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Sample records for evoked glutamate transporter

  1. Down-regulation of astroglial glutamate transporter-1 in the locus coeruleus impairs pain-evoked endogenous analgesia in rats.

    PubMed

    Kimura, Masafumi; Suto, Takashi; Eisenach, James C; Hayashida, Ken-ichiro

    2015-11-01

    Descending noradrenergic inhibition to the spinal cord from the locus coeruleus (LC) is an important endogenous pain-relief mechanism which can be activated by local glutamate signaling. Here we tested whether dysregulation of extracellular glutamate level in the LC induced by down-regulating astroglial glutamate transporter-1(GLT-1) impairs endogenous analgesia. In rats treated with repeated LC injections of GLT-1 selective or non-targeting small interfering RNA (siRNA), a subdermal injection of capsaicin was used to examine noxious stimulation-induced analgesia (NSIA), evoked LC glutamate and spinal noradrenaline release, and evoked LC neuronal activity. LC-injected GLT-1 siRNA reduced expression of GLT-1 in the LC (P=0.02), increased basal activity of LC neurons (P<0.01), and increased basal extracellular concentrations of LC glutamate (P<0.01) and spinal noradrenaline (P<0.01), but did not affect mechanical withdrawal thresholds in the hindpaw (P=0.83), compared to non-targeting siRNA. LC-injected GLT-1 siRNA impaired capsaicin-evoked release of LC glutamate and spinal noradrenaline, capsaicin-evoked LC neuronal activation, and NSIA. These results suggest that astroglial GLT-1 is essential to normal LC function and that increased extracellular glutamate by down-regulating GLT-1 impairs evoked LC activity and NSIA, essentially taking the LC "off-line". PMID:26450532

  2. Activation of glial glutamate transporter via MAPK p38 prevents enhanced and long-lasting non-evoked resting pain after surgical incision in rats.

    PubMed

    Reichl, Sylvia; Segelcke, Daniel; Keller, Viktor; Jonas, Robin; Boecker, Armin; Wenk, Manuel; Evers, Dagmar; Zahn, Peter K; Pogatzki-Zahn, Esther M

    2016-06-01

    Pain after surgery has recently become a major issue not only due to lack of treatment success in the acute phase; even more alarming is the large number of patients developing prolonged pain after surgery. Because spinal glutamate as well as spinal glia plays a major role in acute incisional pain, we investigated the role of the spinal glial glutamate transporters (GT), GLAST, GLT-1, for acute and prolonged pain and hyperalgesia caused by an incision. Spinal administration of the GT-inhibitor DL-TBOA increased non-evoked pain but not evoked pain behavior (hyperalgesia) up to 2 weeks after incision. In accordance, spinal GLAST (and to a lesser degree GLT-1) were upregulated after incision for several days. Long-term incision induced GT upregulation was prevented by long-lasting p38-inhibitor administration but not by long-lasting ERK1/2-inhibition after incision. In accordance, daily treatment with the p38-inhibitor (but not the ERK1/2 inhibitor) prolonged non-evoked but not evoked pain behavior after incision. In electrophysiological experiments, spontaneous activity of high threshold (HT) (but not wide dynamic range (WDR)) neurons known to transmit incision induced non-evoked pain was increased after prolonged treatment with the p38-inhibitor. In conclusion, our findings indicate a new spinal pathway by which non-evoked pain behavior after incision is modulated. The pathway is modality (non-evoked pain) and neuron (HT) specific and disturbance contributes to prolonged long-term pain after surgical incision. This may have therapeutic implications for the treatment of acute and - even more relevant - for prevention of chronic pain after surgery in patients. PMID:26920805

  3. SLC1 Glutamate Transporters

    PubMed Central

    Grewer, Christof; Gameiro, Armanda; Rauen, Thomas

    2014-01-01

    The plasma membrane transporters for the neurotransmitter glutamate belong to the solute carrier 1 (SLC1) family. They are secondary active transporters, taking up glutamate into the cell against a substantial concentration gradient. The driving force for concentrative uptake is provided by the cotransport of Na+ ions and the countertransport of one K+ in a step independent of the glutamate translocation step. Due to eletrogenicity of transport, the transmembrane potential can also act as a driving force. Glutamate transporters are expressed in many tissues, but are of particular importance in the brain, where they contribute to the termination of excitatory neurotransmission. Glutamate transporters can also run in reverse, resulting in glutamate release from cells. Due to these important physiological functions, glutamate transporter expression and, therefore, the transport rate, are tightly regulated. This review summarizes recent literature on the functional and biophysical properties, structure-function relationships, regulation, physiological significance, and pharmacology of glutamate transporters. Particular emphasis is on the insight from rapid kinetic and electrophysiological studies, transcriptional regulation of transporter expression, and reverse transport and its importance for pathophysiological glutamate release under ischemic conditions. PMID:24240778

  4. 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

  5. Glutamate release from platelets: exocytosis versus glutamate transporter reversal.

    PubMed

    Kasatkina, Ludmila A; Borisova, Tatiana A

    2013-11-01

    Platelets express neuronal and glial glutamate transporters EAAT 1-3 in the plasma membrane and vesicular glutamate transporters VGLUT 1,2 in the membrane of secretory granules. This study is focused on the assessment of non-exocytotic glutamate release, that is, the unstimulated release, heteroexchange and glutamate transporter reversal in platelets. Using the glutamate dehydrogenase assay, the absence of unstimulated release of endogenous glutamate from platelets was demonstrated, even after inhibition of glutamate transporters and cytoplasmic enzyme glutamine synthetase by dl-threo-β-benzyloxyaspartate and methionine sulfoximine, respectively. Depolarization of the plasma membrane by exposure to elevated [K(+)] did not induce the release of glutamate from platelets that was shown using the glutamate dehydrogenase assay and radiolabeled l-[(14)C]glutamate. Glutamate efflux by means of heteroexchange with transportable inhibitor of glutamate transporters dl-threo-β-hydroxyaspartate (dl-THA) was not observed. Furthermore, the protonophore cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and inhibitor of V-type H(+)-ATPase bafilomycin A1 also failed to stimulate the release of glutamate from platelets. However, exocytotic release of glutamate from secretory granules in response to thrombin stimulation was not prevented by elevated [K(+)], dl-THA, FCCP and bafilomycin A1. In contrast to nerve terminals, platelets cannot release glutamate in a non-exocytotic manner. Heteroexchange, transporter-mediated and unstimulated release of glutamate are not inherent to platelets. Therefore, platelets may be used as a peripheral marker/model for the analysis of glutamate uptake by brain nerve terminals only (direct function of transporters), whereas the mechanisms of glutamate release are different in platelets and nerve terminals. Glutamate is released by platelets exclusively by means of exocytosis. Also, reverse function of vesicular glutamate transporters of platelets is

  6. 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

  7. Temperature Differentially Facilitates Spontaneous but Not Evoked Glutamate Release from Cranial Visceral Primary Afferents

    PubMed Central

    Fawley, Jessica A.; Hofmann, Mackenzie E.; Largent-Milnes, Tally M.; Andresen, Michael C.

    2015-01-01

    Temperature is fundamentally important to all biological functions including synaptic glutamate release. Vagal afferents from the solitary tract (ST) synapse on second order neurons in the nucleus of the solitary tract, and glutamate release at this first central synapse controls autonomic reflex function. Expression of the temperature-sensitive Transient Receptor Potential Vanilloid Type 1 receptor separates ST afferents into C-fibers (TRPV1+) and A-fibers (TRPV1-). Action potential-evoked glutamate release is similar between C- and A-fiber afferents, but TRPV1 expression facilitates a second form of synaptic glutamate release in C-fibers by promoting substantially more spontaneous glutamate release. The influence of temperature on different forms of glutamate release is not well understood. Here we tested how temperature impacts the generation of evoked and spontaneous release of glutamate and its relation to TRPV1 expression. In horizontal brainstem slices of rats, activation of ST primary afferents generated synchronous evoked glutamate release (ST-eEPSCs) at constant latency whose amplitude reflects the probability of evoked glutamate release. The frequency of spontaneous EPSCs in these same neurons measured the probability of spontaneous glutamate release. We measured both forms of glutamate from each neuron during ramp changes in bath temperature of 4–5°C. Spontaneous glutamate release from TRPV1+ closely tracked with these thermal changes indicating changes in the probability of spontaneous glutamate release. In the same neurons, temperature changed axon conduction registered as latency shifts but ST-eEPSC amplitudes were constant and independent of TRPV1 expression. These data indicate that TRPV1-operated glutamate release is independent of action potential-evoked glutamate release in the same neurons. Together, these support the hypothesis that evoked and spontaneous glutamate release originate from two pools of vesicles that are independently

  8. Transport dynamics in a glutamate transporter homologue

    PubMed Central

    Akyuz, Nurunisa; Altman, Roger B.; Blanchard, Scott C.; Boudker, Olga

    2013-01-01

    Summary Glutamate transporters are integral membrane proteins that catalyze neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons1. Their mechanism involves transitions between extracellular- (outward-) and intracellular- (inward-) facing conformations, whereby substrate binding sites become accessible to the opposite sides of the membrane2. This process has been proposed to entail trans-membrane movements of three discrete transport domains within a trimeric scaffold3. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging4, we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters for the first time. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods5,6. We suggest that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent trans-membrane movements and may be rate determining in the transport cycle. PMID:23792560

  9. 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.

  10. Transport-mediated release of endogenous glutamate in the vertebrate retina.

    PubMed

    Maguire, G; Simko, H; Weinreb, R N; Ayoub, G

    1998-08-01

    In the present study we measured calcium-dependent, vesicular glutamate release, and calcium-independent, transport-mediated glutamate release patterns in the vertebrate retina to better understand the sources of elevated glutamate in neural tissue under ischemic conditions. A potassium concentration of 40 mM, which mimics the extracellular potassium concentration in the central nervous system during ischemia, was applied to the bathing medium of a retinal slice prepared from zebrafish. High external potassium evoked release of endogenous glutamate that was measured using a glutamate-specific fluorometric assay applied to the bath. The slice was visualized under 668 nm light using Normarski optics and fluorescent images were captured using a cooled charge-coupled device (CCD) camera. Following the elevation of external potassium to 40 mM several bands of glutamate fluorescence, reflecting the spatial distribution of glutamate release, were observed. A calcium-dependent cloud of glutamate was observed in the inner plexiform layer, that was antagonized by bath-applied nifedipine. A relatively dense glutamate cloud (1-10 microM) was observed over the ganglion cell layer, which was blocked by dihydrokainate, a glutamate transport antagonist. In contrast, nifedipine, an inhibitor of calcium-dependent neurotransmitter release in the retina, failed to block the cloud of released glutamate in the ganglion cell layer. These data suggest that under pathological conditions in the eye where glutamate levels are elevated surrounding retinal ganglion cells, such as observed in some forms of glaucoma, a possible source of the elevated glutamate is through a glutamate transporter operating in a reversed direction. A likely candidate for mediating this reversed transport of glutamate is the retinal Muller cell. PMID:9644233

  11. 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

  12. Transport mechanism of a glutamate transporter homologue GltPh.

    PubMed

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

    2016-06-15

    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

  13. 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

  14. 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

  15. 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.

  16. Nonvesicular Release of Glutamate by Glial xCT Transporters Suppresses Glutamate Receptor Clustering In Vivo

    PubMed Central

    Augustin, Hrvoje; Grosjean, Yael; Chen, Kaiyun; Sheng, Qi; Featherstone, David E.

    2008-01-01

    We hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, we identified and functionally characterized a novel Drosophila xCT gene, which we subsequently named “genderblind” (gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia. Genetic elimination of gb causes a 50% reduction in extracellular glutamate concentration, demonstrating that xCT transporters are important regulators of extracellular glutamate. Consistent with previous studies showing that extracellular glutamate regulates postsynaptic glutamate receptor clustering, gb mutants show a large (200–300%) increase in the number of postsynaptic glutamate receptors. This increase in postsynaptic receptor abundance is not accompanied by other obvious synaptic changes and is completely rescued when synapses are cultured in wild-type levels of glutamate. Additional in situ pharmacology suggests that glutamate-mediated suppression of glutamate receptor clustering depends on receptor desensitization. Together, our results suggest that (1) xCT transporters are critical for regulation of ambient extracellular glutamate in vivo; (2) ambient extracellular glutamate maintains some receptors constitutively desensitized in vivo; and (3) constitutive desensitization of ionotropic glutamate receptors suppresses their ability to cluster at synapses. PMID:17202478

  17. Evidence for evoked release of adenosine and glutamate from cultured cerebellar granule cells

    SciTech Connect

    Schousboe, A.; Frandsen, A.; Drejer, J. )

    1989-09-01

    Evoked release of ({sup 3}H)-D-aspartate which labels the neurotransmitter glutamate pool in cultured cerebellar granule cells was compared with evoked release of adenosine from similar cultures. It was found that both adenosine and (3H)-D-aspartate could be released from the neurons in a calcium dependent manner after depolarization of the cells with either 10-100 microM glutamate or 50 mM KCl. Cultures of cerebellar granule cells treated with 50 microM kainate to eliminate GABAergic neurons behaved in the same way. This together with the observation that cultured astrocytes did not exhibit a calcium dependent, potassium stimulated adenosine release strongly suggest that cerebellar granule cells release adenosine in a neurotransmitter-like fashion together with glutamate which is the classical neurotransmitter of these neurons. Studies of the metabolism of adenosine showed that in the granule cells adenosine is rapidly metabolized to ATP, ADP, and AMP, but in spite of this, adenosine was found to be released preferential to ATP.

  18. Glutamate transporters and presynaptic metabotropic glutamate receptors protect neocortical Cajal-Retzius cells against over-excitation.

    PubMed

    Dvorzhak, Anton; Unichenko, Petr; Kirischuk, Sergei

    2012-08-01

    Cajal-Retzius (CR) cells, early generated neurons in the marginal zone of developing neocortex, are reported to be highly vulnerable to excitotoxic damage. Because extracellular glutamate concentration in the central nervous system is mainly controlled by glutamate transporters (EAATs), we studied the effects of EAAT blockade on CR cells. DL: -TBOA, a specific EAAT antagonist, induced NMDA receptor-dependent bursting discharges in layer 2/3 pyramidal neurons, indicating that EAATs operate in the uptake mode and their blockade leads to elevation of extracellular glutamate concentration. In CR cells, however, DL: -TBOA failed to change either the membrane resistance or holding current, and moreover, it reduced the frequency of spontaneous GABAergic postsynaptic currents. DL: -TBOA decreased the mean amplitude and increased paired-pulse ratio of evoked GABAergic postsynaptic currents, indicating the presynaptic locus of its action. Indeed, LY379268, a specific agonist of group II metabotropic glutamate receptors (mGluR-II), mimicked the DL: -TBOA-mediated effects, and LY341495, an unspecific mGluR antagonist, eliminated the DL: -TBOA-induced effects. As dihydrokainic acid, a specific EAAT2 blocker, failed to affect evoked GABAergic postsynaptic currents, whereas TFB-TBOA, a selective blocker of EAAT1 and EAAT2, produced effects similar to that of DL: -TBOA, extracellular glutamate concentration in the marginal zone is mainly controlled by EAAT1 (GLAST). Thus, even though CR cells are highly vulnerable to excitotoxic damage, a number of mechanisms serve to protect them against excessive extracellular glutamate concentration including a lack of functional glutamatergic synapses, Mg(2+) blockade of NMDA receptors, and presynaptic mGluRs that inhibit transmission at GABAergic synapses. PMID:22665047

  19. Cannabinoid 1 and transient receptor potential vanilloid 1 receptors discretely modulate evoked glutamate separately from spontaneous glutamate transmission.

