in the context of primary afferent neurons. We will focus on the role of VGLUTs in pain triggered by noxious stimuli, peripheral nerve injury, and tissue inflammation, as mostly explored in transgenic mice. The possible interplay between glutamate biosynthesis and VGLUT-dependent packaging in synaptic vesicles, and its potential impact in various pain states will be presented.

  18. Reduced plasma membrane surface expression of GLAST mediates decreased glutamate regulation in the aged striatum.

    PubMed

    Nickell, Justin; Salvatore, Michael F; Pomerleau, Francois; Apparsundaram, Subbu; Gerhardt, Greg A

    2007-11-01

    Extracellular L-glutamate poses a severe excitotoxic threat to neurons and glia when unregulated, therefore low synaptic levels of this neurotransmitter must be maintained via a rapid and robust transport system. A recent study from our laboratory showed a reduced glutamate uptake rate in the striatum of the aged Fischer 344 (F344) rat, yet the mechanism underlying this phenomenon is unknown. The current study utilized in vivo electrochemical recordings, immunoblotting and biotinylation in young (6 months), late-middle aged (18 months) and aged (24 months) F344 rats to elucidate the potential role that glutamate transporters (GLT-1, GLAST, and EAAC1) may play in this mechanism. Here we show that the time necessary to clear glutamate from the late-middle aged and aged striatum is significantly prolonged in comparison to the young striatum. In addition, an analysis of various sub-regions of the striatum revealed a marked dorsoventral gradient in terms of glutamate clearance times in the aged striatum, a phenomenon which was not present in the striatum of the animals of the remaining age groups. We also found that the decreased glutamate clearance time observed in the late-middle aged and aged rats is not due to a decrease in the production of total transporter protein among these three transporters. Rather, a significant reduction in the amount of GLAST expressed on the plasma membrane surface in the aged animals (approximately 55% when compared to young rats) may contribute to this phenomenon. These age-related alterations in extracellular l-glutamate regulation may be key contributors to the increased susceptibility of the aged brain to excitotoxic insults such as stroke and hypoxia.

  19. The glutamate homeostasis hypothesis of addiction.

    PubMed

    Kalivas, Peter W

    2009-08-01

    Addiction is associated with neuroplasticity in the corticostriatal brain circuitry that is important for guiding adaptive behaviour. The hierarchy of corticostriatal information processing that normally permits the prefrontal cortex to regulate reinforcement-seeking behaviours is impaired by chronic drug use. A failure of the prefrontal cortex to control drug-seeking behaviours can be linked to an enduring imbalance between synaptic and non-synaptic glutamate, termed glutamate homeostasis. The imbalance in glutamate homeostasis engenders changes in neuroplasticity that impair communication between the prefrontal cortex and the nucleus accumbens. Some of these pathological changes are amenable to new glutamate- and neuroplasticity-based pharmacotherapies for treating addiction. PMID:19571793

  20. Transportation.

    ERIC Educational Resources Information Center

    Crank, Ron

    This instructional unit is one of 10 developed by students on various energy-related areas that deals specifically with transportation and energy use. Its objective is for the student to be able to discuss the implication of energy usage as it applies to the area of transportation. Some topics covered are efficiencies of various transportation…

  1. Metabotropic glutamate receptors as targets of neuromodulatory influence of nitric oxide.

    PubMed

    Ryzhova, I V; Nozdrachev, A D; Tobias, T V; Orlov, I V; Chikhman, V N; Solnushkin, S D

    2016-07-01

    A possible effect of nitric oxide (NO) on metabotropic glutamate receptor (mGluR) function in the amino acid afferent synapse was investigated in the isolated labyrinth of the frog Rana temporaria. The modification of the amplitude of responses of metabotropic glutamate receptor agonist trans-ACPD was analyzed during bath applied NO donor S-nitroso-N-acetyl-DL-penicillamine SNAP (0.1-100 μM) or nitric oxide synthase inhibitor L-NAME. It was shown that NO donor SNAP (1 μM) inhibited mGluR induced responses, and the inhibitor of NO-synthase L-NAME (100 μM) increased the amplitude of trans-ACPD evoked answers. The results suggest that NO can depress mGluR function due to modulation of functions of the endoplasmic reticulum channels. PMID:27595818

  2. Resting GABA and glutamate concentrations do not predict visual gamma frequency or amplitude

    PubMed Central

    Cousijn, Helena; Haegens, Saskia; Wallis, George; Near, Jamie; Stokes, Mark G.; Harrison, Paul J.; Nobre, Anna C.

    2014-01-01

    Gamma band oscillations arise in neuronal networks of interconnected GABAergic interneurons and excitatory pyramidal cells. A previous study found a correlation between visual gamma peak frequency, as measured with magnetoencephalography, and resting GABA levels, as measured with magnetic resonance spectroscopy (MRS), in 12 healthy volunteers. If true, this would allow studies in clinical populations testing modulation of this relationship, but this finding has not been replicated. We addressed this important question by measuring gamma oscillations and GABA, as well as glutamate, in 50 healthy volunteers. Visual gamma activity was evoked using an established gratings paradigm, and we applied a beamformer spatial filtering technique to extract source-reconstructed gamma peak frequency and amplitude from the occipital lobe. We determined gamma peak frequency and amplitude from the location with maximal activation and from the location of the MRS voxel to assess the relationship of GABA with gamma. Gamma peak frequency was estimated from the highest value of the raw spectra and by a Gaussian fit to the spectra. MRS data were acquired from occipital cortex. We did not replicate the previously found correlation between gamma peak frequency and GABA concentration. Calculation of a Bayes factor provided strong evidence in favor of the null hypothesis. We also did not find a correlation between gamma activity and glutamate or between gamma and the ratio of GABA/glutamate. Our results suggest that cortical gamma oscillations do not have a consistent, demonstrable relationship to excitatory/inhibitory network activity as proxied by MRS measurements of GABA and glutamate. PMID:24927588

  3. Resting GABA and glutamate concentrations do not predict visual gamma frequency or amplitude.

    PubMed

    Cousijn, Helena; Haegens, Saskia; Wallis, George; Near, Jamie; Stokes, Mark G; Harrison, Paul J; Nobre, Anna C

    2014-06-24

    Gamma band oscillations arise in neuronal networks of interconnected GABAergic interneurons and excitatory pyramidal cells. A previous study found a correlation between visual gamma peak frequency, as measured with magnetoencephalography, and resting GABA levels, as measured with magnetic resonance spectroscopy (MRS), in 12 healthy volunteers. If true, this would allow studies in clinical populations testing modulation of this relationship, but this finding has not been replicated. We addressed this important question by measuring gamma oscillations and GABA, as well as glutamate, in 50 healthy volunteers. Visual gamma activity was evoked using an established gratings paradigm, and we applied a beamformer spatial filtering technique to extract source-reconstructed gamma peak frequency and amplitude from the occipital lobe. We determined gamma peak frequency and amplitude from the location with maximal activation and from the location of the MRS voxel to assess the relationship of GABA with gamma. Gamma peak frequency was estimated from the highest value of the raw spectra and by a Gaussian fit to the spectra. MRS data were acquired from occipital cortex. We did not replicate the previously found correlation between gamma peak frequency and GABA concentration. Calculation of a Bayes factor provided strong evidence in favor of the null hypothesis. We also did not find a correlation between gamma activity and glutamate or between gamma and the ratio of GABA/glutamate. Our results suggest that cortical gamma oscillations do not have a consistent, demonstrable relationship to excitatory/inhibitory network activity as proxied by MRS measurements of GABA and glutamate. PMID:24927588

  4. Scavenging of blood glutamate for enhancing brain-to-blood glutamate efflux.

    PubMed

    Li, Yunhong; Hou, Xiaolin; Qi, Qi; Wang, Le; Luo, Lan; Yang, Shaoqi; Zhang, Yumei; Miao, Zhenhua; Zhang, Yanli; Wang, Fei; Wang, Hongyan; Huang, Weidong; Wang, Zhenhai; Shen, Ying; Wang, Yin

    2014-01-01

    The presence of excess glutamate in the brain interstitial fluid characterizes several acute pathological conditions of the brain, including traumatic brain injury and stroke. It has been demonstrated that it is possible to eliminate excess glutamate in the brain by decreasing blood glutamate levels and, accordingly, accelerating the brain-to-blood glutamate efflux. It is feasible to accomplish this process by activating blood resident enzymes in the presence of the respective glutamate cosubstrates. In the present study, several glutamate cosubstrates and cofactors were studied in an attempt to identify the optimal conditions to reduce blood glutamate levels. The administration of a mixture of 1 mM pyruvate and oxaloacetate (Pyr/Oxa) for 1 h decreased blood glutamate levels by ≤50%. The addition of lipoamide to this mixture resulted in a further reduction in blood glutamate levels of >80%. In addition, in vivo experiments showed that lipoamide together with Pyr/Oxa is able to decrease blood glutamate levels to a greater extent than Pyr/Oxa alone, and accordingly, this enhances the glutamate efflux from the brain to the blood. These results may outline a novel neuroprotective strategy with increased effectiveness for the removal of excess brain glutamate in various neurodegenerative conditions.

  5. Decreased glial and synaptic glutamate uptake in the striatum of HIV-1 gp120 transgenic mice.

    PubMed

    Melendez, Roberto I; Roman, Cristina; Capo-Velez, Coral M; Lasalde-Dominicci, Jose A

    2016-06-01

    The mechanisms leading to the neurocognitive deficits in humans with immunodeficiency virus type 1 (HIV-1) are not well resolved. A number of cell culture models have demonstrated that the HIV-envelope glycoprotein 120 (gp120) decreases the reuptake of glutamate, which is necessary for learning, memory, and synaptic plasticity. However, the impact of brain HIV-1 gp120 on glutamate uptake systems in vivo remains unknown. Notably, alterations in brain glutamate uptake systems are implicated in a number of neurodegenerative and neurocognitive disorders. We characterized the kinetic properties of system XAG (sodium-dependent) and systems xc- (sodium-independent) [3H]-L-glutamate uptake in the striatum and hippocampus of HIV-1 gp120 transgenic mice, an established model of HIV neuropathology. We determined the kinetic constant Vmax (maximal velocity) and Km (affinity) of both systems XAG and xc- using subcellular preparations derived from neurons and glial cells. We show significant (30-35 %) reductions in the Vmax of systems XAG and xc- in both neuronal and glial preparations derived from the striatum, but not from the hippocampus of gp120 mice relative to wild-type (WT) controls. Moreover, immunoblot analysis showed that the protein expression of glutamate transporter subtype-1 (GLT-1), the predominant brain glutamate transporter, was significantly reduced in the striatum but not in the hippocampus of gp120 mice. These extensive and region-specific deficits of glutamate uptake likely contribute to the development and/or severity of HIV-associated neurocognitive disorders. Understanding the role of striatal glutamate uptake systems in HIV-1 gp120 may advance the development of new therapeutic strategies to prevent neuronal damage and improve cognitive function in HIV patients. PMID:26567011

  6. Glutamate-glutamine cycle and exchange in the placenta-fetus unit during late pregnancy.

    PubMed

    Wu, Xin; Xie, Chunyan; Zhang, Yuzhe; Fan, Zhiyong; Yin, Yulong; Blachier, Francois

    2015-01-01

    The present review focuses on the physiological functions of glutamate-glutamine exchange involving placental amino acid transport and umbilical amino acid uptake in mammals (particularly in sows), with special emphasis on the associated regulating mechanisms. Glutamate plus glutamine are among the most abundant and the most utilized amino acids in fetus during late gestation. During pregnancy, amino acids, notably as precursors of macromolecules including proteins and nucleotides are involved in fetal development and growth. Amino acid concentrations in fetus are generally higher than in the mother. Among amino acids, the transport and metabolism of glutamate and glutamine during fetal development exhibit characteristics that clearly emphasize the importance of the interaction between the placenta and the fetal liver. Glutamate is quite remarkable among amino acids, which originate from the placenta, and is cleared from fetal plasma. In addition, the flux of glutamate through the placenta from the fetal plasma is highly correlated with the umbilical glutamate delivery rate. Glutamine plays a central role in fetal carbon and nitrogen metabolism and exhibits one of the highest fetal/maternal plasma ratio among all amino acids in human and other mammals. Glutamate is taken up by placenta from the fetal circulation and then converted to glutamine before being released back into the fetal circulation. Works are required on the glutamate-glutamine metabolism during late pregnancy in physiological and pathophysiological situations since such works may help to improve fetal growth and development both in humans and other mammals. Indeed, glutamine supplementation appears to ameliorate fetal growth retardation in sows and reduces preweaning mortality of piglets.