    PubMed

    Fawley, Jessica A; Hofmann, Mackenzie E; Andresen, Michael C

    2014-06-11

    Action potentials trigger synaptic terminals to synchronously release vesicles, but some vesicles release spontaneously. G-protein-coupled receptors (GPCRs) can modulate both of these processes. At cranial primary afferent terminals, the GPCR cannabinoid 1 (CB1) is often coexpressed with transient receptor potential vanilloid 1 (TRPV1), a nonselective cation channel present on most afferents. Here we tested whether CB1 activation modulates synchronous, action potential-evoked (eEPSCs) and/or spontaneous (sEPSCs) EPSCs at solitary tract nucleus neurons. In rat horizontal brainstem slices, activation of solitary tract (ST) primary afferents generated ST-eEPSCs that were rapidly and reversibly inhibited from most afferents by activation of CB1 with arachidonyl-2'-chloroethylamide (ACEA) or WIN 55,212-2 [R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone monomethanesulfonate]. The CB1 antagonist/inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] blocked these responses. Despite profound depression of ST-eEPSCs during CB1 activation, sEPSCs in these same neurons were unaltered. Changes in temperature changed sEPSC frequency only from TRPV1(+) afferents (i.e., thermal sEPSC responses only occurred in TRPV1(+) afferents). CB1 activation failed to alter these thermal sEPSC responses. However, the endogenous arachidonate metabolite N-arachidonyldopamine (NADA) promiscuously activated both CB1 and TRPV1 receptors. NADA inhibited ST-eEPSCs while simultaneously increasing sEPSC frequency, and thermally triggered sEPSC increases in neurons with TRPV1(+) afferents. We found no evidence for CB1/TRPV1 interactions suggesting independent regulation of two separate vesicle pools. Together, these data demonstrate that action potential-evoked synchronous glutamate release is modulated separately from TRPV1-mediated glutamate release despite coexistence

  20. Shifts in striatal responsivity evoked by chronic stimulation of dopamine and glutamate systems.

    PubMed

    Canales, J J; Capper-Loup, C; Hu, D; Choe, E S; Upadhyay, U; Graybiel, A M

    2002-10-01

    Dopamine and glutamate are key neurotransmitters in cortico-basal ganglia loops affecting motor and cognitive function. To examine functional convergence of dopamine and glutamate neurotransmitter systems in the basal ganglia, we evaluated the long-term effects of chronic stimulation of each of these systems on striatal responses to stimulation of the other. First we exposed rats to chronic intermittent cocaine and used early-gene assays to test the responsivity of the striatum to subsequent acute motor cortex stimulation by application of the GABA(A) (gamma-aminobutyric acid alpha subunit) receptor antagonist, picrotoxin. Reciprocally, we studied the effects of chronic intermittent motor cortex stimulation on the capacity for subsequent acute dopaminergic treatments to induce early-gene activation in the striatum. Prior treatment with chronic intermittent cocaine induced motor sensitization and significantly potentiated the striatal expression of Fos-family early genes in response to stimulation of the motor cortex. Contrary to this, chronic intermittent stimulation of the motor cortex down-regulated cocaine-induced gene expression in the striatum, but enhanced striatal gene expression induced by a full D1 receptor agonist (SKF 81297) and did not change the early-gene response elicited by a D2 receptor antagonist (haloperidol). These findings suggests that repeated dopaminergic stimulation produces long-term enhancement of corticostriatal signalling from the motor cortex, amplifying cortically evoked modulation of the basal ganglia. By contrast, persistent stimulation of the motor cortex inhibits cocaine-stimulated signalling in the striatum, but not signalling mediated by individual dopamine receptor sites, suggesting that chronic cortical hyperexcitability produces long-term impairment of dopaminergic activity and compensation at the receptor level. These findings prompt a model of the basal ganglia function as being regulated by opposing homeostatic dopamine-glutamate

  1. Vesicular Glutamate Transport Promotes Dopamine Storage and Glutamate Corelease In Vivo

    PubMed Central

    Hnasko, Thomas S.; Chuhma, Nao; Zhang, Hui; Goh, Germaine Y.; Sulzer, David; Palmiter, Richard D.; Rayport, Stephen; Edwards, Robert H.

    2010-01-01

    SUMMARY Dopamine neurons in the ventral tegmental area (VTA) play an important role in the motivational systems underlying drug addiction, and recent work has suggested that they also release the excitatory neurotransmitter glutamate. To assess a physiological role for glutamate corelease, we disrupted the expression of vesicular glutamate transporter 2 selectively in dopamine neurons. The conditional knockout abolishes glutamate release from midbrain dopamine neurons in culture and severely reduces their excitatory synaptic output in mesoaccumbens slices. Baseline motor behavior is not affected, but stimulation of locomotor activity by cocaine is impaired, apparently through a selective reduction of dopamine stores in the projection of VTA neurons to ventral striatum. Glutamate co-entry promotes monoamine storage by increasing the pH gradient that drives vesicular monoamine transport. Remarkably, low concentrations of glutamate acidify synaptic vesicles more slowly but to a greater extent than equimolar Cl−, indicating a distinct, presynaptic mechanism to regulate quantal size. PMID:20223200

  2. Molecular physiology of vesicular glutamate transporters in the digestive system

    PubMed Central

    Li, Tao; Ghishan, Fayez K.; Bai, Liqun

    2005-01-01

    Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Packaging and storage of glutamate into glutamatergic neuronal vesicles require ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. Three vesicular glutamate transporters (VGLUT1-3) have been recently identified from neuronal tissue where they play a key role to maintain the vesicular glutamate level. Recently, it has been demonstrated that glutamate signaling is also functional in peripheral neuronal and non-neuronal tissues, and occurs in sites of pituitary, adrenal, pineal glands, bone, GI tract, pancreas, skin, and testis. The glutamate receptors and VGLUTs in digestive system have been found in both neuronal and endocrinal cells. The glutamate signaling in the digestive system may have significant relevance to diabetes and GI tract motility disorders. This review will focus on the most recent update of molecular physiology of digestive VGLUTs. PMID:15793854

  3. 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 ...

  4. 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

  5. K+ depolarization evokes ATP, adenosine and glutamate release from glia in rat hippocampus: a microelectrode biosensor study

    PubMed Central

    Heinrich, A; Andó, RD; Túri, G; Rózsa, B; Sperlágh, B

    2012-01-01

    BACKGROUND AND PURPOSE This study was undertaken to characterize the ATP, adenosine and glutamate outflow evoked by depolarization with high K+ concentrations, in slices of rat hippocampus. EXPERIMENTAL APPROACH We utilized the microelectrode biosensor technique and extracellular electrophysiological recording for the real-time monitoring of the efflux of ATP, adenosine and glutamate. KEY RESULTS ATP, adenosine and glutamate sensors exhibited transient and reversible current during depolarization with 25 mM K+, with distinct kinetics. The ecto-ATPase inhibitor ARL67156 enhanced the extracellular level of ATP and inhibited the prolonged adenosine efflux, suggesting that generation of adenosine may derive from the extracellular breakdown of ATP. Stimulation-evoked ATP, adenosine and glutamate efflux was inhibited by tetrodotoxin, while exposure to Ca2+-free medium abolished ATP and adenosine efflux from hippocampal slices. Extracellular elevation of ATP and adenosine were decreased in the presence of NMDA receptor antagonists, D-AP-5 and ifenprodil, whereas non-NMDA receptor blockade by CNQX inhibited glutamate but not ATP and adenosine efflux. The gliotoxin fluoroacetate and P2X7 receptor antagonists inhibited the K+-evoked ATP, adenosine and glutamate efflux, while carbenoxolone in low concentration and probenecid decreased only the adenosine efflux. CONCLUSIONS AND IMPLICATIONS Our results demonstrated activity-dependent gliotransmitter release in the hippocampus in response to ongoing neuronal activity. ATP and glutamate were released by P2X7 receptor activation into extracellular space. Although the increased extracellular levels of adenosine did derive from released ATP, adenosine might also be released directly via pannexin hemichannels. LINKED ARTICLE This article is commented on by Sershen, pp. 1000–1002 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02072.x PMID:22394324

  6. 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

  7. 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. PMID:25935421

  8. 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

  9. 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

  10. 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

  11. 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…

  12. [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

  13. Molecular pharmacology of glutamate transporters, EAATs and VGLUTs.

    PubMed

    Shigeri, Yasushi; Seal, Rebecca P; Shimamoto, Keiko

    2004-07-01

    L-Glutamate serves as a major excitatory neurotransmitter in the mammalian central nervous system (CNS) and is stored in synaptic vesicles by an uptake system that is dependent on the proton electrochemical gradient (VGLUTs). Following its exocytotic release, glutamate activates fast-acting, excitatory ionotropic receptors and slower-acting metabotropic receptors to mediate neurotransmission. Na+-dependent glutamate transporters (EAATs) located on the plasma membrane of neurons and glial cells rapidly terminate the action of glutamate and maintain its extracellular concentration below excitotoxic levels. Thus far, five Na+-dependent glutamate transporters (EAATs 1-5) and three vesicular glutamate transporters (VGLUTs 1-3) have been identified. Examination of EAATs and VGLUTs in brain preparations and by heterologous expression of the various cloned subtypes shows these two transporter families differ in many of their functional properties including substrate specificity and ion requirements. Alterations in the function and/or expression of these carriers have been implicated in a range of psychiatric and neurological disorders. EAATs have been implicated in cerebral stroke, epilepsy, Alzheimer's disease, HIV-associated dementia, Huntington's disease, amyotrophic lateral sclerosis (ALS) and malignant glioma, while VGLUTs have been implicated in schizophrenia. To examine the physiological role of glutamate transporters in more detail, several classes of transportable and non-transportable inhibitors have been developed, many of which are derivatives of the natural amino acids, aspartate and glutamate. This review summarizes the development of these indispensable pharmacological tools, which have been critical to our understanding of normal and abnormal synaptic transmission. PMID:15210307

  14. A novel glutamate transport system in poly(γ-glutamic acid)-producing strain Bacillus subtilis CGMCC 0833.

    PubMed

    Wu, Qun; Xu, Hong; Zhang, Dan; Ouyang, Pingkai

    2011-08-01

    Bacillus subtilis CGMCC 0833 is a poly(γ-glutamic acid) (γ-PGA)-producing strain. It has the capacity to tolerate high concentration of extracellular glutamate and to utilize glutamate actively. Such a high uptake capacity was owing to an active transport system for glutamate. Therefore, a specific transport system for L-glutamate has been observed in this strain. It was a novel transport process in which glutamate was symported with at least two protons, and an inward-directed sodium gradient had no stimulatory effect on it. K(m) and V(m) for glutamate transport were estimated to be 67 μM and 152 nmol⁻¹ min⁻¹ mg⁻¹ of protein, respectively. The transport system showed structural specificity and stereospecificity and was strongly dependent on extracellular pH. Moreover, it could be stimulated by Mg²⁺, NH₄⁺, and Ca²⁺. In addition, the glutamate transporter in this strain was studied at the molecular level. As there was no important mutation of the transporter protein, it appeared that the differences of glutamate transporter properties between this strain and other B. subtilis strains were not due to the differences of the amino acid sequence and the structure of transporter protein. This is the first extensive report on the properties of glutamate transport system in γ-PGA-producing strain. PMID:21437781

  15. NEURONAL ACTIVITY REGULATES GLUTAMATE TRANSPORTER DYNAMICS IN DEVELOPING ASTROCYTES

    PubMed Central

    Benediktsson, A.M.; Marrs, G.S.; Tu, J.C.; Worley, P.F.; Rothstein, J.D.; Bergles, D.E.; Dailey, M.E.

    2011-01-01

    Glutamate transporters maintain a low ambient level of glutamate in the CNS and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here we examined the subcellular distribution and dynamic remodeling of the predominant glutamate transporter GLT-1 (EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT-1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin-1 positive synapses. GFP-GLT-1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine-like structures. Time-lapse 3D confocal imaging in tissue slices revealed that GFP-GLT-1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine-like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT-1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT-1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of glutamate transporters in developing astrocyte membranes and their position relative to synapses. PMID:22052455

  16. 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

  17. 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

    2016-01-01

    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

  18. 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

  19. Flow Synthesis and Biological Studies of an Analgesic Adamantane Derivative That Inhibits P2X7-Evoked Glutamate Release

    PubMed Central

    2013-01-01

    We report the biological evaluation of a class of adamantane derivatives, which were achieved via modified telescoped machine-assisted flow procedure. Among the series of compounds tested in this work, 5 demonstrated outstanding analgesic properties. This compound showed that its action was not mediated through direct interaction with opioid and/or cannabinoid receptors. Moreover, it did not display any significant anti-inflammatory properties. Experiments carried out on rat cerebrocortical purified synaptosomes indicated that 5 inhibits the P2X7-evoked glutamate release, which may contribute to its antinociceptive properties. Nevertheless, further experiments are ongoing to characterize the pharmacological properties and mechanism of action of this molecule. PMID:24900736

  20. Systemic pregabalin attenuates facial hypersensitivity and noxious stimulus-evoked release of glutamate in medullary dorsal horn in a rodent model of trigeminal neuropathic pain.

    PubMed

    Kumar, Naresh; Cherkas, Pavel S; Varathan, Vidya; Miyamoto, Makiko; Chiang, Chen Yu; Dostrovsky, Jonathan O; Sessle, Barry J; Coderre, Terence J

    2013-05-01

    Pregabalin is effective in treating many neuropathic pain conditions. However, the mechanisms of its analgesic effects remain poorly understood. The aim of the present study was to determine whether pregabalin suppresses facial mechanical hypersensitivity and evoked glutamate release in the medullary dorsal horn (MDH) in a rodent model of trigeminal neuropathic pain. Nociceptive mechanical sensitivity was assessed pre-operatively, and then post-operatively 1h following pregabalin or vehicle (saline) treatment on post-operative days 2 and 5 following infraorbital nerve transection (IONX). In addition, an in vivo microdialysis probe was inserted into the exposed medulla post-operatively and dialysate samples were collected. Glutamate release was then evoked by mustard oil (MO) application to the tooth pulp, and the effects of pregabalin or vehicle were examined on the MDH glutamate release. Glutamate concentrations in the dialysated samples were determined by HPLC, and data analyzed by ANOVA. IONX animals (but not control animals) showed facial mechanical hypersensitivity for several days post-operatively. In addition, tooth pulp stimulation with MO evoked a transient release of glutamate in the MDH of IONX animals. Compared to vehicle, administration of pregabalin significantly attenuated the facial mechanical hypersensitivity as well as the MO-evoked glutamate release in MDH. This study provides evidence in support of recent findings pointing to the usefulness of pregabalin in the treatment of orofacial neuropathic pain. PMID:23454190

  1. 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

  2. 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%.

  3. Distribution of vesicular glutamate transporters in the human brain

    PubMed Central

    Vigneault, Érika; Poirel, Odile; Riad, Mustapha; Prud'homme, Josée; Dumas, Sylvie; Turecki, Gustavo; Fasano, Caroline; Mechawar, Naguib; El Mestikawy, Salah

    2015-01-01

    Glutamate is the major excitatory transmitter in the brain. Vesicular glutamate transporters (VGLUT1-3) are responsible for uploading glutamate into synaptic vesicles. VGLUT1 and VGLUT2 are considered as specific markers of canonical glutamatergic neurons, while VGLUT3 is found in neurons previously shown to use other neurotransmitters than glutamate. Although there exists a rich literature on the localization of these glutamatergic markers in the rodent brain, little is currently known about the distribution of VGLUT1-3 in the human brain. In the present study, using subtype specific probes and antisera, we examined the localization of the three vesicular glutamate transporters in the human brain by in situ hybridization, immunoautoradiography and immunohistochemistry. We found that the VGLUT1 transcript was highly expressed in the cerebral cortex, hippocampus and cerebellum, whereas VGLUT2 mRNA was mainly found in the thalamus and brainstem. VGLUT3 mRNA was localized in scarce neurons within the cerebral cortex, hippocampus, striatum and raphe nuclei. Following immunoautoradiographic labeling, intense VGLUT1- and VGLUT2-immunoreactivities were observed in all regions investigated (cerebral cortex, hippocampus, caudate-putamen, cerebellum, thalamus, amygdala, substantia nigra, raphe) while VGLUT3 was absent from the thalamus and cerebellum. This extensive mapping of VGLUT1-3 in human brain reveals distributions that correspond for the most part to those previously described in rodent brains. PMID:25798091

  4. Glial glutamate transporter and glutamine synthetase regulate GABAergic synaptic strength in the spinal dorsal horn.

    PubMed

    Jiang, Enshe; Yan, Xisheng; Weng, Han-Rong

    2012-05-01

    Decreased GABAergic synaptic strength ('disinhibition') in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory post-synaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or miniature IPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate-glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial-neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions. PMID:22339645

  5. EFFECTS OF PERINATAL MONOSODIUM GLUTAMATE ADMINISTRATIONON VISUAL EVOKED POTENTIALS OF JUVENILE AND ADULT RATS

    EPA Science Inventory

    Administration of high doses of monosodium glutamate (MSG) to rats during the first postnatal week results in severe losses of retinal ganglion cells and interneurons in the retina. his study was conducted to determine what effect this severe retinal damage would have upon the on...

  6. 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

  7. 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

  8. Antipsychotic treatment modulates glutamate transport and NMDA receptor expression.

    PubMed

    Zink, Mathias; Englisch, Susanne; Schmitt, Andrea

    2014-11-01

    Schizophrenia patients often suffer from treatment-resistant cognitive and negative symptoms, both of which are influenced by glutamate neurotransmission. Innovative therapeutic strategies such as agonists at metabotropic glutamate receptors or glycin reuptake inhibitors try to modulate the brain's glutamate network. Interactions of amino acids with monoamines have been described on several levels, and first- and second-generation antipsychotic agents (FGAs, SGAs) are known to exert modulatory effects on the glutamatergic system. This review summarizes the current knowledge on effects of FGAs and SGAs on glutamate transport and receptor expression derived from pharmacological studies. Such studies serve as a control for molecular findings in schizophrenia brain tissue and are clinically relevant. Moreover, they may validate animal models for psychosis, foster basic research on antipsychotic substances and finally lead to a better understanding of how monoaminergic and amino acid neurotransmissions are intertwined. In the light of these results, important differences dependent on antipsychotic substances, dosage and duration of treatment became obvious. While some post-mortem findings might be confounded with multifold drug effects, others are unlikely to be influenced by antipsychotic treatment and could represent important markers of schizophrenia pathophysiology. In similarity to the convergence of toxic and psychotomimetic effects of dopaminergic, serotonergic and anti-glutamatergic substances, the therapeutic mechanisms of SGAs might merge on a yet to be defined molecular level. In particular, serotonergic effects of SGAs, such as an agonism at 5HT1A receptors, represent important targets for further clinical research. PMID:25214389

  9. The role of spinal nitric oxide and glutamate in nociceptive behaviour evoked by high-dose intrathecal morphine in rats.

    PubMed

    Watanabe, Chizuko; Sakurada, Tsukasa; Okuda, Kazuhiro; Sakurada, Chikai; Ando, Ryuichiro; Sakurada, Shinobu

    2003-12-01

    Injection of high-dose of morphine into the spinal lumbar intrathecal (i.t.) space of rats elicits a nociceptive behavioural syndrome characterized by periodic bouts of spontaneous agitation and severe vocalization. The induced behavioural response such as vocalization and agitation was observed dose-dependently by i.t. administration of morphine (125-500 nmol). Pretreatment with naloxone (s.c. and i.t.), an opioid receptor antagonist, failed to reverse the morphine-induced behavioural response. The excitatory effect of morphine was inhibited dose-dependently by pretreatment with 3-((+)2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist and MK-801, a non-competitive NMDA receptor antagonist. The non-selective nitric oxide (NO) synthase inhibitor N(G)-nitro L-arginine methyl ester (L-NAME) inhibited dose-dependently the behavioural response to high-dose i.t. morphine (500 nmol), whereas D-NAME was without affecting the response to high-dose i.t. morphine. In the present study, we measured NO metabolites (nitrite/nitrate) in the extracellular fluid of rat dorsal spinal cord using in vivo microdialysis. The i.t. injection of morphine (500 nmol) evoked significant increases in NO metabolites and glutamate from the spinal cord. Not only NO metabolites but also glutamate released by high-dose morphine were reduced significantly by pretreatment with L-NAME (400 nmol). Pretreatment with CPP and MK-801 showed a significant reduction of the NO metabolites and glutamate levels elevated by high-dose i.t. morphine. These results suggest that the excitatory action of high-dose i.t. morphine may be mediated by an NMDA-NO cascade in the spinal cord. PMID:14659510

  10. Time Course Analysis of the Effects of Botulinum Neurotoxin Type A on Pain and Vasomotor Responses Evoked by Glutamate Injection into Human Temporalis Muscles

    PubMed Central

    Bittencourt da Silva, Larissa; Kulas, Dolarose; Karshenas, Ali; Cairns, Brian E.; Bach, Flemming W.; Arendt-Nielsen, Lars; Gazerani, Parisa

    2014-01-01

    The effect of botulinum neurotoxin type A (BoNTA) on glutamate-evoked temporalis muscle pain and vasomotor responses was investigated in healthy men and women over a 60 day time course. Subjects participated in a pre-BoNTA session where their responses to injection of glutamate (1 M, 0.2 mL) and saline (0.2 mL) into the temporalis muscles were assessed. On Day 1, BoNTA (5 U) was injected into one temporalis muscle and saline into the contralateral temporalis muscle, in a randomized order. Subjects then received intramuscular injections of glutamate (1 M, 0.2 mL) into the left and right temporalis muscles at 3 h and subsequently 7, 30 and 60 days post-injection of BoNTA. Pain intensity, pain area, and neurogenic inflammation (skin temperature and skin blood perfusion) were recorded. Prior to BoNTA treatment, glutamate evoked significantly greater pain and vasomotor reactions (P < 0.001) than saline. BoNTA significantly reduced glutamate-evoked pain intensity (P < 0.05), pain area (P < 0.01), skin blood perfusion (P < 0.05), and skin temperature (P < 0.001). The inhibitory effect of BoNTA was present at 3 h after injection, peaked after 7 days and returned to baseline by 60 days. Findings from the present study demonstrated a rapid action of BoNTA on glutamate-evoked pain and neurogenic inflammation, which is in line with animal studies. PMID:24517906

  11. Impairment of Neuronal Glutamate Uptake and Modulation of the Glutamate Transporter GLT-1 Induced by Retinal Ischemia

    PubMed Central

    Varano, Giuseppe Pasquale; Milanese, Marco; Adornetto, Annagrazia; Nucci, Carlo; Bonanno, Giambattista; Morrone, Luigi Antonio; Corasaniti, Maria Tiziana; Bagetta, Giacinto

    2013-01-01

    Excitotoxicity has been implicated in the retinal neuronal loss in several ocular pathologies including glaucoma. Dysfunction of Excitatory Amino Acid Transporters is often a key component of the cascade leading to excitotoxic cell death. In the retina, glutamate transport is mainly operated by the glial glutamate transporter GLAST and the neuronal transporter GLT-1. In this study we evaluated the expression of GLAST and GLT-1 in a rat model of acute glaucoma based on the transient increase of intraocular pressure (IOP) and characterized by high glutamate levels during the reperfusion that follows the ischemic event associated with raised IOP. No changes were reported in GLAST expression while, at neuronal level, a reduction of glutamate uptake and of transporter reversal-mediated glutamate release was observed in isolated retinal synaptosomes. This was accompanied by modulation of GLT-1 expression leading to the reduction of the canonical 65 kDa form and upregulation of a GLT-1-related 38 kDa protein. These results support a role for neuronal transporters in glutamate accumulation observed in the retina following an ischemic event and suggest the presence of a GLT-1 neuronal new alternative splice variant, induced in response to the detrimental stimulus. PMID:23936321

  12. 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

  13. 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

  14. Dynamic regulation of glycine–GABA co-transmission at spinal inhibitory synapses by neuronal glutamate transporter

    PubMed Central

    Ishibashi, Hitoshi; Yamaguchi, Junya; Nakahata, Yoshihisa; Nabekura, Junichi

    2013-01-01

    Fast inhibitory neurotransmission in the central nervous system is mediated by γ-aminobutyric acid (GABA) and glycine, which are accumulated into synaptic vesicles by a common vesicular inhibitory amino acid transporter (VIAAT) and are then co-released. However, the mechanisms that control the packaging of GABA + glycine into synaptic vesicles are not fully understood. In this study, we demonstrate the dynamic control of the GABA–glycine co-transmission by the neuronal glutamate transporter, using paired whole-cell patch recording from monosynaptically coupled cultured spinal cord neurons derived from VIAAT-Venus transgenic rats. Short step depolarization of presynaptic neurons evoked unitary (cell-to-cell) inhibitory postsynaptic currents (IPSCs). Under normal conditions, the fractional contribution of postsynaptic GABA or glycine receptors to the unitary IPSCs did not change during a 1 h recording. Intracellular loading of GABA or glycine via a patch pipette enhanced the respective components of inhibitory transmission, indicating the importance of the cytoplasmic concentration of inhibitory transmitters. Raised extracellular glutamate levels increased the amplitude of GABAergic IPSCs but reduced glycine release by enhancing glutamate uptake. Similar effects were observed when presynaptic neurons were intracellularly perfused with glutamate. Interestingly, high-frequency trains of stimulation decreased glycinergic IPSCs more than GABAergic IPSCs, and repetitive stimulation occasionally failed to evoke glycinergic but not GABAergic IPSCs. The present results suggest that the enhancement of GABA release by glutamate uptake may be advantageous for rapid vesicular refilling of the inhibitory transmitter at mixed GABA/glycinergic synapses and thus may help prevent hyperexcitability. PMID:23690564

  15. 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

  16. Role of astrocytic glutamate transporter in alcohol use disorder.

    PubMed

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

    2016-03-22

    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

  17. L-Leucine prevents ammonia-induced changes in glutamate receptors in the brain and in visual evoked potentials in the rabbit.

    PubMed

    Ferenci, P; Pappas, C S; Jones, E A

    1984-01-01

    The effect of L-leucine on glutamate receptors in the brain and on visual evoked potentials was studied in hyperammonemic rabbits. Hyperammonemia was induced by the iv infusion of 2.1 mmol NH4Cl/h over 3 hr. Hyperammonemia was followed by a 116% increase in the specific binding of 3H-glutamate to synaptic membranes prepared from the hippocampus. This increase was due to both an increase in the affinity and in the density of the glutamate receptor. The simultaneous infusion of L-leucine (6.7 mmol/hr) completely prevented the ammonia-induced increase in the specific glutamate binding, whereas L-valine and D-leucine had no effect. Hyperammonemia was also associated with typical, reproducible, and reversible changes in visual evoked potentials. The amplitudes of the first negative and the second positive peak decreased, whereas the latencies of these peaks remained unchanged. The simultaneous infusion of L-leucine completely prevented these changes. These findings indicate (1) that L-leucine prevents ammonia-induced changes in the glutamatergic excitatory neurotransmitter system and (2) that pharmacologic doses of L-leucine modulate the effects of hyperammonemia on central neurotransmission as assessed by visual evoked potentials. A causal relationship between the effects of L-leucine on ammonia-induced changes in glutamate receptors and in visual evoked potentials cannot be inferred with confidence. These findings provide a potential alternative explanation for the apparent beneficial effects of infusions of branched-chain amino acids on hepatic encephalography in patients with chronic liver disease. PMID:6151602

  18. Cortical Metabotropic Glutamate Receptors Contribute to Habituation of a Simple Odor-Evoked Behavior

    PubMed Central

    Best, Aaron R.; Thompson, Jason V.; Fletcher, Max L.; Wilson, Donald A.

    2008-01-01

    Defining the circuits that are involved in production and cessation of specific behaviors is an ultimate goal of neuroscience. Short-term behavioral habituation is the response decrement observed in many behaviors that occurs during repeated presentation of non-reinforced stimuli. Within a number of invertebrate models of short-term behavioral habituation, depression of a defined synapse has been implicated as the mechanism. However, the synaptic mechanisms of short-term behavioral habituation have not been identified within mammals. We have shown previously that a presynaptic metabotropic glutamate receptor (mGluR)-dependent depression of synapses formed by olfactory bulb afferents to the piriform (olfactory) cortex significantly contributes to adaptation of cortical odor responses. Here we show that blockade of mGluRs within the olfactory cortex of awake, behaving rats diminishes habituation of a simple odor-induced behavior, strongly implicating a central mechanism for sensory gating in olfaction. PMID:15758159

  19. 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. PMID:26071109

  20. Regional brain glutamate transport in rats at normal and raised concentrations of circulating glutamate.

    PubMed

    Hawkins, R A; DeJoseph, M R; Hawkins, P A

    1995-08-01

    The permeability of the blood-brain barrier to glutamate was measured by quantitative autoradiography in brains of control rats (average plasma glutamate concentration of 95 microns) and rats infused with glutamate (average plasma glutamate concentration of 837 microns). Measurements of glutamate permeability were initiated by the injection of [14C]glutamate and stopped at 1 min to avoid the accumulation of [14C]glutamate metabolites. Glutamate entered the brain at a slow rate, with an average permeability-surface area product of 7 microliters.min-g-1, except in those areas known to have fenestrated capillaries. Glutamate accumulated in the choroid plexus of ventricles, but did not seem to enter the cerebrospinal fluid in detectable amounts regardless of the circulating concentration. Glutamate accumulated in circumventricular organs, such as the median eminence, where the radioactivity was localized without detectable spread. Infusion of glutamate to create high plasma concentrations did not result in greater spread of [14C]glutamate beyond the immediate vicinity of the circumventricular organs. PMID:7648616

  1. Glutamate-evoked release of endogenous brain dopamine: inhibition by an excitatory amino acid antagonist and an enkephalin analogue.