  7. Simulation of Postsynaptic Glutamate Receptors Reveals Critical Features of Glutamatergic Transmission

    PubMed Central

    Greget, Renaud; Pernot, Fabien; Bouteiller, Jean-Marie C.; Ghaderi, Viviane; Allam, Sushmita; Keller, Anne Florence; Ambert, Nicolas; Legendre, Arnaud; Sarmis, Merdan; Haeberle, Olivier; Faupel, Michel; Bischoff, Serge; Berger, Theodore W.; Baudry, Michel

    2011-01-01

    Activation of several subtypes of glutamate receptors contributes to changes in postsynaptic calcium concentration at hippocampal synapses, resulting in various types of changes in synaptic strength. Thus, while activation of NMDA receptors has been shown to be critical for long-term potentiation (LTP) and long term depression (LTD) of synaptic transmission, activation of metabotropic glutamate receptors (mGluRs) has been linked to either LTP or LTD. While it is generally admitted that dynamic changes in postsynaptic calcium concentration represent the critical elements to determine the direction and amplitude of the changes in synaptic strength, it has been difficult to quantitatively estimate the relative contribution of the different types of glutamate receptors to these changes under different experimental conditions. Here we present a detailed model of a postsynaptic glutamatergic synapse that incorporates ionotropic and mGluR type I receptors, and we use this model to determine the role of the different receptors to the dynamics of postsynaptic calcium with different patterns of presynaptic activation. Our modeling framework includes glutamate vesicular release and diffusion in the cleft and a glutamate transporter that modulates extracellular glutamate concentration. Our results indicate that the contribution of mGluRs to changes in postsynaptic calcium concentration is minimal under basal stimulation conditions and becomes apparent only at high frequency of stimulation. Furthermore, the location of mGluRs in the postsynaptic membrane is also a critical factor, as activation of distant receptors contributes significantly less to calcium dynamics than more centrally located ones. These results confirm the important role of glutamate transporters and of the localization of mGluRs in postsynaptic sites in their signaling properties, and further strengthen the notion that mGluR activation significantly contributes to postsynaptic calcium dynamics only following

  8. Hypothyroidism Affects Olfactory Evoked Potentials

    PubMed Central

    Świdziński, Teodor; Czerniejewska-Wolska, Hanna; Wiskirska-Woźnica, Bożena; Owecki, Maciej; Głowacka, Maria Danuta; Frankowska, Anna; Łącka, Katarzyna; Glapiński, Mariusz; Maciejewska-Szaniec, Zofia; Świdziński, Piotr

    2016-01-01

    Background. Objective electrophysiological methods for investigations of the organ of smell consist in recordings of olfactory cortex responses to specific, time restricted odor stimuli. In hypothyroidism have impaired sense of smell. Material and Methods. Two groups: control of 31 healthy subjects and study group of 21 with hypothyroidism. The inclusion criterion for the study group was the TSH range from 3.54 to 110 μIU/mL. Aim. Assessment of the latency time of evoked responses from the olfactory nerve N1 and the trigeminal nerve N5 using two smells of mint and anise in hypothyroidism. Results. The smell perception in subjective olfactory tests was normal in 85% of the hypothyroid group. Differences were noticed in the objective tests. The detailed intergroup analysis of latency times of recorded cortical responses PN5 and PN1 performed by means between the groups of patients with overt clinical hypothyroidism versus subclinical hypothyroidism demonstrated a significant difference (p < 0.05) whereas no such differences were found between the control group versus subclinical hypothyroidism group (p > 0.05). Conclusion. We can conclude that registration of cortex potentials at irritation of olfactory and trigeminal nerves offers possibilities for using this method as an objective indicator of hypothyroidism severity and prognostic process factor. PMID:27656655

  9. [Intraoperative Visual Evoked Potential Monitoring].

    PubMed

    Hayashi, Hironobu; Kawaguchi, Masahiko

    2015-05-01

    Visual evoked potential (VEP) is recorded from the back of the head, which is elicited by retinal stimulation transmitted through optic nerve, optic chiasm, optic tract lateral geniculate body, optic radiation and finally cortical visual area. VEP monitoring did not prevail since 1990s because marked intra-individual difference and instability of VEP recording limited the clinical usefulness under inhalation anesthetic management and techniques of VEP monitoring at the time. However, recent advances in techniques including a new light-stimulating device consisting of high-luminosity LEDs and induction of electroretinography to ascertain the arrival of the stimulus at the retina provided better conditions for stable VEP recording under general anesthesia. In addition, the introduction of total intravenous anesthesia using propofol is important for the successful VEP recordings because inhaled anesthetics have suppressive effect on VEP waveform. Intraoperative VEP has been considered to monitor the functional integrity of visual function during neurosurgical procedures, in which the optic pathway is at a risk of injury. Intraoperative VEP monitoring may allow us to detect reversible damage to the visual pathway intraoperatively and enable us to prevent permanent impairment.

  10. Hypothyroidism Affects Olfactory Evoked Potentials

    PubMed Central

    Świdziński, Teodor; Czerniejewska-Wolska, Hanna; Wiskirska-Woźnica, Bożena; Owecki, Maciej; Głowacka, Maria Danuta; Frankowska, Anna; Łącka, Katarzyna; Glapiński, Mariusz; Maciejewska-Szaniec, Zofia; Świdziński, Piotr

    2016-01-01

    Background. Objective electrophysiological methods for investigations of the organ of smell consist in recordings of olfactory cortex responses to specific, time restricted odor stimuli. In hypothyroidism have impaired sense of smell. Material and Methods. Two groups: control of 31 healthy subjects and study group of 21 with hypothyroidism. The inclusion criterion for the study group was the TSH range from 3.54 to 110 μIU/mL. Aim. Assessment of the latency time of evoked responses from the olfactory nerve N1 and the trigeminal nerve N5 using two smells of mint and anise in hypothyroidism. Results. The smell perception in subjective olfactory tests was normal in 85% of the hypothyroid group. Differences were noticed in the objective tests. The detailed intergroup analysis of latency times of recorded cortical responses PN5 and PN1 performed by means between the groups of patients with overt clinical hypothyroidism versus subclinical hypothyroidism demonstrated a significant difference (p < 0.05) whereas no such differences were found between the control group versus subclinical hypothyroidism group (p > 0.05). Conclusion. We can conclude that registration of cortex potentials at irritation of olfactory and trigeminal nerves offers possibilities for using this method as an objective indicator of hypothyroidism severity and prognostic process factor.

  11. Regulation of brain glutamate metabolism by nitric oxide and S-nitrosylation

    PubMed Central

    Raju, Karthik; Doulias, Paschalis-Thomas; Evans, Perry; Krizman, Elizabeth N.; Jackson, Joshua G.; Horyn, Oksana; Daikhin, Yevgeny; Nissim, Ilana; Yudkoff, Marc; Nissim, Itzhak; Sharp, Kim A.; Robinson, Michael B.; Ischiropoulos, Harry

    2016-01-01

    Nitric oxide (NO) is a signaling intermediate during glutamatergic neurotransmission in the central nervous system (CNS). NO signaling is in part accomplished through cysteine S-nitrosylation, a posttranslational modification by which NO regulates protein function and signaling. In our investigation of the protein targets and functional impact of S-nitrosylation in the CNS under physiological conditions, we identified 269 S-nitrosocysteine residues in 136 proteins in the wild-type mouse brain. The number of sites was significantly reduced in the brains of mice lacking endothelial nitric oxide synthase (eNOS−/−) or neuronal nitric oxide synthase (nNOS−/−). In particular, nNOS−/− animals showed decreased S-nitrosylation of proteins that participate in the glutamate/glutamine cycle, a metabolic process by which synaptic glutamate is recycled or oxidized to provide energy. 15N-glutamine–based metabolomic profiling and enzymatic activity assays indicated that brain extracts from nNOS−/− mice converted less glutamate to glutamine and oxidized more glutamate than those from mice of the other genotypes. GLT1 [also known as EAAT2 (excitatory amino acid transporter 2)], a glutamate transporter in astrocytes, was S-nitrosylated at Cys373 and Cys561 in wild-type and eNOS−/− mice, but not in nNOS−/− mice. A form of rat GLT1 that could not be S-nitrosylated at the equivalent sites had increased glutamate uptake compared to wild-type GLT1 in cells exposed to an S-nitrosylating agent. Thus, NO modulates glutamatergic neurotransmission through the selective, nNOS-dependent S-nitrosylation of proteins that govern glutamate transport and metabolism. PMID:26152695

  12. Behavioral History of Withdrawal Influences Regulation of Cocaine Seeking by Glutamate Re-Uptake

    PubMed Central

    Zhou, Luyi; Andersen, Haley; Arreola, Adrian C.; Turner, Jill R.; Ortinski, Pavel I.

    2016-01-01

    Withdrawal from cocaine regulates expression of distinct glutamate re-uptake transporters in the nucleus accumbens (NAc). In this study, we examined the cumulative effect of glutamate re-uptake by multiple excitatory amino acid transporters (EAATs) on drug-seeking at two different stages of withdrawal from self-administered cocaine. Rats were trained on fixed ratio 1 (FR1), progressing to FR5 schedule of reinforcement. After one day of withdrawal, microinfusion of a broad non-transportable EAAT antagonist, DL-threo-beta-benzyloxyaspartate (DL-TBOA), into the NAc shell dose-dependently attenuated self-administration of cocaine. Sucrose self-administration was not affected by DL-TBOA, indicating an effect specific to reinforcing properties of cocaine. The attenuating effect on cocaine seeking was not due to suppression of locomotor response, as DL-TBOA was found to transiently increase spontaneous locomotor activity. Previous studies have established a role for EAAT2-mediated re-uptake on reinstatement of cocaine seeking following extended withdrawal and extinction training. We found that blockade of NAc shell EAATs did not affect cocaine-primed reinstatement of cocaine seeking. These results indicate that behavioral history of withdrawal influences the effect of re-uptake mediated glutamate clearance on cocaine seeking. Dynamic regulation of glutamate availability by re-uptake mechanisms may impact other glutamate signaling pathways to account for such differences. PMID:27685834

  13. Glutamate: Tastant and Neuromodulator in Taste Buds.

    PubMed

    Vandenbeuch, Aurelie; Kinnamon, Sue C

    2016-07-01

    In taste buds, glutamate plays a double role as a gustatory stimulus and neuromodulator. The detection of glutamate as a tastant involves several G protein-coupled receptors, including the heterodimer taste receptor type 1, member 1 and 3 as well as metabotropic glutamate receptors (mGluR1 and mGluR4). Both receptor types participate in the detection of glutamate as shown with knockout animals and selective antagonists. At the basal part of taste buds, ionotropic glutamate receptors [N-methyl-d-aspartate (NMDA) and non-NMDA] are expressed and participate in the modulation of the taste signal before its transmission to the brain. Evidence suggests that glutamate has an efferent function on taste cells and modulates the release of other neurotransmitters such as serotonin and ATP. This short article reviews the recent developments in the field with regard to glutamate receptors involved in both functions as well as the influence of glutamate on the taste signal. PMID:27422519

  14. Auditory evoked potentials and transcendental meditation.

    PubMed

    Barwood, T J; Empson, J A; Lister, S G; Tilley, A J

    1978-11-01

    Auditory evoked potentials to tone stimuli were recorded from 8 practised meditators before, during, and after meditation, and also during light sleep. No consistent changes were noted between baseline and meditating AEPs, or between meditating and sleep AEPs.

  15. Effects of inhibiting glutamine synthetase and blocking glutamate uptake on b-wave generation in the isolated rat retina

    PubMed Central

    WINKLER, BARRY S.; KAPOUSTA-BRUNEAU, NATALIA; ARNOLD, MATTHEW J.; GREEN, DANIEL G.

    2006-01-01

    The purpose of the present experiments was to evaluate the contribution of the glutamate-glutamine cycle in retinal glial (Müller) cells to photoreceptor cell synaptic transmission. Dark-adapted isolated rat retinas were superfused with oxygenated bicarbonate-buffered media. Recordings were made of the b-wave of the electroretinogram as a measure of light-induced photoreceptor to ON-bipolar neuron transmission. L-methionine sulfoximine (1–10 mM) was added to superfusion media to inhibit glutamine synthetase, a Müller cell specific enzyme, by more than 99% within 5–10 min, thereby disrupting the conversion of glutamate to glutamine in the Müller cells. Threo-hydroxyaspartic acid and D-aspartate were used to block glutamate transporters. The amplitude of the b-wave was well maintained for 1–2 h provided 0.25 mM glutamate or 0.25 mM glutamine was included in the media. Without exogenous glutamate or glutamine the amplitude of the b-wave declined by about 70% within 1 h. Inhibition of glutamate transporters led to a rapid (2–5 min) reversible loss of the b-wave in the presence and absence of the amino acids. In contrast, inhibition of glutamine synthetase did not alter significantly either the amplitude of the b-wave in the presence of glutamate or glutamine or the rate of decline of the b-wave found in the absence of these amino acids. Excellent recovery of the b-wave was found when 0.25 mM glutamate was resupplied to L-methionine sulfoximine–treated retinas. The results suggest that in the isolated rat retina uptake of released glutamate into photoreceptors plays a more important role in transmitter recycling than does uptake of glutamate into Müller cells and its subsequent conversion to glutamine. PMID:10367968

  16. The somatosensory evoked magnetic fields.

    PubMed

    Kakigi, R; Hoshiyama, M; Shimojo, M; Naka, D; Yamasaki, H; Watanabe, S; Xiang, J; Maeda, K; Lam, K; Itomi, K; Nakamura, A

    2000-08-01

    Averaged magnetoencephalography (MEG) following somatosensory stimulation, somatosensory evoked magnetic field(s) (SEF), in humans are reviewed. The equivalent current dipole(s) (ECD) of the primary and the following middle-latency components of SEF following electrical stimulation within 80-100 ms are estimated in area 3b of the primary somatosensory cortex (SI), the posterior bank of the central sulcus, in the hemisphere contralateral to the stimulated site. Their sites are generally compatible with the homunculus which was reported by Penfield using direct cortical stimulation during surgery. SEF to passive finger movement is generated in area 3a or 2 of SI, unlike with electrical stimulation. Long-latency components with peaks of approximately 80-120 ms are recorded in the bilateral hemispheres and their ECD are estimated in the secondary somatosensory cortex (SII) in the bilateral hemispheres. We also summarized (1) the gating effects on SEF by interference tactile stimulation or movement applied to the stimulus site, (2) clinical applications of SEF in the fields of neurosurgery and neurology and (3) cortical plasticity (reorganization) of the SI. SEF specific to painful stimulation is also recorded following painful stimulation by CO(2) laser beam. Pain-specific components are recorded over 150 ms after the stimulus and their ECD are estimated in the bilateral SII and the limbic system. We introduced a newly-developed multi (12)-channel gradiometer system with the smallest and highest quality superconducting quantum interference device (micro-SQUID) available to non-invasively detect the magnetic fields of a human peripheral nerve. Clear nerve action fields (NAFs) were consistently recorded from all subjects.

  17. Ethanol directly depresses AMPA and NMDA glutamate currents in spinal cord motor neurons independent of actions on GABAA or glycine receptors.

    PubMed

    Wang, M Y; Rampil, I J; Kendig, J J

    1999-07-01

    Ethanol is a general anesthetic agent as defined by abolition of movement in response to noxious stimulation. This anesthetic endpoint is due to spinal anesthetic actions. This study was designed to test the hypothesis that ethanol acts directly on motor neurons to inhibit excitatory synaptic transmission at glutamate receptors. Whole cell recordings were made in visually identified motor neurons in spinal cord slices from 14- to 23-day-old rats. Currents were evoked by stimulating a dorsal root fragment or by brief pulses of glutamate. Ethanol at general anesthetic concentrations (50-200 mM) depressed both responses. Ethanol also depressed glutamate-evoked responses in the presence of tetrodotoxin (300 nM), showing that its actions are postsynaptic. Block of inhibitory gamma-aminobutyric acidA and glycine receptors by bicuculline (50 microM) and strychnine (5 microM), respectively, did not significantly reduce the effects of ethanol on glutamate currents. Ethanol also depressed glutamate-evoked currents when the inhibitory receptors were blocked and either D, L-2-amino-5-phosphonopentanoic acid (40 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (10 microM) were applied to block N-methyl-D-aspartate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors, respectively. The results show that ethanol exerts direct depressant effects on both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate glutamate currents in motor neurons. Enhancement of gamma-aminobutyric acidA and glycine inhibition is not required for this effect. Direct depression of glutamatergic excitatory transmission by a postsynaptic action on motor neurons thus may contribute to general anesthesia as defined by immobility in response to a noxious stimulus. PMID:10381800

  18. Growth differentiation factor-15 promotes glutamate release in medial prefrontal cortex of mice through upregulation of T-type calcium channels

    PubMed Central

    Liu, Dong-Dong; Lu, Jun-Mei; Zhao, Qian-Ru; Hu, Changlong; Mei, Yan-Ai

    2016-01-01

    Growth differentiation factor-15 (GDF-15) has been implicated in ischemic brain injury and synapse development, but its involvement in modulating neuronal excitability and synaptic transmission remain poorly understood. In this study, we investigated the effects of GDF-15 on non-evoked miniature excitatory post-synaptic currents (mEPSCs) and neurotransmitter release in the medial prefrontal cortex (mPFC) in mice. Incubation of mPFC slices with GDF-15 for 60 min significantly increased the frequency of mEPSCs without effect on their amplitude. GDF-15 also significantly elevated presynaptic glutamate release, as shown by HPLC. These effects were blocked by dual TGF-β type I receptor (TβRI) and TGF-β type II receptor (TβRII) antagonists, but not by a TβRI antagonist alone. Meanwhile, GDF-15 enhanced pERK level, and inhibition of MAPK/ERK activity attenuated the GDF-15-induced increases in mEPSC and glutamate release. Blocking T-type calcium channels reduced the GDF-15 induced up-regulation of synaptic transmission. Membrane-protein extraction and use of an intracellular protein-transport inhibitor showed that GDF-15 promoted CaV3.1 and CaV3.3 α-subunit expression by trafficking to the membrane. These results confirm previous findings in cerebellar granule neurons, in which GDF-15 induces its neurobiological effects via TβRII and activation of the ERK pathway, providing novel insights into the mechanism of GDF-15 function in cortical neurons. PMID:27353765

  19. Effects of free fatty acids, ethanol and development on gamma-aminobutyric acid and glutamate fluxes in rat nerve endings.

    PubMed

    Hitzemann, R; Mark, C; Panini, A

    1982-12-15

    The effects of type A (cis-unsaturated) and type B (trans-unsaturated and saturated) fatty acids, 1% and 3% ethanol (v/v), and development (7 days) on the thermodynamics of glutamate and gamma-aminobutyric acid (GABA) transport into cortical rat brain nerve endings were examined. The effects of the various manipulations, which are known to affect membrane fluidity, may be summarized. Three percent ethanol and oleic acid increased delta S degrees and delta S+ for glutamate transport and decreased delta H degrees and delta H+. Type B fatty acids had the opposite effects. In comparison to glutamate transport, GABA transport was less affected by the various manipulations and showed less specificity in terms of the fatty acid effects. Similarly, the effects of development on the thermodynamic parameters for glutamate and GABA transport were not consistent. Glutamate transport into 7-day nerve endings showed thermodynamic behavior similar to that seen when type A fatty acids were incorporated into adult nerve endings. In contrast, GABA transport into 7-day nerve endings had the character of adult nerve endings into which type B fatty acids were incorporated.

  20. Genes involved in Drosophila glutamate receptor expression and localization

    PubMed Central

    Liebl, Faith LW; Featherstone, David E

    2005-01-01

    Background A clear picture of the mechanisms controlling glutamate receptor expression, localization, and stability remains elusive, possibly due to an incomplete understanding of the proteins involved. We screened transposon mutants generated by the ongoing Drosophila Gene Disruption Project in an effort to identify the different types of genes required for glutamate receptor cluster development. Results To enrich for non-silent insertions with severe disruptions in glutamate receptor clustering, we identified and focused on homozygous lethal mutants in a collection of 2185 BG and KG transposon mutants generated by the BDGP Gene Disruption Project. 202 lethal mutant lines were individually dissected to expose glutamatergic neuromuscular junctions, stained using antibodies that recognize neuronal membrane and the glutamate receptor subunit GluRIIA, and viewed using laser-scanning confocal microscopy. We identified 57 mutants with qualitative differences in GluRIIA expression and/or localization. 84% of mutants showed loss of receptors and/or clusters; 16% of mutants showed an increase in receptors. Insertion loci encode a variety of protein types, including cytoskeleton proteins and regulators, kinases, phosphatases, ubiquitin ligases, mucins, cell adhesion proteins, transporters, proteins controlling gene expression and protein translation, and proteins of unknown/novel function. Expression pattern analyses and complementation tests, however, suggest that any single mutant – even if a mutant gene is uniquely tagged – must be interpreted with caution until the mutation is validated genetically and phenotypically. Conclusion Our study identified 57 transposon mutants with qualitative differences in glutamate receptor expression and localization. Despite transposon tagging of every insertion locus, extensive validation is needed before one can have confidence in the role of any individual gene. Alternatively, one can focus on the types of genes identified, rather

  1. Glutamate metabolism of astrocytes during hyperbaric oxygen exposure and its effects on central nervous system oxygen toxicity.