    PubMed Central

    Jhamandas, K.; Marien, M.

    1987-01-01

    The present study examined the effect of a selective delta-opioid receptor agonist [D-Ala2-D-Leu5] enkephalin (DADL) on the spontaneous and the L-glutamic acid (L-Glu)-evoked release of endogenous dopamine from superfused slices of rat caudate-putamen. The amount of dopamine in slice superfusates was measured by a sensitive method employing high-performance liquid chromatography with electrochemical detection (h.p.l.c.-e.d.) after a two-step separation procedure. The spontaneous release of endogenous dopamine was partially dependent on Ca2+, enhanced in Mg2+-free superfusion medium, partially reduced by tetrodotoxin (TTX, 0.3 microM), partially reduced by the putative excitatory amino acid receptor antagonist DL-2-amino-7-phosphonoheptanoic acid (DL-APH, 1 mM), and increased 10 fold by the dopamine uptake blocker, nomifensine (10 microM). DADL (5 and 50 nM) did not significantly affect spontaneous dopamine release. L-Glu (0.1-10 mM) produced a concentration-dependent release of endogenous dopamine from slices of caudate-putamen. This effect was Ca2+-dependent, strongly inhibited by 1.2 mM Mg2+, attenuated by DL-APH (1 mM), attenuated by TTX (0.3 microM), and enhanced by nomifensine (10 microM). In the presence of nomifensine DADL (50 nM) reduced significantly the L-Glu-evoked release of endogenous dopamine by 20%. The inhibitory effect of DADL was blocked by 10 microM naloxone. These results indicate that L-Glu stimulates the Ca2+-dependent release of endogenous dopamine in the caudate-putamen by activation of N-methy-D-aspartate-type of excitatory amino acid receptors. This release can be selectively modified by the delta-opioid agonist DADL in a naloxone-sensitive manner. PMID:2884003

  2. 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

  3. 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.

  4. 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

  5. Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment.

    PubMed

    MacNamee, Sarah E; Liu, Kendra E; Gerhard, Stephan; Tran, Cathy T; Fetter, Richard D; Cardona, Albert; Tolbert, Leslie P; Oland, Lynne A

    2016-07-01

    Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron-glia interactions. Here, we show the first electrophysiological recordings from Drosophila astrocytes and characterize their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic premotor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via excitatory amino acid transporter 1 (Eaat1), and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory postsynaptic currents in motor neurons. The neuronal synapses were always located within 1 μm of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters. J. Comp. Neurol. 524:1979-1998, 2016. © 2016 Wiley Periodicals, Inc. PMID:27073064

  6. 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

  7. 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

  8. 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.

  9. Retinal Glial Cell Glutamate Transporter is Coupled to an Anionic Conductance

    NASA Astrophysics Data System (ADS)

    Eliasof, Scott; Jahr, Craig E.

    1996-04-01

    Application of L-glutamate to retinal glial (Muller) cells results in an inwardly rectifying current due to the net influx of one positive charge per molecule of glutamate transported into the cell. However, at positive potentials an outward current can be elicited by glutamate. This outward current is eliminated by removal of external chloride ions. Substitution of external chloride with the anions thiocyanate, perchlorate, nitrate, and iodide, which are known to be more permeant at other chloride channels, results in a considerably larger glutamate-elicited outward current at positive potentials. The large outward current in external nitrate has the same ionic dependence, apparent affinity for L-glutamate, and pharmacology as the glutamate transporter previously reported to exist in these cells. Varying the concentration of external nitrate shifts the reversal potential in a manner consistent with a conductance permeable to nitrate. Together, these results suggest that the glutamate transporter in retinal glial cells is associated with an anionic conductance. This anionic conductance may be important for preventing a reduction in the rate of transport due the depolarization that would otherwise occur as a result of electrogenic glutamate uptake.

  10. Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3.

    PubMed

    Seal, Rebecca P; Akil, Omar; Yi, Eunyoung; Weber, Christopher M; Grant, Lisa; Yoo, Jong; Clause, Amanda; Kandler, Karl; Noebels, Jeffrey L; Glowatzki, Elisabeth; Lustig, Lawrence R; Edwards, Robert H

    2008-01-24

    The expression of unconventional vesicular glutamate transporter VGLUT3 by neurons known to release a different classical transmitter has suggested novel roles for signaling by glutamate, but this distribution has raised questions about whether the protein actually contributes to glutamate release. We now report that mice lacking VGLUT3 are profoundly deaf due to the absence of glutamate release from hair cells at the first synapse in the auditory pathway. The early degeneration of some cochlear ganglion neurons in knockout mice also indicates an important developmental role for the glutamate released by hair cells before the onset of hearing. In addition, the mice exhibit primary, generalized epilepsy that is accompanied by remarkably little change in ongoing motor behavior. The glutamate release conferred by expression of VGLUT3 thus has an essential role in both function and development of the auditory pathway, as well as in the control of cortical excitability. PMID:18215623

  11. The high-mobility group box 1 cytokine induces transporter-mediated release of glutamate from glial subcellular particles (gliosomes) prepared from in situ-matured astrocytes.

    PubMed

    Bonanno, Giambattista; Raiteri, Luca; Milanese, Marco; Zappettini, Simona; Melloni, Edon; Pedrazzi, Marco; Passalacqua, Mario; Tacchetti, Carlo; Usai, Cesare; Sparatore, Bianca

    2007-01-01

    The multifunctional protein high-mobility group box 1 (HMGB1) is expressed in restricted areas of adult brain where it can act as a proinflammatory cytokine. We report here that HMGB1 affects CNS transmission by inducing glutamatergic release from glial (gliosomes) but not neuronal (synaptosomes) resealed subcellular particles isolated from mouse cerebellum and hippocampus. Confocal microscopy showed that gliosomes are enriched with glia-specific proteins such as GFAP and S-100, but not with neuronal proteins such as PSD-95, MAP-2, and beta-tubulin III. Furthermore, gliosomes exhibit labeling neither for integrin-alphaM nor for myelin basic protein, specific for microglia and oligodendrocytes, respectively. The gliosomal fraction contains proteins of the exocytotic machinery coexisting with GFAP. Consistent with ultrastructural analysis, several approximately 30-nm nonclustered vesicles are present in the gliosome cytoplasm. Finally, gliosomes represent functional organelles that actively export glutamate when subjected to releasing stimuli, such as ionomycin or ATP, by mechanisms involving extracellular Ca(2+) and Ca(2+) release from intracellular stores. HMGB1-induced release of the stable glutamate analogue [(3)H]d-aspartate and endogenous glutamate form gliosomes, whereas nerve terminals were insensitive to the protein. The HMGB1-evoked release of glutamate was independent on modifications of cytosolic Ca(2+) concentration, but it was blocked by dl-threo-beta-benzyloxyaspartate, suggesting the involvement of transporter-mediated release mechanisms. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 does not block the HMGB1 effect, indicating a role for the glial glutamate-aspartate transporter (GLAST) subtype in this response. HMGB1 bind to gliosomes but not to synaptosomes and can physically interact with GLAST and receptor for advanced glycation end products (RAGE). Taken together, these results suggest that the HMGB1 cytokine

  12. 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

  13. Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

    PubMed Central

    Rinehart, C A; Hubbard, J S

    1976-01-01

    When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate. PMID:956126

  14. 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

  15. 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. PMID:26858177

  16. Exposure to altered gravity conditions results in hypoxia-related enhancement of the presynaptic transporter-mediated release of glutamate.

    NASA Astrophysics Data System (ADS)

    Borisova, Tatiana

    High-affinity Na+-dependent glutamate transporters locate in the plasma membrane and maintain the low concentration of glutamate in synaptic cleft by the uptake of glutamate into neurons. Under hypoxic conditions glutamate transporters contribute to the glutamate release due to functioning in reverse mode. The release of glutamate via reverse-operated Na+-dependent glutamate transporters was investigated in brain synaptosomes under conditions of centrifugeinduced hypergravity. Flow cytometric analisis revealed similarity in the size and cytoplasmic granularity of control and hypergravity synaptosomes. Protonophore FCCP dissipates the proton gradient across synaptic vesicle thus synaptic vesicles are not able to keep glutamate inside. 1 microM FCCP induced the release of 4. 8 ±1. 0 % and 8. 0 ±1. 0 % of total accumulated synaptosomal label in control and G-loaded animals, respectively. Ca 2+-independent high- KCl stimulated L-[14C]glutamate release from synaptosomes preliminary treated with FCCP increased considerably from 27. 0 ± 2. 2 % to 35. 0 ± 2. 3 % after centrifuge-induced hypergravity. No-transportable inhibitor of glutamate transporter DL-threo-beta-benzyloxyaspartate was found to inhibit high-KCl and FCCP-stimulated release of L-[14C]glutamate, thus the release was concluded to occur due to reversal of glutamate transporters. We have also found the inhibition of the activity of Na \\ K ATPase in the plasma membrane of synaptosomes after hypergravity that might also contribute to the enhancement of the transporter-mediated release of glutamate. These hypergravity-induced alterations in the transporter-mediated release of glutamate were suggested to correlate with the hypoxic injury of neurons. The changes we have revealed for the transporter-mediated release of glutamate may lead to mental disorders, upcoming seizures and neurotoxicity under hypergravity conditions.

  17. 1,25-Dihydroxyvitamin D induces the glutamate transporter SLC1A1 and alters glutamate handling in non-transformed mammary cells.

    PubMed

    Beaudin, Sarah; Welsh, JoEllen

    2016-03-15

    Genomic profiling of immortalized human mammary epithelial (hTERT-HME1) cells identified several metabolic genes, including the membrane glutamate transporter, SLC1A1, as 1,25-dihydroxyvitamin D3 (1,25D) regulated. In these studies we have surveyed the effects of 1,25D on known glutamate transporters and evaluated its impact on cellular glutamate handling. We confirm that expression of SLC1A1 and all of its known transcript variants are significantly upregulated in hTERT-HME1 cells following 1,25D treatment. Expression of the full-length cognate protein, EAAT3, is correspondingly increased in 1,25D treated hTERT-HME1 cells. Under the same conditions, the expression of two other glutamate transporters--SLC1A6 (EAAT4) and SLC1A2 (EAAT2 or GLT-1)--is enhanced by 1,25D while that of SLC1A3 (EAAT1 or GLAST) and SLC7A11 (xCT) is decreased. Glutamate is not essential for growth of hTERT-HME1 cells, and supplemental glutamate (up to 0.5 mM) does not abrogate the growth inhibitory effects of 1,25D. These data suggest that extracellular glutamate is not a major contributor to cellular energy metabolism in hTERT-HME1 cells under basal conditions and that the growth inhibitory effects of 1,25D are not secondary to its effects on glutamate handling. Instead, the effects of 1,25D on glutamate transporters translated to a decrease in cellular glutamate concentration and an increase in media glutamate concentration, suggesting that one or more of these transporters functions to export glutamate in response to 1,25D exposure. The reduced cellular glutamate concentration may also reflect its incorporation into the cellular glutathione (GSH) pool, which is increased upon 1,25D treatment. In support of this concept, the expression of GCLC (which codes for the rate-limiting enzyme in GSH synthesis) and genes which generate reducing equivalents in the form of NADPH (ie, G6PD, PGD, IDH2) are elevated in 1,25D-treated cells. Taken together, these data identify 1,25D as a physiological

  18. 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. PMID:26037264

  19. P301L tau expression affects glutamate release and clearance in the hippocampal trisynaptic pathway.

    PubMed

    Hunsberger, Holly C; Rudy, Carolyn C; Batten, Seth R; Gerhardt, Greg A; Reed, Miranda N

    2015-01-01

    Individuals at risk of developing Alzheimer's disease (AD) often exhibit hippocampal hyperexcitability. A growing body of evidence suggests that perturbations in the glutamatergic tripartite synapse may underlie this hyperexcitability. Here, we used a tau mouse model of AD (rTg(TauP301L)4510) to examine the effects of tau pathology on hippocampal glutamate regulation. We found a 40% increase in hippocampal vesicular glutamate transporter, which packages glutamate into vesicles, and has previously been shown to influence glutamate release, and a 40% decrease in hippocampal glutamate transporter 1, the major glutamate transporter responsible for removing glutamate from the extracellular space. To determine whether these alterations affected glutamate regulation in vivo, we measured tonic glutamate levels, potassium-evoked glutamate release, and glutamate uptake/clearance in the dentate gyrus, cornu ammonis 3(CA3), and cornu ammonis 1(CA1) regions of the hippocampus. P301L tau expression resulted in a 4- and 7-fold increase in potassium-evoked glutamate release in the dentate gyrus and CA3, respectively, and significantly decreased glutamate clearance in all three regions. Both release and clearance correlated with memory performance in the hippocampal-dependent Barnes maze task. Alterations in mice expressing P301L were observed at a time when tau pathology was subtle and before readily detectable neuron loss. These data suggest novel mechanisms by which tau may mediate hyperexcitability. Pre-synaptic vesicular glutamate transporters (vGLUTs) package glutamate into vesicles before exocytosis into the synaptic cleft. Once in the extracellular space, glutamate acts on glutamate receptors. Glutamate is removed from the extracellular space by excitatory amino acid transporters, including GLT-1, predominantly localized to glia. P301L tau expression increases vGLUT expression and glutamate release, while also decreasing GLT-1 expression and glutamate clearance. PMID

  20. On the potential role of glutamate transport in mental fatigue.

    PubMed

    Rönnbäck, Lars; Hansson, Elisabeth

    2004-11-01

    Mental fatigue, with decreased concentration capacity, is common in neuroinflammatory and neurodegenerative diseases, often appearing prior to other major mental or physical neurological symptoms. Mental fatigue also makes rehabilitation more difficult after a stroke, brain trauma, meningitis or encephalitis. As increased levels of proinflammatory cytokines are reported in these disorders, we wanted to explore whether or not proinflammatory cytokines could induce mental fatigue, and if so, by what mechanisms.It is well known that proinflammatory cytokines are increased in major depression, "sickness behavior" and sleep deprivation, which are all disorders associated with mental fatigue. Furthermore, an influence by specific proinflammatory cytokines, such as interleukin (IL)-1, on learning and memory capacities has been observed in several experimental systems. As glutamate signaling is crucial for information intake and processing within the brain, and due to the pivotal role for glutamate in brain metabolism, dynamic alterations in glutamate transmission could be of pathophysiological importance in mental fatigue. Based on this literature and observations from our own laboratory and others on the role of astroglial cells in the fine-tuning of glutamate neurotransmission we present the hypothesis that the proinflammatory cytokines tumor necrosis factor-alpha, IL-1beta and IL-6 could be involved in the pathophysiology of mental fatigue through their ability to attenuate the astroglial clearance of extracellular glutamate, their disintegration of the blood brain barrier, and effects on astroglial metabolism and metabolic supply for the neurons, thereby attenuating glutamate transmission. To test whether our hypothesis is valid or not, brain imaging techniques should be applied with the ability to register, over time and with increasing cognitive loading, the extracellular concentrations of glutamate and potassium (K+) in humans suffering from mental fatigue. At

  1. Differential expression of vesicular glutamate transporters by vagal afferent terminals in rat nucleus of the solitary tract: projections from the heart preferentially express vesicular glutamate transporter 1.