    PubMed

    Chen, Yu-Liang; Li, Dan; Wang, Zhong-Zhuang; Xu, Wei-Gang; Li, Run-Ping; Zhang, Jun-Dong

    2016-01-20

    Hyperbaric oxygen (HBO) has been used widely in many underwater missions and clinical work. However, exposure to extremely high oxygen pressure may cause central nervous system oxygen toxicity (CNS-OT). The regulation of astrocyte glutamate metabolism is closely related to epilepsy. This study aimed to observe the effects of HBO exposure on glutamate metabolism in astrocytes and confirm the role of glutamate metabolism in CNS-OT. Anesthetized rats were exposed to 5 atmosphere absolute HBO for 80 min and microdialysis samples of brain interstitial fluid were continuously collected. Extracellular glutamate and glutamine concentrations were also detected. Freely moving rats were exposed to HBO of the same pressure for 20 min and glutamine synthetase (GS) activity in brain tissues was measured. Finally, we observed the effects of different doses of drugs related to glutamate metabolism on the latency of CNS-OT. Results showed that HBO exposure significantly increased glutamate content, whereas glutamine content was significantly reduced. Moreover, HBO exposure significantly reduced GS activity. Glutamate transporter-1 (GLT-1) selective antagonist ceftriaxone prolonged CNS-OT latency, whereas GLT-1 selective inhibitor dihydrokainate shortened CNS-OT latency. In summary, HBO exposure improved glutamate concentration and reduced glutamine concentration by inhibition of GS activity. GLT-1 activation also participated in the prevention of HBO-induced CNS-OT. Our research will provide a potential new target to terminate or attenuate CNS-OT. PMID:26619231

  2. Glutamate metabolism of astrocytes during hyperbaric oxygen exposure and its effects on central nervous system oxygen toxicity.

    PubMed

    Chen, Yu-Liang; Li, Dan; Wang, Zhong-Zhuang; Xu, Wei-Gang; Li, Run-Ping; Zhang, Jun-Dong

    2016-01-20

    Hyperbaric oxygen (HBO) has been used widely in many underwater missions and clinical work. However, exposure to extremely high oxygen pressure may cause central nervous system oxygen toxicity (CNS-OT). The regulation of astrocyte glutamate metabolism is closely related to epilepsy. This study aimed to observe the effects of HBO exposure on glutamate metabolism in astrocytes and confirm the role of glutamate metabolism in CNS-OT. Anesthetized rats were exposed to 5 atmosphere absolute HBO for 80 min and microdialysis samples of brain interstitial fluid were continuously collected. Extracellular glutamate and glutamine concentrations were also detected. Freely moving rats were exposed to HBO of the same pressure for 20 min and glutamine synthetase (GS) activity in brain tissues was measured. Finally, we observed the effects of different doses of drugs related to glutamate metabolism on the latency of CNS-OT. Results showed that HBO exposure significantly increased glutamate content, whereas glutamine content was significantly reduced. Moreover, HBO exposure significantly reduced GS activity. Glutamate transporter-1 (GLT-1) selective antagonist ceftriaxone prolonged CNS-OT latency, whereas GLT-1 selective inhibitor dihydrokainate shortened CNS-OT latency. In summary, HBO exposure improved glutamate concentration and reduced glutamine concentration by inhibition of GS activity. GLT-1 activation also participated in the prevention of HBO-induced CNS-OT. Our research will provide a potential new target to terminate or attenuate CNS-OT.

  3. Targeting glutamate uptake to treat alcohol use disorders

    PubMed Central

    Rao, P.S.S.; Bell, Richard L.; Engleman, Eric A.; Sari, Youssef

    2015-01-01

    Alcoholism is a serious public health concern that is characterized by the development of tolerance to alcohol's effects, increased consumption, loss of control over drinking and the development of physical dependence. This cycle is often times punctuated by periods of abstinence, craving and relapse. The development of tolerance and the expression of withdrawal effects, which manifest as dependence, have been to a great extent attributed to neuroadaptations within the mesocorticolimbic and extended amygdala systems. Alcohol affects various neurotransmitter systems in the brain including the adrenergic, cholinergic, dopaminergic, GABAergic, glutamatergic, peptidergic, and serotonergic systems. Due to the myriad of neurotransmitter and neuromodulator systems affected by alcohol, the efficacies of current pharmacotherapies targeting alcohol dependence are limited. Importantly, research findings of changes in glutamatergic neurotransmission induced by alcohol self- or experimenter-administration have resulted in a focus on therapies targeting glutamatergic receptors and normalization of glutamatergic neurotransmission. Glutamatergic receptors implicated in the effects of ethanol include the ionotropic glutamate receptors (AMPA, Kainate, and NMDA) and some metabotropic glutamate receptors. Regarding glutamatergic homeostasis, ceftriaxone, MS-153, and GPI-1046, which upregulate glutamate transporter 1 (GLT1) expression in mesocorticolimbic brain regions, reduce alcohol intake in genetic animal models of alcoholism. Given the hyperglutamatergic/hyperexcitable state of the central nervous system induced by chronic alcohol abuse and withdrawal, the evidence thus far indicates that a restoration of glutamatergic concentrations and activity within the mesocorticolimbic system and extended amygdala as well as multiple memory systems holds great promise for the treatment of alcohol dependence. PMID:25954150

  4. Huntington disease and Tourette syndrome. II. Uptake of glutamic acid and other amino acids by fibroblasts.

    PubMed

    Comings, D E; Goetz, I E; Holden, J; Holtz, J

    1981-03-01

    Injection of kainic acid, a rigid analog of the excitatory neurotransmitter glutamic acid (glu), into the neostriatum of rats produces a condition that mimics Huntington disease (HD) in at least 12 different morphological and biochemical parameters. These results suggested that one of the possible basic mechanisms in HD is a defect in the presynaptic of glial uptake of glu, resulting in chronic hyperstimulation and death of a specific set of neurons. To test this hypothesis, the uptake of glu was studied in 12 carefully matched sets of control-HD pairs and two lines of Tourette syndrome fibroblasts. Although the first six sets suggested a glutamate transport defect in HD cells, examination of 12 sets indicated that there were no significant differences between control and HD cells. The fibroblasts showed both a high and low affinity uptake of glutamic acid. Sodium dependent uptake of L-glutamate (L-glu) minus D-glutamate (D-glu) at 100, 1,000, and 10,000 Micrometers glutamate was normal in HD and Tourette syndrome cells.

  5. Maternal inflammation leads to impaired glutamate homeostasis and up-regulation of glutamate carboxypeptidase II in activated microglia in the fetal/newborn rabbit brain.

    PubMed

    Zhang, Zhi; Bassam, Bassam; Thomas, Ajit G; Williams, Monica; Liu, Jinhuan; Nance, Elizabeth; Rojas, Camilo; Slusher, Barbara S; Kannan, Sujatha

    2016-10-01

    Astrocyte dysfunction and excessive activation of glutamatergic systems have been implicated in a number of neurologic disorders, including periventricular leukomalacia (PVL) and cerebral palsy (CP). However, the role of chorioamnionitis on glutamate homeostasis in the fetal and neonatal brains is not clearly understood. We have previously shown that intrauterine endotoxin administration results in intense microglial 'activation' and increased pro-inflammatory cytokines in the periventricular region (PVR) of the neonatal rabbit brain. In this study, we assessed the effect of maternal inflammation on key components of the glutamate pathway and its relationship to astrocyte and microglial activation in the fetal and neonatal New Zealand white rabbit brain. We found that intrauterine endotoxin exposure at gestational day 28 (G28) induced acute and prolonged glutamate elevation in the PVR of fetal (G29, 1day post-injury) and postnatal day 1 (PND1, 3days post-injury) brains along with prominent morphological changes in the astrocytes (soma hypertrophy and retracted processes) in the white matter tracts. There was a significant increase in glutaminase and N-Methyl-d-Aspartate receptor (NMDAR) NR2 subunit expression along with decreased glial L-glutamate transporter 1 (GLT-1) in the PVR at G29, that would promote acute dysregulation of glutamate homeostasis. This was accompanied with significantly decreased TGF-β1 at PND1 in CP kits indicating ongoing neuroinflammation. We also show for the first time that glutamate carboxypeptidase II (GCPII) was significantly increased in the activated microglia at the periventricular white matter area in both G29 and PND1 CP kits. This was confirmed by in vitro studies demonstrating that LPS activated primary microglia markedly upregulate GCPII enzymatic activity. These results suggest that maternal intrauterine endotoxin exposure results in early onset and long-lasting dysregulation of glutamate homeostasis, which may be mediated by

  6. Evidence for the participation of glutamate in reflexes involving afferent, substance P-containing nerve fibres in the rat.

    PubMed

    Juránek, I; Lembeck, F

    1996-01-01

    almost completely blocked by SR-140333 (100 micrograms kg-1, i.v.), but no inhibition was observed with MK-801, indicating that capsaicin had brought about a release of substance P from peripheral nerve terminals. Colchicine did not influence heat dissipation induced by i.v. capsaicin. 5. When capsaicin was injected i.c., the rise in paw skin temperature in colchicine- and SR-140333-pretreated groups did not differ from that of the control group. MK-801 totally prevented the heat loss reaction to i.c. capsaicin administration. Colchicine did not change the effects of i.v. or i.c. injected capsaicin: this excludes the involvement of a mechanism dependent on axonal transport of neurotransmitters. 6. The reduction of axonal transport by colchicine reduced plasma extravasation induced by mustard oil and antidromic sciatic nerve stimulation (peripheral functions) and depressor reflexes evoked by i.a. capsaicin and colon distension (central functions). It can be argued that afferent stimulation of the sciatic nerve includes the stimulation of A-fibres, which might be less sensitive to colchicine. SR-140333 was effective only on peripherally mediated responses. 7. The recent evidence for the concomitant release of glutamate and substance P from central terminals of afferent C-fibres, known to mediate reflexes abolished after capsaicin treatment allows the following conclusions: (a) the inhibition by MK-801 indicates an essential role for glutamate in the central transmission of these reflexes; (b) tachykinin antagonists such as SR-140333 do not affect these responses when administered systemically. Centrally released substance P could be involved in functions of the CNS other than those investigated here unless the access of neurokinin antagonists to their receptors in the CNS is insufficient.

  7. Selective potentiation of (α4)3(β2)2 nicotinic acetylcholine receptors augments amplitudes of prefrontal acetylcholine- and nicotine-evoked glutamatergic transients in rats.

    PubMed

    Grupe, Morten; Paolone, Giovanna; Jensen, Anders A; Sandager-Nielsen, Karin; Sarter, Martin; Grunnet, Morten

    2013-11-15

    Prefrontal glutamate release evoked through activation of α4β2* nicotinic acetylcholine receptors (nAChRs) situated on thalamic glutamatergic afferents mediates cue detection processes and thus contributes to attentional performance. However, little is known about the respective contributions of the high sensitivity and low sensitivity (LS) stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, to these processes. In the present study we employed glutamate-sensitive microelectrodes and the (α4)3(β2)2-selective positive allosteric modulator (PAM) NS9283 to investigate the importance of the LS α4β2 nAChR for glutamate release in the rat medial prefrontal cortex (mPFC). Firstly, the signaling evoked by physiologically relevant ACh concentrations through the (α4)3(β2)2 nAChR in HEK293 cells was potentiated by NS9283, consistent with the classification of NS9283 as a PAM. In urethane-anesthetized rats, intra-prefrontal pressure ejections of NS9283 evoked glutamatergic transients. Importantly, this glutamate release was attenuated by removal of cholinergic projections to the recording area. This finding indicates that the effects of NS9283 depend on endogenous ACh, again consistent with effects of a PAM. We then conducted microdialysis to demonstrate the presence of extracellular ACh in urethane-anesthetized control rats. While detectable, those levels were significantly lower than in awake rats. Finally, the amplitudes of glutamatergic transients evoked by local pressure ejections of a low concentration of nicotine were significantly augmented following systemic administration of NS9283 (3.0mg/kg). In conclusion, our results indicate that a LS α4β2 nAChR PAM such as NS9283 may enhance the cholinergic modulation of glutamatergic neurotransmission in the cortex, thereby perhaps alleviating the attentional impairments common to a range of brain disorders.

  8. Selective potentiation of (α4)3(β2)2 nicotinic acetylcholine receptors augments amplitudes of prefrontal acetylcholine- and nicotine-evoked glutamatergic transients in rats

    PubMed Central

    Grupe, Morten; Paolone, Giovanna; Jensen, Anders A.; Sandager-Nielsen, Karin; Sarter, Martin; Grunnet, Morten

    2013-01-01

    Prefrontal glutamate release evoked through activation of α4β2* nicotinic acetylcholine receptors (nAChRs) situated on thalamic glutamatergic afferents mediates cue detection processes and thus contributes to attentional performance. However, little is known about the respective contributions of the high sensitivity and low sensitivity (LS) stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, to these processes. In the present study we employed glutamate-sensitive microelectrodes and the (α4)3(β2)2-selective positive allosteric modulator (PAM) NS9283 to investigate the importance of the LS α4β2 nAChR for glutamate release in the rat medial prefrontal cortex (mPFC). Firstly, the signaling evoked by physiologically relevant ACh concentrations through the (α4)3(β2)2 nAChR in HEK293 cells was potentiated by NS9283, consistent with the classification of NS9238 as a PAM. In urethane-anesthetized rats, intra-prefrontal pressure ejections of NS9283 evoked glutamatergic transients. Importantly, this glutamate release was attenuated by removal of cholinergic projections to the recording area. This finding indicates that the effects of NS9283 depend on endogenous ACh, again consistent with effects of a PAM. We then conducted microdialysis to demonstrate the presence of extracellular ACh in urethane-anesthetized control rats. While detectable, those levels were significantly lower than in awake rats. Finally, the amplitudes of glutamatergic transients evoked by local pressure ejections of a low concentration of nicotine were significantly augmented following systemic administration of NS9283 (3.0 mg/kg). In conclusion, our results indicate that a LS α4β2 nAChR PAMs such as NS9283 may enhance the cholinergic modulation of glutamatergic neurotransmission in the cortex, thereby perhaps alleviating the attentional impairments common to a range of brain disorders. PMID:24051136

  9. Modification of Glutamate Receptor Channels: Molecular Mechanisms and Functional Consequences

    NASA Astrophysics Data System (ADS)

    Hatt, Hanns

    Of the many possible mechanisms for modulating the efficiency of ion channels, the phosphorylation of receptor channel proteins may be the primary one. Changes in the set of molecular subunits of which the channels are composed are also important, especially for long-term regulation. In the central nervous system synaptic plasticity may be altered by modulating the ligand-activated neuronal ion channels involved in synaptic transmission; among them are channels gated directly by glutamate, the regulation of which we are only beginning to understand. This paper focuses on modulation of these channels [α-amino-3-hydroxy-5-methyl-4-isoxazoleprionic acid (AMPA), kainate, and N-methyl-d-aspartate (NMDA) types] by phosphorylation and changes in subunit composition. AMPA- and kainate-activated receptors are modulated by adenosine 3, 5-monophosphate (cAMP) dependent protein kinase A (PKA) coupled via D1 dopamine receptors. An increase in the intracellular concentration of cAMP and protein kinase A potentiates kainate-activated currents in α-motoneurons of the spinal cord by increasing the affinity of the ligand (glutamate) for the phosphorylated receptor protein (GluR6 and 7). The rapid desensitization of AMPA-evoked currents normally observed in horizontal cells of the retina is completely blocked by increasing the intracellular concentration of cAMP. The effects of changes in subunit composition were examined in rat hippocampal neurons. The subunit composition of the NMDA receptor determines the kinetic properties of synaptic currents and can be regulated by the type of innervating neuron. Similar changes also occur during development. An important determinant here is the activity of the system. Dynamic regulation of excitatory receptors by both mechanisms may well be associated with some forms of learning and memory in the mammalian brain.

  10. Convergence of dopamine and glutamate signaling onto striatal ERK activation in response to drugs of abuse

    PubMed Central

    Cahill, Emma; Salery, Marine; Vanhoutte, Peter; Caboche, Jocelyne

    2014-01-01

    Despite their distinct targets, all addictive drugs commonly abused by humans evoke increases in dopamine (DA) concentration within the striatum. The main DA Guanine nucleotide binding protein couple receptors (GPCRs) expressed by medium-sized spiny neurons of the striatum are the D1R and D2R, which are positively and negatively coupled to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling, respectively. These two DA GPCRs are largely segregated into distinct neuronal populations, where they are co-expressed with glutamate receptors in dendritic spines. Direct and indirect interactions between DA GPCRs and glutamate receptors are the molecular basis by which DA modulates glutamate transmission and controls striatal plasticity and behavior induced by drugs of abuse. A major downstream target of striatal D1R is the extracellular signal-regulated kinase (ERK) kinase pathway. ERK activation by drugs of abuse behaves as a key integrator of D1R and glutamate NMDAR signaling. Once activated, ERK can trigger chromatin remodeling and induce gene expression that permits long-term cellular alterations and drug-induced morphological and behavioral changes. Besides the classical cAMP/PKA pathway, downstream of D1R, recent evidence implicates a cAMP-independent crosstalk mechanism by which the D1R potentiates NMDAR-mediated calcium influx and ERK activation. The mounting evidence of reciprocal modulation of DA and glutamate receptors adds further intricacy to striatal synaptic signaling and is liable to prove relevant for addictive drug-induced signaling, plasticity, and behavior. Herein, we review the evidence that built our understanding of the consequences of this synergistic signaling for the actions of drugs of abuse. PMID:24409148

  11. [The effect of metabotropic glutamate receptors on longitude of posttetanic reaction in spinal motoneurons of frog].

    PubMed

    Mel'ian, Z E; Kozhanov, V M; Clamann, H P

    2001-01-01

    Effects of metabotropic glutamate receptors of the duration of posttetanic changes in monosynaptic excitatory postsynaptic potentials (mEPSP), evoked by afferent and reticulospinal input stimulation, were investigated in lumbar motoneurons of the frog isolated spinal cord. It was found that application of MAP4 (25 microM), a selective antagonist of group III of these receptors, prolonged posttetanic potentiation and depression of synaptic transmission, whereas activation of this group of metabotropic glutamate receptors by L-AP4 (1 mM), a selective agonist of these receptors, suppressed the amplitude of synaptic responses, but did not affect the dynamics of development of posttetanic changes. The NMDA receptor antagonist AP5 (50 microM), added to the perfusing solution, blocked completely the effects produced by MAP4. Neither selective antagonist MCCG (400 microM), nor agonist tACPD (50 microM) of group II metabotropic glutamate receptors affected the terms of mEPSP posttetanic potentiation and depression, although the latter, in contrast to the antagonist, in most cases increased the synaptic potential amplitude. The data obtained permit to suggest that group III metabotropic receptors may control the duration of posttetanic changes of synaptic transmission in the frog spinal motoneurons. The long-term changes in the investigated synapses seem to be mediated by activation of postsynaptic metabotropic glutamate receptors (most likely, of group I receptors), which is normally masked with activation of group III presynaptic autoreceptors. The mechanism of such an induction essentially depends on activation of NMDA type of inotropic glutamate receptors.

  12. Methylmalonate impairs mitochondrial respiration supported by NADH-linked substrates: involvement of mitochondrial glutamate metabolism.

    PubMed

    Melo, Daniela R; Mirandola, Sandra R; Assunção, Nilson A; Castilho, Roger F

    2012-06-01

    The neurodegeneration that occurs in methylmalonic acidemia is proposed to be associated with impairment of mitochondrial oxidative metabolism resulting from methylmalonate (MMA) accumulation. The present study evaluated the effects of MMA on oxygen consumption by isolated rat brain mitochondria in the presence of NADH-linked substrates (α-ketoglutarate, citrate, isocitrate, glutamate, malate, and pyruvate). Respiration supported either by glutamate or glutamate plus malate was significantly inhibited by MMA (1-10 mM), whereas no inhibition was observed when a cocktail of NADH-linked substrates was used. Measurements of glutamate transport revealed that the inhibitory effect of MMA on respiration maintained by this substrate is not due to inhibition of its mitochondrial uptake. In light of this result, the effect of MMA on the activity of relevant enzymes involved in mitochondrial glutamate metabolism was investigated. MMA had minor inhibitory effects on glutamate dehydrogenase and aspartate aminotransferase, whereas α-ketoglutarate dehydrogenase was significantly inhibited by this metabolite (K(i) = 3.65 mM). Moreover, measurements of α-ketoglutarate transport and mitochondrial MMA accumulation indicated that MMA/α-ketoglutarate exchange depletes mitochondria from this substrate, which may further contribute to the inhibition of glutamate-sustained respiration. To study the effect of chronic in vivo MMA treatment on mitochondrial function, young rats were intraperitoneally injected with MMA. No significant difference was observed in respiration between isolated brain mitochondria from control and MMA-treated rats, indicating that in vivo MMA treatment did not lead to permanent mitochondrial respiratory defects. Taken together, these findings indicate that the inhibitory effect of MMA on mitochondrial oxidative metabolism can be ascribed to concurrent inhibition of specific enzymes and lower availability of respiratory substrates. PMID:22488725

  13. Peripheral nerve injury produces a sustained shift in the balance between glutamate release and uptake in the dorsal horn of the spinal cord

    PubMed Central

    Inquimbert, Perrine; Bartels, Karsten; Babaniyi, Olusegun B.; Barrett, Lee B.; Tegeder, Irmgard; Scholz, Joachim

    2012-01-01

    Peripheral nerve injury provokes heightened excitability of primary sensory afferents including nociceptors, and elicits ectopic activity in lesioned and neighboring intact nerve fibers. The major transmitter released by sensory afferents in the superficial dorsal horn of the spinal cord is glutamate. Glutamate is critically involved in nociceptive signaling and the development of neuropathic pain. We recorded miniature excitatory postsynaptic currents (mEPSCs) from neurons in lamina II of the rat dorsal horn to assess spontaneous synaptic activity after spared nerve injury (SNI), a model of chronic neuropathic pain. Following SNI, the frequency of mEPSCs doubled, indicating heightened glutamate release from primary afferents or spinal interneurons. Consistent with this finding, glutamate concentrations in the cerebrospinal fluid were elevated at one and four weeks after SNI. Transmitter uptake was insufficient to prevent the rise in extracellular glutamate as the expression of glutamate transporters remained unchanged or decreased. 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), an antagonist of metabotropic glutamate receptor 5 (mGluR5), reduced the frequency of mEPSCs to its preinjury level, suggesting a positive feedback mechanism that involves facilitation of transmitter release by mGluR5 activation in the presence of high extracellular glutamate. Treatment with the β-lactam antibiotic ceftriaxone increased the expression of glutamate transporter 1 (Glt1) in the dorsal horn after SNI, raised transmitter uptake and lowered extracellular glutamate. Improving glutamate clearance prevented the facilitation of transmitter release by mGluR5 and attenuated neuropathic pain-like behavior. Balancing glutamate release and uptake after nerve injury should be an important target in the management of chronic neuropathic pain. PMID:23021150

  14. LY354740 is a potent and highly selective group II metabotropic glutamate receptor agonist in cells expressing human glutamate receptors.