    PubMed

    Corbett, E K A; Sinfield, J K; McWilliam, P N; Deuchars, J; Batten, T F C

    2005-01-01

    The central projections and neurochemistry of vagal afferent neurones supplying the heart in the rat were investigated by injecting cholera toxin B-subunit into the pericardium. Transganglionically transported cholera toxin B-subunit was visualized in the medulla oblongata in axons and varicosities that were predominantly aggregated in the dorsomedial, dorsolateral, ventrolateral and commissural subnuclei of the caudal nucleus of the solitary tract. Unilateral vagal section in control rats prevented cholera toxin B-subunit labeling on the ipsilateral side of the nucleus of the solitary tract. Fluorescent and electron microscopic dual labeling showed colocalization of immunoreactivity for vesicular glutamate transporter 1, but only rarely vesicular glutamate transporters 2 or 3 with cholera toxin B-subunit in terminals in nucleus of the solitary tract, suggesting that cardiac vagal axons release glutamate as a neurotransmitter. In contrast, populations of vagal afferent fibers labeled by injection of cholera toxin B-subunit, tetra-methylrhodamine dextran or biotin dextran amine into the aortic nerve, stomach or nodose ganglion colocalized vesicular glutamate transporter 2 more frequently than vesicular glutamate transporter 1. The presence of other neurochemical markers of primary afferent neurones was examined in nucleus of the solitary tract axons and nodose ganglion cells labeled by pericardial cholera toxin B-subunit injections. Immunoreactivity for a 200-kDa neurofilament protein in many large, cholera toxin B-subunit-labeled nodose ganglion cells indicated that the cardiac afferent fibers labeled are mostly myelinated, whereas binding of Griffonia simplicifolia isolectin B4 to fewer small cholera toxin B-subunit-labeled ganglion cells suggested that tracer was also taken up by some non-myelinated axons. A few labeled nucleus of the solitary tract axons and ganglion cells were positive for substance P and calcitonin gene-related peptide, which are considered as

  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. PMID:26416546

  3. Functional and morphological characterization of glutamate transporters in the rat locus coeruleus

    PubMed Central

    Medrano, M C; Gerrikagoitia, I; Martínez-Millán, L; Mendiguren, A; Pineda, J

    2013-01-01

    Background and Purpose Excitatory amino acid transporters (EAATs) in the CNS contribute to the clearance of glutamate released during neurotransmission. The aim of this study was to explore the role of EAATs in the regulation of locus coeruleus (LC) neurons by glutamate. Experimental Approach We measured the effect of different EAAT subtype inhibitors/enhancers on glutamate- and KCl-induced activation of LC neurons in rat slices. EAAT2–3 expression in the LC was also characterized by immunohistochemistry. Key Results The EAAT2–5 inhibitor DL-threo-β-benzyloxaspartic acid (100 μM), but not the EAAT2, 4, 5 inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (100 μM) or the EAAT2 inhibitor dihydrokainic acid (DHK; 100 μM), enhanced the glutamate- and KCl-induced activation of the firing rate of LC neurons. These effects were blocked by ionotropic, but not metabotrobic, glutamate receptor antagonists. DHK (100 μM) was the only EAAT inhibitor that increased the spontaneous firing rate of LC cells, an effect that was due to inhibition of EAAT2 and subsequent AMPA receptor activation. Chronic treatment with ceftriaxone (200 mg·kg−1 i.p., once daily, 7 days), an EAAT2 expression enhancer, increased the actions of glutamate and DHK, suggesting a functional impact of EAAT2 up-regulation on the glutamatergic system. Immuhistochemical data revealed the presence of EAAT2 and EAAT3 surrounding noradrenergic neurons and EAAT2 on glial cells in the LC. Conclusions and Implications These results remark the importance of EAAT2 and EAAT3 in the regulation of rat LC by glutamate. Neuronal EAAT3 would be responsible for terminating the action of synaptically released glutamate, whereas glial EAAT2 would regulate tonic glutamate concentrations in this nucleus. PMID:23638698

  4. 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

  5. Increased extracellular glutamate evoked by 1-methyl-4-phenylpyridinium [MPP(+)] in the rat striatum is not essential for dopaminergic neurotoxicity and is not derived from released glutathione.

    PubMed

    Foster, S B; Tang, H; Miller, K E; Dryhurst, G

    2005-01-01

    A number of studies have implicated the interactions of the excitatory amino acid L-glutamate (Glu) with its ionotropic and metabotropic receptors as important components of the mechanism underlying the dopaminergic neurotoxicity of 1-methyl-4-phenylpyridinium [MPP(+)]. Furthermore, microdialysis experiments have demonstrated that perfusion of relatively high concentrations of MPP(+) into the rat striatum evoke a delayed, massive release of Glu. Interestingly, perfusion of MPP(+) also mediates a similar release of glutathione (GSH). Together, these observations raise the possibility that the rise of extracellular Glu mediated by MPP(+) may be the result of hydrolysis of released GSH by gamma-glutamyl transpeptidase (gamma-GT). In the present investigation it is demonstrated that perfusions of solutions of 0.7 and 1.3 mM MPP(+) dissolved in artificial cerebrospinal fluid into the rat striatum evoke neurotoxic damage to dopaminergic terminals, assessed by both a two-day test/challenge procedure and tyrosine hydroxylase immunoreactivity, but without the release of Glu. Perfusions of 2.5 mM MPP(+) cause more extensive dopaminergic neurotoxicity and a dose-dependent release of Glu. However, neither this release of Glu nor MPP(+)-induced dopaminergic neurotoxicity are blocked by the irreversible gamma-GT inhibitor acivicin. Together, these observations indicate that a rise of extracellular levels of Glu is not essential for the dopaminergic neurotoxicity of MPP(+). Furthermore, the rise of extracellular Glu caused by perfusion of 2.5 mM MPP(+) is not the result of the gamma-GT-mediated hydrolysis of released GSH. It is possible that the rise of extracellular levels of Glu, L-aspartate, L-glycine and L-taurine evoked by perfusions of 2.5 mM MPP(+) into the rat striatum may reflect, at least in part, the release of these amino acids from astrocytes. PMID:16179262

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

    PubMed

    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

  7. 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

  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. The nuclear receptor FXR regulates hepatic transport and metabolism of glutamine and glutamate.

    PubMed

    Renga, Barbara; Mencarelli, Andrea; Cipriani, Sabrina; D'Amore, Claudio; Zampella, Angela; Monti, Maria Chiara; Distrutti, Eleonora; Fiorucci, Stefano

    2011-11-01

    Hepatic transport and metabolism of glutamate and glutamine are regulated by intervention of several proteins. Glutamine is taken up by periportal hepatocytes and is the major source of ammonia for urea synthesis and glutamate for N-acetylglutamate (NAG) synthesis, which is catalyzed by the N-acetylglutamate synthase (NAGS). Glutamate is taken up by perivenous hepatocytes and is the main source for the synthesis of glutamine, catalyzed by glutamine synthase (GS). Accumulation of glutamate and ammonia is a common feature of chronic liver failure, but mechanism that leads to failure of the urea cycle in this setting is unknown. The Farnesoid X Receptor (FXR) is a bile acid sensor in hepatocytes. Here, we have investigated its role in the regulation of the metabolism of both glutamine and glutamate. In vitro studies in primary cultures of hepatocytes from wild type and FXR(-/-) mice and HepG2 cells, and in vivo studies, in FXR(-/-) mice as well as in a rodent model of hepatic liver failure induced by carbon tetrachloride (CCl(4)), demonstrate a role for FXR in regulating this metabolism. Further on, promoter analysis studies demonstrate that both human and mouse NAGS promoters contain a putative FXRE, an ER8 sequence. EMSA, ChIP and luciferase experiments carried out to investigate the functionality of this sequence demonstrate that FXR is essential to induce the expression of NAGS. In conclusion, FXR activation regulates glutamine and glutamate metabolism and FXR ligands might have utility in the treatment of hyperammonemia states. PMID:21757002

  10. 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...

  11. Tuning the ion selectivity of glutamate transporter-associated uncoupled conductances.

    PubMed

    Cater, Rosemary J; Vandenberg, Robert J; Ryan, Renae M

    2016-07-01

    The concentration of glutamate within a glutamatergic synapse is tightly regulated by excitatory amino acid transporters (EAATs). In addition to their primary role in clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl(-) conductance. This conductance is activated by the binding of substrate and Na(+), but the direction of Cl(-) flux is independent of the rate or direction of substrate transport; thus, the two processes are thermodynamically uncoupled. A recent molecular dynamics study of the archaeal EAAT homologue GltPh (an aspartate transporter from Pyrococcus horikoshii) identified an aqueous pore at the interface of the transport and trimerization domains, through which anions could permeate, and it was suggested that an arginine residue at the most restricted part of this pathway might play a role in determining anion selectivity. In this study, we mutate this arginine to a histidine in the human glutamate transporter EAAT1 and investigate the role of the protonation state of this residue on anion selectivity and transporter function. Our results demonstrate that a positive charge at this position is crucial for determining anion versus cation selectivity of the uncoupled conductance of EAAT1. In addition, because the nature of this residue influences the turnover rate of EAAT1, we reveal an intrinsic link between the elevator movement of the transport domain and the Cl(-) channel. PMID:27296367

  12. Homeostasis of the astrocytic glutamate transporter GLT-1 is altered in mouse models of Lafora disease.

    PubMed

    Muñoz-Ballester, Carmen; Berthier, Arnaud; Viana, Rosa; Sanz, Pascual

    2016-06-01

    Lafora disease (LD, OMIM 254780) is a fatal rare disorder characterized by epilepsy and neurodegeneration. Although in recent years a lot of information has been gained on the molecular basis of the neurodegeneration that accompanies LD, the molecular basis of epilepsy is poorly understood. Here, we present evidence indicating that the homeostasis of glutamate transporter GLT-1 (EAAT2) is compromised in mouse models of LD. Our results indicate that primary astrocytes from LD mice have reduced capacity of glutamate transport, probably because they present a reduction in the levels of the glutamate transporter at the plasma membrane. On the other hand, the overexpression in cellular models of laforin and malin, the two proteins related to LD, results in an accumulation of GLT-1 (EAAT2) at the plasma membrane and in a severe reduction of the ubiquitination of the transporter. All these results suggest that the laforin/malin complex slows down the endocytic recycling of the GLT-1 (EAAT2) transporter. Since, defects in the function of this transporter lead to excitotoxicity and epilepsy, we suggest that the epilepsy that accompanies LD could be due, at least in part, to deficiencies in the function of the GLT-1 (EAAT2) transporter. PMID:26976331

  13. I(2)01810 is a novel type of glutamate transporter that is responsible for megamitochondrial formation

    PubMed Central

    Shim, Myoung Sup; Kim, Jin Young; Lee, Kwang Hee; Jung, Hee Kyoung; Carlson, Bradley A.; Xu, Xue-Ming; Hatfield, Dolph L.; Lee, Byeong Jae

    2012-01-01

    l(2)01810 causes glutamine-dependent megamitochondrial formation when it is overexpressed in Drosophila cells. In the present study, we elucidated the function of l(2)01810 during megamitochondrial formation. The overexpression of l(2)01810 and the inhibition of glutamine synthesis showed that l(2)01810 is involved in the accumulation of glutamate. l(2)01810 was predicted to contain transmembrane domains and was found to be localized to the plasma membrane. By using 14C-labelled glutamate, l(2)01810 was confirmed to uptake glutamate into Drosophila cells with high affinity (Km =69.4 µM). Also, l(2)01810 uptakes glutamate in a Na+ -independent manner. Interestingly, however, this uptake was not inhibited by cystine, which is a competitive inhibitor of Na+ -independent glutamate transporters, but by aspartate. A signal peptide consisting of 34 amino acid residues targeting to endoplasmic reticulum was predicted at the N-terminus of l(2)01810 and this signal peptide is essential for the protein’s localization to the plasma membrane. In addition, l(2)01810 has a conserved functional domain of a vesicular-type glutamate transporter, and Arg146 in this domain was found to play a key role in glutamate transport and megamitochondrial formation. These results indicate that l(2)01810 is a novel type of glutamate transporter and that glutamate uptake is a rate-limiting step for megamitochondrial formation. PMID:21728998

  14. In vitro evidence for the brain glutamate efflux hypothesis: brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate.

    PubMed

    Helms, Hans Christian; Madelung, Rasmus; Waagepetersen, Helle Sønderby; Nielsen, Carsten Uhd; Brodin, Birger

    2012-05-01

    The concentration of the excitotoxic amino acid, L-glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood-brain barrier endothelium in brain L-glutamate homeostasis. Transendothelial transport- and accumulation studies of (3) H-L-glutamate, (3) H-L-aspartate, and (3) H-D-aspartate in an electrically tight bovine endothelial/rat astrocyte blood-brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 ± 70 Ω cm(2) , and (14) C-D-mannitol permeability values of 0.88 ± 0.13 × 10(-6) cm s(-1) . Unidirectional flux studies showed that L-aspartate and L-glutamate, but not D-aspartate, displayed polarized transport in the brain-to-blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a K(m) of 69 ± 15 μM and a J(max) of 44 ± 3.1 pmol min(-1) cm(-2) for L-aspartate and a K(m) of 138 ± 49 μM and J(max) of 28 ± 3.1 pmol min(-1) cm(-2) for L-glutamate. The EAAT inhibitor, DL-threo-ß-Benzyloxyaspartate, inhibited transendothelial brain-to-blood fluxes of L-glutamate and L-aspartate. Expression of EAAT-1 (Slc1a3), -2 (Slc1a2), and -3 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT-1 and -3 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood-brain barrier itself may participate in regulating brain L-glutamate concentrations. PMID:22392649

  15. 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. PMID:9419055

  16. UBIQUITINATION-MEDIATED INTERNALIZATION AND DEGRADATION OF THE ASTROGLIAL GLUTAMATE TRANSPORTER, GLT-1

    PubMed Central

    Sheldon, Amanda L.; González, Marco I.; Krizman-Genda, Elizabeth N.; Susarla, Bala T.S.; Robinson, Michael B.