    PubMed

    Schoepp, D D; Johnson, B G; Wright, R A; Salhoff, C R; Mayne, N G; Wu, S; Cockerman, S L; Burnett, J P; Belegaje, R; Bleakman, D; Monn, J A

    1997-01-01

    The novel compound LY354740 is a conformationally constrained analog of glutamate, which was designed for interaction at metabotropic glutamate (mGlu) receptors. In this paper the selectivity of LY354740 for recombinant human mGlu receptor subtypes expressed in non-neuronal (RGT) cells is described. At human mGlu2 receptors, LY354740 produced > 90% suppression of forskolin-stimulated cAMP formation with an EC50 of 5.1 +/- 0.3 nM. LY354740 was six-fold less potent in activating human mGlu3 receptors (EC50 = 24.3 +/- 0.5 nM). LY354740 inhibition of forskolin-stimulated cAMP formation in human mGlu2 receptor-expressing cells was blocked by competitive mGlu receptor antagonists, including (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) and LY307452 ((2S,4S)-2-amino-4-(4,4-diphenylbut-1-yl)-pentane-1,5-dioic acid). LY354740 had no agonist or antagonist activities at cells expressing human mGlu4 or mGlu7 (group III mGlu receptors) (EC50 > 100,000 nM). When tested at group I phosphoinositide-coupled human mGlu receptors (mGlu1a and mGlu5a), LY354740 did not activate or inhibit mGlu receptor agonist-evoked phosphoinositide hydrolysis at up to 100,000 nM. Electrophysiological experiments also demonstrated that LY354740 also had no appreciable activity in cells expressing human recombinant AMPA (GluR4) and kainate (GluR6) receptors. Thus, LY354740 is a highly potent, efficacious and selective group II (mGlu2/3) receptor agonist, useful to explore the functions of these receptors in situ. PMID:9144636

  15. Inhibition of breast cancer-cell glutamate release with sulfasalazine limits cancer-induced bone pain.

    PubMed

    Ungard, Robert G; Seidlitz, Eric P; Singh, Gurmit

    2014-01-01

    Cancer in bone is frequently a result of metastases from distant sites, particularly from the breast, lung, and prostate. Pain is a common and often severe pathological feature of cancers in bone, and is a significant impediment to the maintenance of quality of life of patients living with bone metastases. Cancer cell lines have been demonstrated to release significant amounts of the neurotransmitter and cell-signalling molecule l-glutamate via the system xC(-) cystine/glutamate antiporter. We have developed a novel mouse model of breast cancer bone metastases to investigate the impact of inhibiting cancer cell glutamate transporters on nociceptive behaviour. Immunodeficient mice were inoculated intrafemorally with the human breast adenocarcinoma cell line MDA-MB-231, then treated 14days later via mini-osmotic pumps inserted intraperitoneally with sulfasalazine, (S)-4-carboxyphenylglycine, or vehicle. Both sulfasalazine and (S)-4-carboxyphenylglycine attenuated in vitro cancer cell glutamate release in a dose-dependent manner via the system xC(-) transporter. Animals treated with sulfasalazine displayed reduced nociceptive behaviours and an extended time until the onset of behavioural evidence of pain. Animals treated with a lower dose of (S)-4-carboxyphenylglycine did not display this reduction in nociceptive behaviour. These results suggest that a reduction in glutamate secretion from cancers in bone with the system xC(-) inhibitor sulfasalazine may provide some benefit for treating the often severe and intractable pain associated with bone metastases.

  16. A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance.

    PubMed

    Ageta-Ishihara, Natsumi; Yamazaki, Maya; Konno, Kohtarou; Nakayama, Hisako; Abe, Manabu; Hashimoto, Kenji; Nishioka, Tomoki; Kaibuchi, Kozo; Hattori, Satoko; Miyakawa, Tsuyoshi; Tanaka, Kohichi; Huda, Fathul; Hirai, Hirokazu; Hashimoto, Kouichi; Watanabe, Masahiko; Sakimura, Kenji; Kinoshita, Makoto

    2015-01-01

    The small GTPase-effector proteins CDC42EP1-5/BORG1-5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4(-/-) mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (γDGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1-5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance. PMID:26657011

  17. A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance

    PubMed Central

    Ageta-Ishihara, Natsumi; Yamazaki, Maya; Konno, Kohtarou; Nakayama, Hisako; Abe, Manabu; Hashimoto, Kenji; Nishioka, Tomoki; Kaibuchi, Kozo; Hattori, Satoko; Miyakawa, Tsuyoshi; Tanaka, Kohichi; Huda, Fathul; Hirai, Hirokazu; Hashimoto, Kouichi; Watanabe, Masahiko; Sakimura, Kenji; Kinoshita, Makoto

    2015-01-01

    The small GTPase-effector proteins CDC42EP1-5/BORG1–5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4−/− mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (γDGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1–5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance. PMID:26657011

  18. Supplementing glutamate to partial enteral nutrition slows gastric emptying rate in preterm pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Premature infants frequently present with gastroduodenal motor dysfunction, which is manifest clinically as feeding intolerance resulting from slow gastric emptying. Glutamate (GLU) is the major excitatory neurotransmitter in the body and multiple GLU receptors and transporters have been found in th...

  19. Emerging aspects of dietary glutamate metabolism in the developing gut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutamate is a major constituent of dietary protein and is also consumed in many prepared foods as a flavour additive in the form of monosodium glutamate (MSG). Evidence from human and animal studies indicates that glutamate is the major oxidative fuel for the gut and that dietary glutamate is exten...

  20. Glutamate protects against Ca(2+) paradox-induced injury and inhibits calpain activity in isolated rat hearts.

    PubMed

    Zhang, Jian-Ying; Kong, Ling-Heng; Lai, Dong; Jin, Zhen-Xiao; Gu, Xiao-Ming; Zhou, Jing-Jun

    2016-10-01

    This study determined the effects of glutamate on the Ca(2+) paradoxical heart, which is a model for Ca(2+) overload-induced injury during myocardial ischaemia and reperfusion, and evaluated its effect on a known mediator of injury, calpain. An isolated rat heart was retrogradely perfused in a Langendorff apparatus. Ca(2+) paradox was elicited via perfusion with a Ca(2+) -free Krebs-Henseleit (KH) solution for 3 minutes followed by Ca(2+) -containing normal KH solution for 30 minutes. The Ca(2+) paradoxical heart exhibited almost no viable tissue on triphenyltetrazolium chloride staining and markedly increased LDH release, caspase-3 activity, cytosolic cytochrome c content, and apoptotic index. These hearts also displayed significantly increased LVEDP and a disappearance of LVDP. Glutamate (5 and 20 mmol/L) significantly alleviated Ca(2+) paradox-induced injury. In contrast, 20 mmol/L mannitol had no effect on Ca(2+) paradox. Ca(2+) paradox significantly increased the extent of the translocation of μ-calpain to the sarcolemmal membrane and the proteolysis of α-fodrin, which suggests calpain activation. Glutamate also blocked these effects. A non-selective inhibitor of glutamate transporters, dl-TBOA (10 μmol/L), had no effect on control hearts, but it reversed glutamate-induced cardioprotection and reduction in calpain activity. Glutamate treatment significantly increased intracellular glutamate content in the Ca(2+) paradoxical heart, which was also blocked by dl-TBOA. We conclude that glutamate protects the heart against Ca(2+) overload-induced injury via glutamate transporters, and the inhibition of calpain activity is involved in this process.

  1. Activation of presynaptic oxytocin receptors enhances glutamate release in the ventral hippocampus of prenatally restraint stressed rats.

    PubMed

    Mairesse, Jérôme; Gatta, Eleonora; Reynaert, Marie-Line; Marrocco, Jordan; Morley-Fletcher, Sara; Soichot, Marion; Deruyter, Lucie; Camp, Gilles Van; Bouwalerh, Hammou; Fagioli, Francesca; Pittaluga, Anna; Allorge, Delphine; Nicoletti, Ferdinando; Maccari, Stefania

    2015-12-01

    Oxytocin receptors are known to modulate synaptic transmission and network activity in the hippocampus, but their precise function has been only partially elucidated. Here, we have found that activation of presynaptic oxytocin receptor with the potent agonist, carbetocin, enhanced depolarization-evoked glutamate release in the ventral hippocampus with no effect on GABA release. This evidence paved the way for examining the effect of carbetocin treatment in "prenatally restraint stressed" (PRS) rats, i.e., the offspring of dams exposed to repeated episodes of restraint stress during pregnancy. Adult PRS rats exhibit an anxious/depressive-like phenotype associated with an abnormal glucocorticoid feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis, and, remarkably, with a reduced depolarization-evoked glutamate release in the ventral hippocampus. Chronic systemic treatment with carbetocin (1mg/kg, i.p., once a day for 2-3 weeks) in PRS rats corrected the defect in glutamate release, anxiety- and depressive-like behavior, and abnormalities in social behavior, in the HPA response to stress, and in the expression of stress-related genes in the hippocampus and amygdala. Of note, carbetocin treatment had no effect on these behavioral and neuroendocrine parameters in prenatally unstressed (control) rats, with the exception of a reduced expression of the oxytocin receptor gene in the amygdala. These findings disclose a novel function of oxytocin receptors in the hippocampus, and encourage the use of oxytocin receptor agonists in the treatment of stress-related psychiatric disorders in adult life.

  2. Regulation of Synaptic Transmission by Ambient Extracellular Glutamate

    PubMed Central

    FEATHERSTONE, DAVID E.; SHIPPY, SCOTT A.

    2008-01-01

    Many neuroscientists assume that ambient extracellular glutamate concentrations in the nervous system are biologically negligible under nonpathological conditions. This assumption is false. Hundreds of studies over several decades suggest that ambient extracellular glutamate levels in the intact mammalian brain are ~0.5 to ~5 μM. This has important implications. Glutamate receptors are desensitized by glutamate concentrations significantly lower than needed for receptor activation; 0.5 to 5 μM of glutamate is high enough to cause constitutive desensitization of most glutamate receptors. Therefore, most glutamate receptors in vivo may be constitutively desensitized, and ambient extracellular glutamate and receptor desensitization may be potent but generally unrecognized regulators of synaptic transmission. Unfortunately, the mechanisms regulating ambient extracellular glutamate and glutamate receptor desensitization remain poorly understood and understudied. PMID:17947494

  3. Construction of a potentiometric glutamate biosensor for determination of glutamate in some real samples.

    PubMed

    Y Lmaz, Demet; Karaku, Emine

    2011-12-01

    The potentiometric glutamate biosensor based on ammonium-selective poly(vinylchloride) (PVC) membrane electrode was constructed by chemically immobilizing glutamate oxidase. Ammonium ions produced after an enzymatic reaction were determined potentiometrically. We determined the optimum working conditions of the biosensor such as buffer concentration, buffer pH, lifetime, response time, linear working range, kinetic constants (K(m) and V(max)) of glutamate oxidase enzyme used for biosensor construction values, and other response characteristics. Additionally, glutamate assay in some real samples such as chicken bullion, healthy human serum, and commercial multipower amino acid mixture were also successfully carried out. The results showed good agreement with previously reported values.

  4. Brain glutamate decarboxylase and pyrroloquinoline quinone.

    PubMed

    Choi, S Y; Khemlani, L S; Churchich, J E

    1992-01-01

    Porcine brain glutamate decarboxylase was examined for the presence of covalently bound pyrroloquinoline quinone (PQQ). HPLC analysis of pure glutamate decarboxylase subjected to the hexanol extraction procedure gave negative results when monitored at 320 nm, the maximum of absorbance of 4-hydroxy-5-hexoxy-PQQ. Resolved glutamate decarboxylase exhibits a structureless absorption band at wavelengths longer than 300 nm which cannot be attributed to PQQ. The holoenzyme is not a pyridoxal-quinoprotein; its catalytic mechanism involves the participation of only one cofactor, i.e. pyridoxal-5-P. Free PQQ is a strong inhibitor of the decarboxylase (Ki = 13 microM) and the reaction with the protein results in spectral changes resembling those of polylysine treated with PQQ. If the concentration of free PQQ in some regions of the brain reaches the micromolar level, then PQQ might play a role in the regulation of glutamate decarboxylase activity.

  5. Mechanism for the activation of glutamate receptors

    Cancer.gov

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  6. DNA nanopore translocation in glutamate solutions

    NASA Astrophysics Data System (ADS)

    Plesa, C.; van Loo, N.; Dekker, C.

    2015-08-01

    Nanopore experiments have traditionally been carried out with chloride-based solutions. Here we introduce silver/silver-glutamate-based electrochemistry as an alternative, and study the viscosity, conductivity, and nanopore translocation characteristics of potassium-, sodium-, and lithium-glutamate solutions. We show that it has a linear response at typical voltages and can be used to detect DNA translocations through a nanopore. The glutamate anion also acts as a redox-capable thickening agent, with high-viscosity solutions capable of slowing down the DNA translocation process by up to 11 times, with a corresponding 7 time reduction in signal. These results demonstrate that glutamate can replace chloride as the primary anion in nanopore resistive pulse sensing.

  7. Genetic labeling of both the axons of transduced, glutamatergic neurons in rat postrhinal cortex and their postsynaptic neurons in other neocortical areas by herpes simplex virus vectors that coexpress an axon-targeted β-galactosidase and wheat germ agglutinin from a vesicular glutamate transporter-1 promoter.

    PubMed

    Zhang, Guo-rong; Cao, Haiyan; Li, Xu; Zhao, Hua; Geller, Alfred I

    2010-11-18

    Neuronal circuits comprise the foundation for neuronal physiology and synaptic plasticity, and thus for consequent behaviors and learning, but our knowledge of neocortical circuits is incomplete. Mapping neocortical circuits is a challenging problem because these circuits contain large numbers of neurons, a high density of synapses, and numerous classes and subclasses of neurons that form many different types of synapses. Expression of specific genetic tracers in small numbers of specific subclasses of neocortical neurons has the potential to map neocortical circuits. Suitable genetic tracers have been established in neurons in subcortical areas, but application to neocortical circuits has been limited. Enabling this approach, Herpes Simplex Virus (HSV-1) plasmid (amplicon) vectors can transduce small numbers of neurons in a specific neocortical area. Further, expression of a particular genetic tracer can be restricted to specific subclasses of neurons; in particular, the vesicular glutamate transporter-1 (VGLUT1) promoter supports expression in VGLUT1-containing glutamatergic neurons in rat postrhinal (POR) cortex. Here, we show that expression of an axon-targeted β-galactosidase (β-gal) from such vectors supports mapping specific commissural and associative projections of the transduced neurons in POR cortex. Further, coexpression of wheat germ agglutinin (WGA) and an axon-targeted β-gal supports mapping both specific projections of the transduced neurons and identifying specific postsynaptic neurons for the transduced neurons. The neocortical circuit mapping capabilities developed here may support mapping specific neocortical circuits that have critical roles in cognitive learning.

  8. Glutamic Acid Decarboxylation in Chlorella12

    PubMed Central

    Lane, T. R.; Stiller, Mary

    1970-01-01

    The decarboxylation of endogenous free glutamic acid by Chlorella pyrenoidosa, Marburg strain, was induced by a variety of metabolic poisons, by anaerobic conditions, and by freezing and thawing the cells. The rate of decarboxylation was proportional to the concentration of inhibitor present. Possible mechanisms which relate the effects of the various conditions on glutamate decarboxylation and oxygen consumption by Chlorella are discussed. Images PMID:5429350

  9. [Glutamate neurotransmission, stress and hormone secretion].

    PubMed

    Jezová, D; Juránková, E; Vigas, M

    1995-11-01

    Glutamate neurotransmission has been investigated in relation to several physiological processes (learning, memory) as well as to neurodegenerative and other disorders. Little attention has been paid to its involvement in neuroendocrine response during stress. Penetration of excitatory amino acids from blood to the brain is limited by the blood-brain barrier. As a consequence, several toxic effects but also bioavailability for therapeutic purposes are reduced. A free access to circulating glutamate is possible only in brain structures lacking the blood-brain barrier or under conditions of its increased permeability. Excitatory amino acids were shown to stimulate the pituitary hormone release, though the mechanism of their action is still not fully understood. Stress exposure in experimental animals induced specific changes in mRNA levels coding the glutamate receptor subunits in the hippocampus and hypothalamus. The results obtained with the use of glutamate receptor antagonists indicate that a number of specific receptor subtypes contribute to the stimulation of ACTH release during stress. The authors provided also data on the role of NMDA receptors in the control of catecholamine release, particularly in stress-induced secretion of epinephrine. These results were the first piece of evidence on the involvement of endogenous excitatory amino acids in neuroendocrine activation during stress. Neurotoxic effects of glutamate in animals are well described, especially after its administration in the neonatal period. In men, glutamate toxicity and its use as a food additive are a continuous subject of discussions. The authors found an increase in plasma cortisol and norepinephrine, but not epinephrine and prolactin, in response to the administration of a high dose of glutamate. It cannot be excluded that these effects might be induced even by lower doses in situations with increased vulnerability to glutamate action (age, individual variability). (Tab. 1, Fig. 6, Ref. 44

  10. A high-fat diet impairs cooling-evoked brown adipose tissue activation via a vagal afferent mechanism.

    PubMed

    Madden, Christopher J; Morrison, Shaun F

    2016-08-01

    In dramatic contrast to rats on a control diet, rats maintained on a high-fat diet (HFD) failed to activate brown adipose tissue (BAT) during cooling despite robust increases in their BAT activity following direct activation of their BAT sympathetic premotor neurons in the raphe pallidus. Cervical vagotomy or blockade of glutamate receptors in the nucleus of the tractus solitarii (NTS) reversed the HFD-induced inhibition of cold-evoked BAT activity. Thus, a HFD does not prevent rats from mounting a robust, centrally driven BAT thermogenesis; however, a HFD does alter a vagal afferent input to NTS neurons, thereby preventing the normal activation of BAT thermogenesis to cooling. These results, paralleling the absence of cooling-evoked glucose uptake in the BAT of obese humans, reveal a neural mechanism through which consumption of a HFD contributes to reduced energy expenditure and thus to weight gain. PMID:27354235

  11. Arctigenin reduces neuronal responses in the somatosensory cortex via the inhibition of non-NMDA glutamate receptors.

    PubMed

    Borbély, Sándor; Jócsák, Gergely; Moldován, Kinga; Sedlák, Éva; Preininger, Éva; Boldizsár, Imre; Tóth, Attila; Atlason, Palmi T; Molnár, Elek; Világi, Ildikó

    2016-07-01

    Lignans are biologically active phenolic compounds related to lignin, produced in different plants. Arctigenin, a dibenzylbutyrolactone-type lignan, has been used as a neuroprotective agent for the treatment of encephalitis. Previous studies of cultured rat cerebral cortical neurones raised the possibility that arctigenin inhibits kainate-induced excitotoxicity. The aims of the present study were: 1) to analyse the effect of arctigenin on normal synaptic activity in ex vivo brain slices, 2) to determine its receptor binding properties and test the effect of arctigenin on AMPA/kainate receptor activation and 3) to establish its effects on neuronal activity in vivo. Arctigenin inhibited glutamatergic transmission and reduced the evoked field responses. The inhibitory effect of arctigenin on the evoked field responses proved to be substantially dose dependent. Our results indicate that arctigenin exerts its effects under physiological conditions and not only on hyper-excited neurons. Furthermore, arctigenin can cross the blood-brain barrier and in the brain it interacts with kainate sensitive ionotropic glutamate receptors. These results indicate that arctigenin is a potentially useful new pharmacological tool for the inhibition of glutamate-evoked responses in the central nervous system in vivo.

  12. Arctigenin reduces neuronal responses in the somatosensory cortex via the inhibition of non-NMDA glutamate receptors.

    PubMed

    Borbély, Sándor; Jócsák, Gergely; Moldován, Kinga; Sedlák, Éva; Preininger, Éva; Boldizsár, Imre; Tóth, Attila; Atlason, Palmi T; Molnár, Elek; Világi, Ildikó

    2016-07-01

    Lignans are biologically active phenolic compounds related to lignin, produced in different plants. Arctigenin, a dibenzylbutyrolactone-type lignan, has been used as a neuroprotective agent for the treatment of encephalitis. Previous studies of cultured rat cerebral cortical neurones raised the possibility that arctigenin inhibits kainate-induced excitotoxicity. The aims of the present study were: 1) to analyse the effect of arctigenin on normal synaptic activity in ex vivo brain slices, 2) to determine its receptor binding properties and test the effect of arctigenin on AMPA/kainate receptor activation and 3) to establish its effects on neuronal activity in vivo. Arctigenin inhibited glutamatergic transmission and reduced the evoked field responses. The inhibitory effect of arctigenin on the evoked field responses proved to be substantially dose dependent. Our results indicate that arctigenin exerts its effects under physiological conditions and not only on hyper-excited neurons. Furthermore, arctigenin can cross the blood-brain barrier and in the brain it interacts with kainate sensitive ionotropic glutamate receptors. These results indicate that arctigenin is a potentially useful new pharmacological tool for the inhibition of glutamate-evoked responses in the central nervous system in vivo. PMID:26972612

  13. [Evoked potentials in patients with secondary headaches].

    PubMed

    Iakupov, E Z; Kuznetsova, E A

    2010-01-01

    Characteristics of brain evoked activity were studied in patients with most frequent variants of secondary headaches: chronic posttraumatic headaches, cervicogenic headaches and vascular headaches in patients with arterial hypertension and chronic brain ischemia. The multimodal registration of evoked potentials (EP) (short-latency brainstem auditory, visual EPs to flash stimulation and cognitive EPs - P300) revealed signs of brainstem dysfunction, decrease of visual analyzer and diminished cognitive functions in most patients with secondary headaches. Based on results obtained, we can recommend a complex therapy of chronic secondary headaches with neuroprotectors and nootropics.

  14. Brain correlates of music-evoked emotions.

    PubMed

    Koelsch, Stefan

    2014-03-01

    Music is a universal feature of human societies, partly owing to its power to evoke strong emotions and influence moods. During the past decade, the investigation of the neural correlates of music-evoked emotions has been invaluable for the understanding of human emotion. Functional neuroimaging studies on music and emotion show that music can modulate activity in brain structures that are known to be crucially involved in emotion, such as the amygdala, nucleus accumbens, hypothalamus, hippocampus, insula, cingulate cortex and orbitofrontal cortex. The potential of music to modulate activity in these structures has important implications for the use of music in the treatment of psychiatric and neurological disorders.