    2008-01-01

    Sodium-dependent glutamate uptake is essential for limiting excitotoxicity, and dysregulation of this process has been implicated in a wide array of neurological disorders. The majority of forebrain glutamate uptake is mediated by the astroglial glutamate transporter, GLT-1. We and others have shown that this transporter undergoes endocytosis and degradation in response to activation of protein kinase C (PKC), however, the mechanisms involved remain unclear. In the current study, transfected C6 glioma cells or primary cortical cultures were used to show that PKC activation results in incorporation of ubiquitin into GLT-1 immunoprecipitates. Mutation of all 11 lysine residues in the amino and carboxyl-terminal domains to arginine (11R) abolished this signal. Selective mutation of the 7 lysine residues in the carboxyl terminus (C7K-R) did not eliminate ubiquitination, but it completely blocked PKC-dependent internalization and degradation. Two families of variants of GLT-1 were prepared with various lysine residues mutated to Arginine. Analyses of these constructs indicated that redundant lysine residues in the carboxyl terminus were sufficient for the appearance of ubiquitinated product and degradation of GLT-1. Together these data define a novel mechanism by which the predominant forebrain glutamate transporter can be rapidly targeted for degradation. PMID:18805448

  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. 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

  19. Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM l-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells

    PubMed Central

    Yanagisawa, Tatsuo; Kim, Kwang Sik; Yokoyama, Shigeyuki; Ohnishi, Makoto

    2015-01-01

    We previously reported that Neisseria meningitidis internalization into human brain microvasocular endothelial cells (HBMEC) was triggered by the influx of extracellular l-glutamate via the GltT-GltM l-glutamate ABC transporter, but the underlying mechanism remained unclear. We found that the ΔgltT ΔgltM invasion defect in assay medium (AM) was alleviated in AM without 10% fetal bovine serum (FBS) [AM(−S)]. The alleviation disappeared again in AM(−S) supplemented with 500 μM glutamate. Glutamate uptake by the ΔgltT ΔgltM mutant was less efficient than that by the wild-type strain, but only upon HBMEC infection. We also observed that both GltT-GltM-dependent invasion and accumulation of ezrin, a key membrane-cytoskeleton linker, were more pronounced when N. meningitidis formed larger colonies on HBMEC under physiological glutamate conditions. These results suggested that GltT-GltM-dependent meningococcal internalization into HBMEC might be induced by the reduced environmental glutamate concentration upon infection. Furthermore, we found that the amount of glutathione within the ΔgltT ΔgltM mutant was much lower than that within the wild-type N. meningitidis strain only upon HBMEC infection and was correlated with intracellular survival. Considering that the l-glutamate obtained via GltT-GltM is utilized as a nutrient in host cells, l-glutamate uptake via GltT-GltM plays multiple roles in N. meningitidis internalization into HBMEC. PMID:26099588

  20. 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. PMID:26677078

  1. GLT-1 Transport Stoichiometry Is Constant at Low and High Glutamate Concentrations when Chloride Is Substituted by Gluconate

    PubMed Central

    Kabakov, Anatoli Y.; Rosenberg, Paul A.

    2015-01-01

    Glutamate is the major excitatory neurotransmitter, but prolonged exposure even at micromolar concentrations causes neuronal death. Extracellular glutamate is maintained at nanomolar level by glutamate transporters, which, however, may reverse transport and release glutamate. If and when the reverse occurs depends on glutamate transport stoichiometry (GTS). Previously we found that in the presence of chloride, the coupled GLT-1 glutamate transporter current and its relationship to radiolabeled glutamate flux significantly decreased when extracellular glutamate concentration increased above 0.2 mM, which implies a change in GTS. Such high concentrations are feasible near GLT-1 expressed close to synaptic release site during excitatory neurotransmission. The aim of this study was to determine GLT-1 GTS at both low (19–75 μM) and high (300–1200 μM) glutamate concentration ranges. GTS experiments were conducted in the absence of chloride to avoid contributions by the GLT-1 uncoupled chloride conductance. Mathematical analysis of the transporter thermodynamic equilibrium allowed us to derive equations revealing the number of a particular type of ion transported per elementary charge based on the measurements of the transporter reversal potential. We found that GLT-1a expressed in COS-7 cells co-transports 1.5 Na+, 0.5 Glu-, 0.5 H+ and counter-transports 0.6 K+ per elementary charge in both glutamate concentration ranges, and at both 37°C and 26°C temperatures. The thermodynamic parameter Q10 = 2.4 for GLT-1 turnover rate of 19 s-1 (37°C, -50 mV) remained constant in the 10 μM–10 mM glutamate concentration range. Importantly, the previously reported decrease in the current/flux ratio at high glutamate concentration was not seen in the absence of chloride in both COS-7 cells and cultured rat neurons. Therefore, only in the absence of chloride, GLT-1 GTS remains constant at all glutamate concentrations. Possible explanations for why apparent GTS might vary in

  2. Characterization of a Glutamate Transporter Operon, glnQHMP, in Streptococcus mutans and Its Role in Acid Tolerance▿ †

    PubMed Central

    Krastel, Kirsten; Senadheera, Dilani B.; Mair, Richard; Downey, Jennifer S.; Goodman, Steven D.; Cvitkovitch, Dennis G.

    2010-01-01

    Glutamate contributes to the acid tolerance response (ATR) of many Gram-negative and Gram-positive bacteria, but its role in the ATR of the oral bacterium Streptococcus mutans is unknown. This study describes the discovery and characterization of a glutamate transporter operon designated glnQHMP (Smu.1519 to Smu.1522) and investigates its potential role in acid tolerance. Deletion of glnQHMP resulted in a 95% reduction in transport of radiolabeled glutamate compared to the wild-type UA159 strain. The addition of glutamate to metabolizing UA159 cells resulted in an increased production of acidic end products, whereas the glnQHMP mutant produced less lactic acid than UA159, suggesting a link between glutamate metabolism and acid production and possible acid tolerance. To investigate this possibility, we conducted a microarray analysis with glutamate and under pH 5.5 and pH 7.5 conditions which showed that expression of the glnQHMP operon was downregulated by both glutamate and mild acid. We also measured the growth kinetics of UA159 and its glnQHMP-negative derivative at pH 5.5 and found that the mutant doubled at a much slower rate than the parent strain but survived at pH 3.5 significantly better than the wild type. Taken together, these findings support the involvement of the glutamate transporter operon glnQHMP in the acid tolerance response in S. mutans. PMID:20023025

  3. BODY TEMPERATURE-DEPENDENT AND INDEPENDENT ACTIONS OF CHLORDIMEFORM ON VISUAL EVOKED POTENTIALS AND AXONAL TRANSPORT IN OPTIC SYSTEM OF RAT

    EPA Science Inventory

    Pattern reversal evoked potentials (PREPs), flash evoked potentials (FEPs), optic nerve axonal transport, and body temperature were measured in hooded rats treated with either saline or the formamidine insecticide/acaricide, chlordimeform (CDM). Rats receiving CDM had low body te...

  4. Glutamate transporter type 3 knockout leads to decreased heart rate possibly via parasympathetic mechanism.

    PubMed

    Deng, Jiao; Li, Jiejie; Li, Liaoliao; Feng, Chenzhuo; Xiong, Lize; Zuo, Zhiyi

    2013-08-01

    Parasympathetic tone is a dominant neural regulator for basal heart rate. Glutamate transporters (EAAT) via their glutamate uptake functions regulate glutamate neurotransmission in the central nervous system. We showed that EAAT type 3 (EAAT3) knockout mice had a slower heart rate than wild-type mice when they were anesthetized. We design this study to determine whether non-anesthetized EAAT3 knockout mice have a slower heart rate and, if so, what may be the mechanism for this effect. Young adult EAAT3 knockout mice had slower heart rates than those of their littermate wild-type mice no matter whether they were awake or anesthetized. This difference was abolished by atropine, a parasympatholytic drug. Carbamylcholine chloride, a parasympathomimetic drug, equally effectively reduced the heart rates of wild-type and EAAT3 knockout mice. Positive immunostaining for EAAT3 was found in the area of nuclei deriving fibers for vagus nerve. There was no positive staining for the EAATs in the sinoatrial node. These results suggest that EAAT3 knockout mice have a slower heart rate at rest. This effect may be caused by an increased parasympathetic tone possibly due to increased glutamate neurotransmission in the central nervous system. These findings indicate that regulation of heart rate, a vital sign, is one of the EAAT biological functions. PMID:23361868

  5. Transplantation of glial progenitors that overexpress glutamate transporter GLT1 preserves diaphragm function following cervical SCI.

    PubMed

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

    2015-03-01

    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

  6. 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

  7. Expression of GFP-tagged neuronal glutamate transporters in cerebellar Purkinje neurons.

    PubMed

    Meera, Pratap; Dodson, Paul D; Karakossian, Movses H; Otis, Thomas S

    2005-11-01

    Of the five excitatory amino acid transporters (EAATs) identified, two genes are expressed by neurons (EAAT3 and EAAT4) and give rise to transporters confined to neuronal cell bodies and dendrites. At an ultrastructural level, EAAT3 and EAAT4 proteins are clustered at the edges of postsynaptic densities of excitatory synapses. This pattern of localization suggests that postsynaptic EAATs may help to limit spillover of glutamate from excitatory synapses. In an effort to study transporter localization in living neurons and ultimately to manipulate uptake at intact synapses, we have developed viral reagents encoding neuronal EAATs tagged with GFP. We demonstrate that these fusion proteins are capable of Na(+)-dependent glutamate uptake, that they generate ionic conductances indistinguishable from their wild-type counterparts, and that GFP does not alter their glutamate dose-dependence. Two-photon microscopy was used to examine fusion protein expression in Purkinje neurons in acute cerebellar slices. Both EAAT3-GFP and EAAT4-GFP were observed at high levels in the dendritic spines of transfected Purkinje neurons. These findings indicate that functional EAAT fusion proteins can be synthesized and appropriately trafficked to postsynaptic compartments. Furthermore, they validate a powerful system for looking at EAAT function in situ. PMID:16212990

  8. pH-profile of cystine and glutamate transport in normal and cystinotic human fibroblasts.

    PubMed

    Forster, S; Lloyd, J B

    1985-04-11

    In the human recessive condition cystinosis, cystine transport has been reported to be normal in the plasma membrane but defective in the lysosome membrane. A possible explanation is that the transport systems at the two cellular sites are identical and that the defect in cystinosis affects the porter's ability to operate at the low pH of the lysosome. To test this hypothesis the uptake of 3H-labelled cystine and glutamate by normal and cystinotic human skin fibroblasts has been measured in vitro at pH 5.8, 6.5, 7.0, 7.4 and 8.0. Uptake of glutamate was more rapid than that of cystine. Uptake of cystine increased with increasing pH, but uptake of glutamate showed no marked pH-dependence. Transport in cystinotic cells was similar to that in normal cells, and similarly affected by pH. This finding is incompatible with the hypothesis proposed above. It is concluded that the cystine porters of the plasma membrane and the lysosome membrane are probably genetically distinct. PMID:2858219

  9. Activity dependent internalization of the glutamate transporter GLT-1 mediated by β-arrestin 1 and ubiquitination.

    PubMed

    Ibáñez, Ignacio; Díez-Guerra, F Javier; Giménez, Cecilio; Zafra, Francisco

    2016-08-01

    GLT-1 is the main glutamate transporter in the brain and undergoes trafficking processes that control its concentration on the cell surface thereby shaping glutamatergic neurotransmission. We have investigated how the traffic of GLT-1 is regulated by transporter activity. We report that internalization of GLT-1 from the cell surface is accelerated by transportable substrates like glutamate or aspartate, as well as by the transportable inhibitor L-trans-2,4-PDC, but not by the non-substrate inhibitor WAY 213613 in primary mixed cultures and in transiently transfected HEK293 cells. Analysis of the mechanism of endocytosis in HEK293 cells revealed that glutamate promoted the association with the transporter of the adaptor protein β-arrestin and the ubiquitin ligase Nedd4-2. The addition of glutamate is accompanied by an increase in the transporter ubiquitination, and the internalization is suppressed by an ubiquitination inhibitor (PYR41), and in a mutant defective in C-terminal lysines. The glutamate triggered endocytosis was also suppressed by siRNA for β-arrestin. This regulatory mechanism might be relevant in controlling the amount of transporter on the cell surface in conditions such as ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated. PMID:27044663

  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. PMID:26620318

  11. ATP-binding Cassette Subfamily C Member 5 (ABCC5) Functions as an Efflux Transporter of Glutamate Conjugates and Analogs.

    PubMed

    Jansen, Robert S; Mahakena, Sunny; de Haas, Marcel; Borst, Piet; van de Wetering, Koen

    2015-12-18

    The ubiquitous efflux transporter ABCC5 (ATP-binding cassette subfamily C member 5) is present at high levels in the blood-brain barrier, neurons, and glia, but its in vivo substrates and function are not known. Using untargeted metabolomic screens, we show that Abcc5(-/-) mice accumulate endogenous glutamate conjugates in several tissues, but brain in particular. The abundant neurotransmitter N-acetylaspartylglutamate was 2.4-fold higher in Abcc5(-/-) brain. The metabolites that accumulated in Abcc5(-/-) tissues were depleted in cultured cells that overexpressed human ABCC5. In a vesicular membrane transport assay, ABCC5 also transported exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid, and NMDA; the therapeutic glutamate analog ZJ43; and, as previously shown, the anti-cancer drug methotrexate. Glutamate conjugates and analogs are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter in the brain. After CO2 asphyxiation, several immediate early genes were expressed at lower levels in Abcc5(-/-) brains than in wild type brains, suggesting altered glutamate signaling. Our results show that ABCC5 is a general glutamate conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins, and drugs. PMID:26515061

  12. 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. PMID:25639143

  13. [Glutamate and malignant gliomas, from epilepsia to biological aggressiveness: therapeutic implications].

    PubMed

    Blecic, Serge; Rynkowski, Michal; De Witte, Olivier; Lefranc, Florence

    2013-09-01

    In this review article, we describe the unrecognized roles of glutamate and glutamate receptors in malignant glioma biology. The neurotransmitter glutamate released from malignant glioma cells in the extracellular matrix is responsible for seizure induction and at higher concentration neuronal cell death. This neuronal cell death will create vacated place for tumor growth. Glutamate also stimulates the growth and the migration of glial tumor cells by means of the activation of glutamate receptors on glioma cells in a paracrine and autocrine manner. The multitude of effects of glutamate in glioma biology supports the rationale for pharmacological targeting of glutamate receptors and transporters in the adjuvant treatment of malignant gliomas in neurology and neuro-oncology. Using the website www.clinicaltrials.gov/ as a reference - a service developed by the National Library of Medicine for the National Health Institute in USA - we have evoked the few clinical trials completed and currently ongoing with therapies targeting the glutamate receptors. PMID:23883552

  14. 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

  15. The glutamate aspartate transporter (GLAST) mediates L-glutamate-stimulated ascorbate-release via swelling-activated anion channels in cultured neonatal rodent astrocytes.