  15. Inhibitors of calcium buffering depress evoked transmitter release at the squid giant synapse.

    PubMed Central

    Adams, D J; Takeda, K; Umbach, J A

    1985-01-01

    Evoked release of transmitter at the squid giant synapse was examined under conditions where the calcium ion concentration in the presynaptic terminal was manipulated by inhibitors of calcium sequestration. Simultaneous intracellular recordings of presynaptic and post-synaptic resting and action potentials were made during bath application of one of the following metabolic inhibitors: sodium cyanide (NaCN), carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP); ruthenium red (RuR) and sodium-free (lithium) sea water. Cyanide and lithium sea water reversibly depressed the post-synaptic potential (p.s.p.) whilst RuR and FCCP blocked the evoked post-synaptic response irreversibly. The progressive reduction of p.s.p. amplitude was accompanied by a reversible increase in synaptic delay. The time course of block of the p.s.p. was similar for different agents and dependent on the rate of presynaptic activity (30-40 min at 0.01 Hz). Recovery of the post-synaptic action potential following block by cyanide and lithium sea water was obtained within 40 min and 5 min respectively. Synaptic depression by the metabolic inhibitors does not result from changes in presynaptic resting or action potentials, nor from a change in post-synaptic receptor sensitivity. The post-synaptic response to the local ionophoresis of L-glutamate was unchanged following inhibition of evoked release of transmitter by cyanide. Injections of EGTA into presynaptic terminals poisoned by cyanide produced transient increases in p.s.p. amplitude, suggesting that cyanide is having its effect through raising intracellular calcium rather than lowering ATP. Control experiments injecting EGTA into unpoisoned nerve terminals showed no apparent effect on evoked transmitter release. PMID:2419546

  16. Ionotropic Glutamate Receptors & CNS Disorders

    PubMed Central

    Bowie, Derek

    2008-01-01

    Disorders of the central nervous system (CNS) are complex disease states that represent a major challenge for modern medicine. Although etiology is often unknown, it is established that multiple factors such as defects in genetics and/or epigenetics, the environment as well as imbalance in neurotransmitter receptor systems are all at play in determining an individual’s susceptibility to disease. Gene therapy is currently not available and therefore, most conditions are treated with pharmacological agents that modify neurotransmitter receptor signaling. Here, I provide a review of ionotropic glutamate receptors (iGluRs) and the roles they fulfill in numerous CNS disorders. Specifically, I argue that our understanding of iGluRs has reached a critical turning point to permit, for the first time, a comprehensive re-evaluation of their role in the cause of disease. I illustrate this by highlighting how defects in AMPA receptor trafficking are important to Fragile X mental retardation and ectopic expression of kainate (KA) receptor synapses contributes to the pathology of temporal lobe epilepsy. Finally, I discuss how parallel advances in studies of other neurotransmitter systems may allow pharmacologists to work towards a cure for many CNS disorders rather than developing drugs to treat their symptoms. PMID:18537642

  17. Medroxyprogesterone acetate exacerbates glutamate excitotoxicity.

    PubMed

    Nilsen, Jon; Morales, Alison; Brinton, Roberta Diaz

    2006-07-01

    We previously demonstrated that progesterone functions as a neuroprotective agent whereas medroxyprogesterone acetate (MPA; Provera) does not. Moreover, MPA antagonized the neuroprotective and neurotrophic outcomes induced by 17beta-estradiol (E2). Towards developing effective hormone therapies for protection against neurodegeneration, we sought to determine whether formulation, chemical features or prevention versus treatment mode of exposure affected the outcome of MPA treatment in survival of primary hippocampal neurons. Results of these analyses indicated that both crystalline MPA and a pharmaceutical formulation (Depo-Provera) lacked neuroprotective efficacy, indicating that the effects were not dependent upon MPA formulation. Likewise, MPA in the prevention and treatment paradigms were equally ineffective at promoting neuronal survival, indicating that timing of MPA administration was not a factor. Further, the detrimental effects of MPA were not due to the presence of the acetate group, as medroxyprogesterone was as ineffective as MPA in promoting neuronal survival. Moreover, MPA pretreatment exacerbated neuron death induced by glutamate excitotoxicity as indicated by a 40% increase in neuron death determined by direct live/dead cell count and a commensurate increase in the number of positive cells by terminal deoxynucleotidyl transferase-mediated nick end-labeling. Collectively these results predict that the progestin formulation of hormone therapy will affect the vulnerability of the central nervous system to degenerative insults.

  18. Involvement of dorsal hippocampus glutamatergic and nitrergic neurotransmission in autonomic responses evoked by acute restraint stress in rats.

    PubMed

    Moraes-Neto, T B; Scopinho, A A; Biojone, C; Corrêa, F M A; Resstel, L B M

    2014-01-31

    The dorsal hippocampus (DH) is a structure of the limbic system that is involved in emotional, learning and memory processes. There is evidence indicating that the DH modulates cardiovascular correlates of behavioral responses to stressful stimuli. Acute restraint stress (RS) is an unavoidable stress situation that evokes marked and sustained autonomic changes, which are characterized by elevated blood pressure (BP), intense heart rate (HR) increase and a decrease in cutaneous temperature. In the present study, we investigated the involvement of an N-methyl-D-aspartate (NMDA) glutamate receptor/nitric oxide (NO) pathway of the DH in the modulation of autonomic (arterial BP, HR and tail skin temperature) responses evoked by RS in rats. Bilateral microinjection of the NMDA receptor antagonist AP-7 (10 nmol/500 nL) into the DH attenuated RS-evoked autonomic responses. Moreover, RS evoked an increase in the content of NO₂/NO₃ in the DH, which are products of the spontaneous oxidation of NO under physiological conditions that can provide an indirect measurement of NO production. Bilateral microinjection of N-propyl-L-arginine (0.1 nmol/500 nL; N-propyl, a neuronal NO synthase (nNOS) inhibitor) or carboxy-PTIO (2 nmol/500 nL; c-PTIO, an NO scavenger) into the DH also attenuated autonomic responses evoked by RS. Therefore, our findings suggest that a glutamatergic system present in the DH is involved in the autonomic modulation during RS, acting via NMDA receptors and nNOS activation. Furthermore, the present results suggest that NMDA receptor/nNO activation has a facilitatory influence on RS-evoked autonomic responses. PMID:24269610

  19. 2-Methylcitric acid impairs glutamate metabolism and induces permeability transition in brain mitochondria.

    PubMed

    Amaral, Alexandre Umpierrez; Cecatto, Cristiane; Castilho, Roger Frigério; Wajner, Moacir

    2016-04-01

    Accumulation of 2-methylcitric acid (2MCA) is observed in methylmalonic and propionic acidemias, which are clinically characterized by severe neurological symptoms. The exact pathogenetic mechanisms of brain abnormalities in these diseases are poorly established and very little has been reported on the role of 2MCA. In the present work we found that 2MCA markedly inhibited ADP-stimulated and uncoupled respiration in mitochondria supported by glutamate, with a less significant inhibition in pyruvate plus malate respiring mitochondria. However, no alterations occurred when α-ketoglutarate or succinate was used as respiratory substrates, suggesting a defect on glutamate oxidative metabolism. It was also observed that 2MCA decreased ATP formation in glutamate plus malate or pyruvate plus malate-supported mitochondria. Furthermore, 2MCA inhibited glutamate dehydrogenase activity at concentrations as low as 0.5 mM. Kinetic studies revealed that this inhibitory effect was competitive in relation to glutamate. In contrast, assays of osmotic swelling in non-respiring mitochondria suggested that 2MCA did not significantly impair mitochondrial glutamate transport. Finally, 2MCA provoked a significant decrease in mitochondrial membrane potential and induced swelling in Ca(2+)-loaded mitochondria supported by different substrates. These effects were totally prevented by cyclosporine A plus ADP or ruthenium red, indicating induction of mitochondrial permeability transition. Taken together, our data strongly indicate that 2MCA behaves as a potent inhibitor of glutamate oxidation by inhibiting glutamate dehydrogenase activity and as a permeability transition inducer, disturbing mitochondrial energy homeostasis. We presume that 2MCA-induced mitochondrial deleterious effects may contribute to the pathogenesis of brain damage in patients affected by methylmalonic and propionic acidemias. We propose that brain glutamate oxidation is disturbed by 2-methylcitric acid (2MCA), which

  20. 2-Methylcitric acid impairs glutamate metabolism and induces permeability transition in brain mitochondria.

    PubMed

    Amaral, Alexandre Umpierrez; Cecatto, Cristiane; Castilho, Roger Frigério; Wajner, Moacir

    2016-04-01

    Accumulation of 2-methylcitric acid (2MCA) is observed in methylmalonic and propionic acidemias, which are clinically characterized by severe neurological symptoms. The exact pathogenetic mechanisms of brain abnormalities in these diseases are poorly established and very little has been reported on the role of 2MCA. In the present work we found that 2MCA markedly inhibited ADP-stimulated and uncoupled respiration in mitochondria supported by glutamate, with a less significant inhibition in pyruvate plus malate respiring mitochondria. However, no alterations occurred when α-ketoglutarate or succinate was used as respiratory substrates, suggesting a defect on glutamate oxidative metabolism. It was also observed that 2MCA decreased ATP formation in glutamate plus malate or pyruvate plus malate-supported mitochondria. Furthermore, 2MCA inhibited glutamate dehydrogenase activity at concentrations as low as 0.5 mM. Kinetic studies revealed that this inhibitory effect was competitive in relation to glutamate. In contrast, assays of osmotic swelling in non-respiring mitochondria suggested that 2MCA did not significantly impair mitochondrial glutamate transport. Finally, 2MCA provoked a significant decrease in mitochondrial membrane potential and induced swelling in Ca(2+)-loaded mitochondria supported by different substrates. These effects were totally prevented by cyclosporine A plus ADP or ruthenium red, indicating induction of mitochondrial permeability transition. Taken together, our data strongly indicate that 2MCA behaves as a potent inhibitor of glutamate oxidation by inhibiting glutamate dehydrogenase activity and as a permeability transition inducer, disturbing mitochondrial energy homeostasis. We presume that 2MCA-induced mitochondrial deleterious effects may contribute to the pathogenesis of brain damage in patients affected by methylmalonic and propionic acidemias. We propose that brain glutamate oxidation is disturbed by 2-methylcitric acid (2MCA), which

  1. Thinking outside the cleft to understand synaptic activity: contribution of the cystine-glutamate antiporter (System xc-) to normal and pathological glutamatergic signaling.

    PubMed

    Bridges, Richard; Lutgen, Victoria; Lobner, Doug; Baker, David A

    2012-07-01

    System x(c)(-) represents an intriguing target in attempts to understand the pathological states of the central nervous system. Also called a cystine-glutamate antiporter, system x(c)(-) typically functions by exchanging one molecule of extracellular cystine for one molecule of intracellular glutamate. Nonvesicular glutamate released during cystine-glutamate exchange activates extrasynaptic glutamate receptors in a manner that shapes synaptic activity and plasticity. These findings contribute to the intriguing possibility that extracellular glutamate is regulated by a complex network of release and reuptake mechanisms, many of which are unique to glutamate and rarely depicted in models of excitatory signaling. Because system x(c)(-) is often expressed on non-neuronal cells, the study of cystine-glutamate exchange may advance the emerging viewpoint that glia are active contributors to information processing in the brain. It is noteworthy that system x(c)(-) is at the interface between excitatory signaling and oxidative stress, because the uptake of cystine that results from cystine-glutamate exchange is critical in maintaining the levels of glutathione, a critical antioxidant. As a result of these dual functions, system x(c)(-) has been implicated in a wide array of central nervous system diseases ranging from addiction to neurodegenerative disorders to schizophrenia. In the current review, we briefly discuss the major cellu