    PubMed

    Lane, Darius J R; Lawen, Alfons

    2013-03-01

    Vitamin C (ascorbate) plays important neuroprotective and neuromodulatory roles in the mammalian brain. Astrocytes are crucially involved in brain ascorbate homeostasis and may assist in regenerating extracellular ascorbate from its oxidised forms. Ascorbate accumulated by astrocytes can be released rapidly by a process that is stimulated by the excitatory amino acid, L-glutamate. This process is thought to be neuroprotective against excitotoxicity. Although of potential clinical interest, the mechanism of this stimulated ascorbate-release remains unknown. Here, we report that primary cultures of mouse and rat astrocytes release ascorbate following initial uptake of dehydroascorbate and accumulation of intracellular ascorbate. Ascorbate-release was not due to cellular lysis, as assessed by cellular release of the cytosolic enzyme lactate dehydrogenase, and was stimulated by L-glutamate and L-aspartate, but not the non-excitatory amino acid L-glutamine. This stimulation was due to glutamate-induced cellular swelling, as it was both attenuated by hypertonic and emulated by hypotonic media. Glutamate-stimulated ascorbate-release was also sensitive to inhibitors of volume-sensitive anion channels, suggesting that the latter may provide the conduit for ascorbate efflux. Glutamate-stimulated ascorbate-release was not recapitulated by selective agonists of either ionotropic or group I metabotropic glutamate receptors, but was completely blocked by either of two compounds, TFB-TBOA and UCPH-101, which non-selectively and selectively inhibit the glial Na(+)-dependent excitatory amino acid transporter, GLAST, respectively. These results suggest that an impairment of astrocytic ascorbate-release may exacerbate neuronal dysfunction in neurodegenerative disorders and acute brain injury in which excitotoxicity and/or GLAST deregulation have been implicated. PMID:22886112

  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. Protonation of a glutamate residue modulates the dynamics of the drug transporter EmrE.

    PubMed

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

    2016-03-01

    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 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 wild-type 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

  18. Cocaine-induced neuroadaptations in the dorsal striatum: glutamate dynamics and behavioral sensitization.

    PubMed

    Parikh, Vinay; Naughton, Sean X; Shi, Xiangdang; Kelley, Leslie K; Yegla, Brittney; Tallarida, Christopher S; Rawls, Scott M; Unterwald, Ellen M

    2014-09-01

    Recent evidence suggests that diminished ability to control cocaine seeking arises from perturbations in glutamate homeostasis in the nucleus accumbens. However, the neurochemical substrates underlying cocaine-induced neuroadaptations in the dorsal striatum and how these mechanisms link to behavioral plasticity is not clear. We employed glutamate-sensitive microelectrodes and amperometry to study the impact of repeated cocaine administration on glutamate dynamics in the dorsolateral striatum of awake freely-moving rats. Depolarization-evoked glutamate release was robustly increased in cocaine-pretreated rats challenged with cocaine. Moreover, the clearance of glutamate signals elicited either by terminal depolarization or blockade of non-neuronal glutamate transporters slowed down dramatically in cocaine-sensitized rats. Repeated cocaine exposure also reduced the neuronal tone of striatal glutamate. Ceftriaxone, a β-lactam antibiotic that activates the astrocytic glutamate transporter, attenuated the effects of repeated cocaine exposure on synaptic glutamate release and glutamate clearance kinetics. Finally, the antagonism of AMPA glutamate receptors in the dorsolateral striatum blocked the development of behavioral sensitization to repeated cocaine administration. Collectively, these data suggest that repeated cocaine exposure disrupts presynaptic glutamate transmission and transporter-mediated clearance mechanisms in the dorsal striatum. Moreover, such alterations produce an over activation of AMPA receptors in this brain region leading to the sensitized behavioral response to repeated cocaine. PMID:24911954

  19. Possible Roles of Glutamate Transporter EAAT5 in Mouse Cone Depolarizing Bipolar Cell Light Responses

    PubMed Central

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

    2015-01-01

    A remarkable feature of neuronal glutamate transporters (EAATs) is their dual functions of classical carriers and ligand-gated chloride (CI−) channels. CI− conductance is rapidly activated by glutamate in subtype EAAT5, which mediates light responses in depolarizing bipolar cells (DBC) in retinae of lower vertebrates. In this study, we examine whether EAAT5 also mediates the DBC light response in mouse. We took advantage of an infrared illuminated micro-injection system, and studied the effects of the EAAT blocker (TBOA) and a glutamate receptor agonist (LAP4) on the mouse electroretinogram (ERG) b-wave responses. Our results showed that TBOA and LAP4 shared similar temporal patterns of inhibition: both inhibited the ERG b-wave shortly after injection and recovered with similar time courses. TBOA inhibited the b-wave completely at mesopic light intensity with an IC50 value about 1 log unit higher than that of LAP4. The inhibitory effects of TBOA and LAP4 were found to be additive in the photopic range. Furthermore, TBOA alone inhibited the b-wave in the cone operative range in knockout mice lacking DBCRs at a low concentration that did not alter synaptic glutamate clearance activity. It also produced a stronger inhibition than that of LAP4 on the cone-driven b-wave measured with a double flash method in wildtype mice. These electrophysiological data suggest a significant role for EAAT5 in mediating cone-driven DBC light responses. Our immunohistochemistry data indicated the presence of postsynaptic EAAT5 on some DBCCs and some DBCRs, providing an anatomical basis for EAAT5’s role in DBC light responses. PMID:24972005

  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. Electrophysiology of glutamate and sodium co-transport in a glial cell of the salamander retina.

    PubMed Central

    Schwartz, E A; Tachibana, M

    1990-01-01

    1. Müller cells were isolated from salamander retinas and their membrane voltage was controlled with a whole-cell voltage clamp. External D-aspartate, L-aspartate and L-glutamate each induced a membrane current. D-Glutamate, kainate, quisqualate and N-methyl-D-aspartate were more than 100x less effective than L-aspartate. Kynurenic acid had no effect on the current produced by L-glutamate, L-aspartate or D-aspartate. 2. The current induced by an acidic amino acid (AAA) was completely dependent on the presence of external Na+. Neither Li+, Cs+, choline nor TEA+ were able to substitute for Na+. The relationship between external Na+ concentration and current amplitude can be explained if the binding of three Na+ ions enabled transport. The apparent affinity constant for Na+ binding was 41 mM. Altering K+, H+ and Cl- concentrations demonstrated that these ions are not required for transport. 3. The shape of the current-voltage relation did not depend on the external amino acid concentration. The relationship between D-aspartate concentration and current amplitude can be described by the binding of D-aspartate to a single site with an apparent affinity constant of 20 microM. 4. Influx and efflux of AAA were not symmetric. Although influx was electrogenic, efflux did not produce a current. Moreover, influx stimulated efflux; but efflux inhibited influx. 5. Removing external Na+ demonstrated that Na+ carried a current in the absence of an AAA. Li+ was a very poor substitute for Na+. This current may be due to the uncoupled movement of Na+ through the transporter. The relationship between the external Na+ concentration and the amplitude of the uncoupled current can be explained if the binding of two or three Na+ ions enabled the translocation of Na+ in the absence of an AAA. The apparent affinity constant for Na+ binding was approximately 90 mM. 6. The temperature dependence of the AAA-induced current had a Q10 between 8 and 18 degrees C of 1.95. The Q10 is consistent

  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. Differential maturation of vesicular glutamate and GABA transporter expression in the mouse auditory forebrain during the first weeks of hearing.

    PubMed

    Hackett, Troy A; Clause, Amanda R; Takahata, Toru; Hackett, Nicholas J; Polley, Daniel B

    2016-06-01

    Vesicular transporter proteins are an essential component of the presynaptic machinery that regulates neurotransmitter storage and release. They also provide a key point of control for homeostatic signaling pathways that maintain balanced excitation and inhibition following changes in activity levels, including the onset of sensory experience. To advance understanding of their roles in the developing auditory forebrain, we tracked the expression of the vesicular transporters of glutamate (VGluT1, VGluT2) and GABA (VGAT) in primary auditory cortex (A1) and medial geniculate body (MGB) of developing mice (P7, P11, P14, P21, adult) before and after ear canal opening (~P11-P13). RNA sequencing, in situ hybridization, and immunohistochemistry were combined to track changes in transporter expression and document regional patterns of transcript and protein localization. Overall, vesicular transporter expression changed the most between P7 and P21. The expression patterns and maturational trajectories of each marker varied by brain region, cortical layer, and MGB subdivision. VGluT1 expression was highest in A1, moderate in MGB, and increased with age in both regions. VGluT2 mRNA levels were low in A1 at all ages, but high in MGB, where adult levels were reached by P14. VGluT2 immunoreactivity was prominent in both regions. VGluT1 (+) and VGluT2 (+) transcripts were co-expressed in MGB and A1 somata, but co-localization of immunoreactive puncta was not detected. In A1, VGAT mRNA levels were relatively stable from P7 to adult, while immunoreactivity increased steadily. VGAT (+) transcripts were rare in MGB neurons, whereas VGAT immunoreactivity was robust at all ages. Morphological changes in immunoreactive puncta were found in two regions after ear canal opening. In the ventral MGB, a decrease in VGluT2 puncta density was accompanied by an increase in puncta size. In A1, perisomatic VGAT and VGluT1 terminals became prominent around the neuronal somata. Overall, the

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

    PubMed

    Al Awabdh, Sana; Gupta-Agarwal, Swati; Sheehan, David F; Muir, James; Norkett, Rosalind; Twelvetrees, Alison E; Griffin, Lewis D; Kittler, Josef T

    2016-07-01

    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

  5. 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

  6. 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 Central

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

    2016-01-01

    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 [DβHCre/0;VGLUT2flox/flox mice (cKO mice)]. We compared the in vivo effects of selectively stimulating RVLM-CA neurons in cKO vs. control mice (DβHCre/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 hydroxy-lase). RVLM-CA neurons had similar morphology and axonal projections in DβHCre/0 and cKO mice. Under urethane anesthesia, photostimulation produced a similar pattern of activation of presumptive ChR2-positive RVLM-CA neurons in DβHCre/0 and cKO mice. Photostimulation in conscious mice produced frequency-dependent respiratory activation in DβHCre/0 mice but no effect in cKO mice. Similarly, photostimulation under urethane anesthesia strongly activated efferent vagal nerve activity in DβHCre/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. PMID:24236954

  7. 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.

  8. 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.

  9. Neuronal influences are necessary to produce mitochondrial co-localization with glutamate transporters in astrocytes

    PubMed Central

    Ugbode, Christopher I; Hirst, Warren D; Rattray, Marcus

    2014-01-01

    Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT-1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co-localization of GLT-1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5-tagged GLT-1, pDsRed1-Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co-localization was quantified using Volocity software. Image analysis of confocal z-stacks revealed no co-localization between mitochondria and GLT-1 in pure astrocyte cultures. Co-culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT-1. This co-localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K+. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT-1/mitochondrial co-localization, however, in co-cultures, Y27632 abolished mitochondrial:GLT-1 co-localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT-1 distribution or GLT-1: mitochondrial co-localization. We conclude that the association between GLT-1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments.Mitochondria have limited co-localization with the glutamate transporter GLT-1 in primary astrocytes in culture. Few mitochondria are in the fine processes where GLT-1 is abundant. It is necessary to culture astrocytes with neurones to drive a significant level of co-localization, but co-localization is not further altered by depolarization, manipulating sodium ion gradients or Na/K ATPase activity. PMID

  10. Glutamate Transporter EAAT2 Expression is Up-Regulated in Reactive Astrocytes in Human Periventricular Leukomalacia

    PubMed Central

    DESILVA, TARA M.; BILLIARDS, SARAID S.; BORENSTEIN, NATALIA S.; TRACHTENBERG, FELICIA L.; VOLPE, JOSEPH J.; KINNEY, HANNAH C.; ROSENBERG, PAUL A.

    2010-01-01

    The major neuropathological correlate of cerebral palsy in premature infants is periventricular leukomalacia (PVL), a disorder of the immature cerebral white matter. Cerebral ischemia leading to excitotoxicity is thought to be important in the pathogenesis of this disorder, implying a critical role for glutamate transporters, the major determinants of extracellular glutamate concentration. Previously, we found that EAAT2 expression is limited primarily to premyelinating oligodendrocytes early in development and is rarely observed in astrocytes until >40 weeks. In this study, we analyzed the expression of EAAT2 in cerebral white matter from PVL and control cases. Western blot analysis suggested an up-regulation of EAAT2 in PVL compared with control cases. Single- and double-label immunocytochemistry showed a significantly higher percentage of EAAT2-immunopositive astrocytes in PVL (51.8% ± 5.6%) compared with control white matter (21.4% ± 5.6%; P = 0.004). Macrophages in the necrotic foci in PVL also expressed EAAT2. Premyelinating oligodendrocytes in both PVL and control cases expressed EAAT2, without qualitative difference in expression. The previously unrecognized up-regulation of EAAT2 in reactive astrocytes and its presence in macrophages in PVL reported here may reflect a response to either hypoxic-ischemic injury or inflammation. PMID:18314905

  11. Pre-ischemic treadmill training for prevention of ischemic brain injury via regulation of glutamate and its transporter GLT-1.

    PubMed

    Yang, Xiaojiao; He, Zhijie; Zhang, Qi; Wu, Yi; Hu, Yongshan; Wang, Xiaolou; Li, Mingfen; Wu, Zhiyuan; Guo, Zhenzhen; Guo, Jingchun; Jia, Jie

    2012-01-01

    Pre-ischemic treadmill training exerts cerebral protection in the prevention of cerebral ischemia by alleviating neurotoxicity induced by excessive glutamate release following ischemic stroke. However, the underlying mechanism of this process remains unclear. Cerebral ischemia-reperfusion injury was observed in a rat model after 2 weeks of pre-ischemic treadmill training. Cerebrospinal fluid was collected using the microdialysis sampling method, and the concentration of glutamate was determined every 40 min from the beginning of ischemia to 4 h after reperfusion with high-performance liquid chromatography (HPLC)-fluorescence detection. At 3, 12, 24, and 48 h after ischemia, the expression of the glutamate transporter-1 (GLT-1) protein in brain tissues was determined by Western blot respectively. The effect of pre-ischemic treadmill training on glutamate concentration and GLT-1 expression after cerebral ischemia in rats along with changes in neurobehavioral score and cerebral infarct volume after 24 h ischemia yields critical information necessary to understand the protection mechanism exhibited by pre-ischemic treadmill training. The results demonstrated that pre-ischemic treadmill training up-regulates GLT-1 expression, decreases extracellular glutamate concentration, reduces cerebral infarct volume, and improves neurobehavioral score. Pre-ischemic treadmill training is likely to induce neuroprotection after cerebral ischemia by regulating GLT-1 expression, which results in re-uptake of excessive glutamate. PMID:22949807

  12. Vesicular glutamate transporter 1 orchestrates recruitment of other synaptic vesicle cargo proteins during synaptic vesicle recycling.

    PubMed

    Pan, Ping-Yue; Marrs, Julia; Ryan, Timothy A

    2015-09-11

    A long standing question in synaptic physiology is how neurotransmitter-filled vesicles are rebuilt after exocytosis. Among the first steps in this process is the endocytic retrieval of the transmembrane proteins that are enriched in synaptic vesicles (SVs). At least six types of transmembrane proteins must be recovered, but the rules for how this multiple cargo selection is accomplished are poorly understood. Among these SV cargos is the vesicular glutamate transporter (vGlut). We show here that vGlut1 has a strong influence on the kinetics of retrieval of half of the known SV cargos and that specifically impairing the endocytosis of vGlut1 in turn slows down other SV cargos, demonstrating that cargo retrieval is a collective cargo-driven process. Finally, we demonstrate that different cargos can be retrieved in the same synapse with different kinetics, suggesting that additional post-endocytic sorting steps likely occur in the nerve terminal. PMID:26224632

  13. 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

  14. Functional analysis of glutamate transporters in excitatory synaptic transmission of GLAST1 and GLAST1/EAAC1 deficient mice.

    PubMed

    Stoffel, Wilhelm; Körner, Rafael; Wachtmann, Dagmar; Keller, Bernhard U

    2004-09-28

    The high affinity, Na(+)-dependent, electrogenic glial L-glutamate transporters GLAST1 and GLT1, and two neuronal EAAC1 and EAAT4, regulate the neurotransmitter concentration in excitatory synapses of the central nervous system. We dissected the function of the individual transporters in the monogenic null allelic mouse lines, glast1(-/-) and eaac1(-/-), and the derived double mutant glast(-/-)eaac1(-/-). Unexpectedly, the biochemical analysis and the behavioral phenotypes of these null allelic mouse lines were inconspicuous. Inhibition studies of the Na(+)-dependent glutamate transport by plasma membrane vesicles and by isolated astrocytes of wt and glast1(-/-) mouse brains indicated the pivotal compensatory role of GLT1 in the absence particularly of GLAST1 and GLAST1 and EAAC1 mutant mice. In electrophysiological studies, the decay rate of excitatory postsynaptic currents (EPSCs) of Purkinje cells (PC) after selective activation of parallel and climbing fibers proved to be similar in wt and eaac1(-/-), but was significantly prolonged in glast1(-/-) PCs. Bath application of the glutamate uptake blocker SYM2081 prolonged EPSC decay profiles in both wt and double mutant glast1(-/-)eaac1(-/-) PCs by 286% and 229%, respectively, indicating a prominent role of compensatory glutamate transport in shaping glast1(-/-)eaac1(-/-) EPSCs. PMID:15363892

  15. Isoforms of the neuronal glutamate transporter gene, SLC1A1/EAAC1, negatively modulate glutamate uptake: relevance to obsessive-compulsive disorder

    PubMed Central

    Porton, B; Greenberg, B D; Askland, K; Serra, L M; Gesmonde, J; Rudnick, G; Rasmussen, S A; Kao, H-T

    2013-01-01

    The SLC1A1 gene, which encodes the neuronal glutamate transporter, EAAC1, has consistently been implicated in obsessive-compulsive disorder (OCD) in genetic studies. Moreover, neuroimaging, biochemical and clinical studies support a role for glutamatergic dysfunction in OCD. Although SLC1A1 is an excellent candidate gene for OCD, little is known about its regulation at the genomic level. Here, we report the identification and characterization of three alternative SLC1A1/EAAC1 mRNAs: a transcript derived from an internal promoter, termed P2 to distinguish it from the transcript generated by the primary promoter (P1), and two alternatively spliced mRNAs: ex2skip, which is missing exon 2, and ex11skip, which is missing exon 11. All isoforms inhibit glutamate uptake from the full-length EAAC1 transporter. Ex2skip and ex11skip also display partial colocalization and interact with the full-length EAAC1 protein. The three isoforms are evolutionarily conserved between human and mouse, and are expressed in brain, kidney and lymphocytes under nonpathological conditions, suggesting that the isoforms are physiological regulators of EAAC1. Moreover, under specific conditions, all SLC1A1 transcripts were differentially expressed in lymphocytes derived from subjects with OCD compared with controls. These initial results reveal the complexity of SLC1A1 regulation and the potential clinical utility of profiling glutamatergic gene expression in OCD and other psychiatric disorders. PMID:23695234

  16. Transport Rates of a Glutamate Transporter Homologue Are Influenced by the Lipid Bilayer*

    PubMed Central

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

    2015-01-01

    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. PMID:25713135

  17. 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

  18. 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

  19. Role of the atypical vesicular glutamate transporter VGLUT3 in l-DOPA-induced dyskinesia.

    PubMed

    Gangarossa, Giuseppe; Guzman, Monica; Prado, Vania F; Prado, Marco A M; Daumas, Stephanie; El Mestikawy, Salah; Valjent, Emmanuel

    2016-03-01

    Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons. The gold standard therapy relies on dopamine (DA) replacement by the administration of levodopa (l-DOPA). However, with time l-DOPA treatment induces severe motor side effects characterized by abnormal and involuntary movements, or dyskinesia. Although earlier studies point to a role of striatal cholinergic interneurons, also known as striatal tonically active neurons (TANs), in l-DOPA-induced dyskinesia (LID), the underlying mechanisms remain to be fully characterized. Here, we find that DA depletion is accompanied by increased expression of choline acetyltransferase (ChAT), the vesicular acetylcholine transporter (VAChT) as well as the atypical vesicular glutamate transporter type 3 (VGLUT3). TANs number and soma size are not changed. In dyskinetic mice, the VAChT levels remain high whereas the expression of VGLUT3 decreases. LID is attenuated in VGLUT3-deficient mice but not in mice bearing selective inactivation of VAChT in TANs. Finally, the absence of VGLUT3 is accompanied by a reduction of l-DOPA-induced phosphorylation of ERK1/2, ribosomal subunit (rpS6) and GluA1. Our results reveal that VGLUT3 plays an important role in the development of LID and should be considered as a potential and promising therapeutic target for prevention of LID. PMID:26711621

  20. 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

  1. NMDA and non-NMDA glutamate receptors in the paraventricular nucleus of the hypothalamus modulate different stages of hemorrhage-evoked cardiovascular responses in rats.

    PubMed

    Busnardo, C; Crestani, C C; Fassini, A; Resstel, L B M; Corrêa, F M A

    2016-04-21

    Here we report the involvement of N-Methyl-d-Aspartate (NMDA) and non-NMDA glutamate receptors from the paraventricular nucleus of the hypothalamus (PVN) in the mediation of cardiovascular changes observed during hemorrhage and post-bleeding periods. In addition, the present study provides further evidence of the involvement of circulating vasopressin and cardiac sympathetic activity in cardiovascular responses to hemorrhage. Systemic treatment with the V1-vasopressin receptor antagonist dTyr(CH2)5(Me)AVP (50 μg/kg, i.v.) increased the latency to the onset of hypotension during hemorrhage and slowed post-bleeding recovery of blood pressure. Systemic treatment with the β1-adrenergic receptor antagonist atenolol (1 mg/kg, i.v.) also increased the latency to the onset of hypotension during hemorrhage. Moreover, atenolol reversed the hemorrhage-induced tachycardia into bradycardia. Bilateral microinjection of the selective NMDA glutamate receptor antagonist LY235959 (2 nmol/100 nL) into the PVN blocked the hypotensive response to hemorrhage and reduced the tachycardia during the post-hemorrhage period. Systemic treatment with dTyr(CH2)5(Me)AVP inhibited the effect of LY235959 on hemorrhage-induced hypotension, without affecting the post-bleeding tachycardia. PVN treatment with the selective non-NMDA receptor antagonist NBQX (2 nmol/100 nL) reduced the recovery of blood pressure to normal levels in the post-bleeding phase and reduced hemorrhage-induced tachycardia. Combined blockade of both NMDA and non-NMDA glutamate receptors in the PVN completely abolished the hypotensive response in the hemorrhage period and reduced the tachycardiac response in the post-hemorrhage period. These results indicate that local PVN glutamate neurotransmission is involved in the neural pathway mediating cardiovascular responses to hemorrhage, via an integrated control involving autonomic nervous system activity and vasopressin release into the circulation. PMID:26861418

  2. 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.

  3. Ceftriaxone ameliorates tau pathology and cognitive decline via restoration of glial glutamate transporter in a mouse model of Alzheimer's disease.

    PubMed

    Zumkehr, Joannee; Rodriguez-Ortiz, Carlos J; Cheng, David; Kieu, Zanett; Wai, Thin; Hawkins, Charlesice; Kilian, Jason; Lim, Siok Lam; Medeiros, Rodrigo; Kitazawa, Masashi

    2015-07-01

    Glial glutamate transporter, GLT-1, is the major Na(+)-driven glutamate transporter to control glutamate levels in synapses and prevent glutamate-induced excitotoxicity implicated in neurodegenerative disorders including Alzheimer's disease (AD). Significant functional loss of GLT-1 has been reported to correlate well with synaptic degeneration and severity of cognitive impairment among AD patients, yet the underlying molecular mechanism and its pathological consequence in AD are not well understood. Here, we find the temporal decrease in GLT-1 levels in the hippocampus of the 3xTg-AD mouse model and that the pharmacological upregulation of GLT-1 significantly ameliorates the age-dependent pathological tau accumulation, restores synaptic proteins, and rescues cognitive decline with minimal effects on Aβ pathology. In primary neuron and astrocyte coculture, naturally secreted Aβ species significantly downregulate GLT-1 steady-state and expression levels. Taken together, our data strongly suggest that GLT-1 restoration is neuroprotective and Aβ-induced astrocyte dysfunction represented by a functional loss of GLT-1 may serve as one of the major pathological links between Aβ and tau pathology. PMID:25964214

  4. Metabotropic glutamate receptors inhibit microglial glutamate release

    PubMed Central

    McMullan, Stephen M; Phanavanh, Bounleut; Guo Li, Gary; Barger, Steven W

    2012-01-01

    Pro-inflammatory stimuli evoke an export of glutamate from microglia that is sufficient to contribute to excitotoxicity in neighbouring neurons. Since microglia also express various glutamate receptors themselves, we were interested in the potential feedback of glutamate on this system. Several agonists of mGluRs (metabotropic glutamate receptors) were applied to primary rat microglia, and the export of glutamate into their culture medium was evoked by LPS (lipopolysaccharide). Agonists of group-II and -III mGluR ACPD [(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] and L-AP4 [L-(+)-2-amino-4-phosphonobutyric acid] were both capable of completely blocking the glutamate export without interfering with the production of NO (nitric oxide); the group-I agonist tADA (trans-azetidine-2,4-dicarboxylic acid) was ineffective. Consistent with the possibility of feedback, inhibition of mGluR by MSPG [(R,S)-α-2-methyl-4sulfonophenylglycine] potentiated glutamate export. As the group-II and -III mGluR are coupled to Gαi-containing G-proteins and the inhibition of adenylate cyclase, we explored the role of cAMP in this effect. Inhibition of cAMP-dependent protein kinase [also known as protein kinase A (PKA)] by H89 mimicked the effect of ACPD, and the mGluR agonist had its actions reversed by artificially sustaining cAMP through the PDE (phosphodiesterase) inhibitor IBMX (isobutylmethylxanthine) or the cAMP mimetic dbcAMP (dibutyryl cAMP). These data indicate that mGluR activation attenuates a potentially neurotoxic export of glutamate from activated microglia and implicate cAMP as a contributor to this aspect of microglial action. PMID:22770428

  5. Synaptic connections of amacrine cells containing vesicular glutamate transporter 3 in baboon retinas

    PubMed Central

    MARSHAK, DAVID W.; CHUANG, ALICE Z.; DOLINO, DREW M.; JACOBY, ROY A.; LIU, WEILEY S.; LONG, YE; SHERMAN, MICHAEL B.; SUH, JAE M.; VILA, ALEJANDRO; MILLS, STEPHEN L.

    2016-01-01

    The goals of these experiments were to describe the morphology and synaptic connections of amacrine cells in the baboon retina that contain immunoreactive vesicular glutamate transporter 3 (vGluT3). These amacrine cells had the morphology characteristic of knotty bistratified type 1 cells, and their dendrites formed two plexuses on either side of the center of the inner plexiform layer. The primary dendrites received large synapses from amacrine cells, and the higher-order dendrites were both pre- and postsynaptic to other amacrine cells. Based on light microscopic immunolabeling results, these include AII cells and starburst cells, but not the polyaxonal amacrine cells tracer-coupled to ON parasol ganglion cells. The vGluT3 cells received input from ON bipolar cells at ribbon synapses and made synapses onto OFF bipolar cells, including the diffuse DB3a type. Many synapses from vGluT3 cells onto retinal ganglion cells were observed in both plexuses. At synapses where vGluT3 cells were presynaptic, two types of postsynaptic densities were observed; there were relatively thin ones characteristic of inhibitory synapses and relatively thick ones characteristic of excitatory synapses. In the light microscopic experiments with Neurobiotin-injected ganglion cells, vGluT3 cells made contacts with midget and parasol ganglion cells, including both ON and OFF types. Puncta containing immunoreactive gephyrin, an inhibitory synapse marker, were found at appositions between vGluT3 cells and each of the four types of labeled ganglion cells. The vGluT3 cells did not have detectable levels of immunoreactive γ-aminobutyric acid (GABA) or immunoreactive glycine transporter 1. Thus, the vGluT3 cells would be expected to have ON responses to light and make synapses onto neurons in both the ON and the OFF pathways. Taken with previous results, these findings suggest that vGluT3 cells release glycine at some of their output synapses and glutamate at others. PMID:26241195

  6. Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells.

    PubMed

    Jiang, Nan-Wei; Wang, De-Juan; Xie, Ya-Jun; Zhou, Liang; Su, Li-Da; Li, Huashun; Wang, Qin-Wen; Shen, Ying

    2016-06-01

    Excitatory amino acid transporter 4 (EAAT4) is believed to be critical to the synaptic activity of cerebellar Purkinje cells by limiting extracellular glutamate concentrations and facilitating the induction of long-term depression. However, the modulation of EAAT4 expression has not been elucidated. It has been shown that Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling plays essential roles in the regulation of protein translation, cell size, and cell growth. In addition, we previously found that a cascade including mTOR suppression and Akt activation induces increased expression of EAAT2 in astrocytes. In the present work, we explored whether Rheb/mTOR signaling is involved in the regulation of EAAT4 expression using conditional Rheb1 knockout mice. Our results demonstrated that Rheb1 deficiency resulted in the downregulation of EAAT4 expression, as well as decreased activity of mTOR and increased activity of Akt. The downregulation of EAAT4 was also confirmed by reduced EAAT4 currents and slowed kinetics of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor-mediated currents. On the other hand, conditional knockout of Rheb1 did not alter the morphology of Purkinje cell layer and the number of Purkinje cells. Overall, our findings suggest that small GTPase Rheb1 is a modulator in the expression of EAAT4 in Purkinje cells. PMID:26194056

  7. The Neuroprotective Effect of the Association of Aquaporin-4/Glutamate Transporter-1 against Alzheimer's Disease

    PubMed Central

    Lan, Yu-Long; Zou, Shuang; Chen, Jian-Jiao; Zhao, Jie; Li, Shao

    2016-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by memory loss and cognitive dysfunction. Aquaporin-4 (AQP4), which is primarily expressed in astrocytes, is the major water channel expressed in the central nervous system (CNS). This protein plays an important role in water and ion homeostasis in the normal brain and in various brain pathological conditions. Emerging evidence suggests that AQP4 deficiency impairs learning and memory and that this may be related to the expression of glutamate transporter-1 (GLT-1). Moreover, the colocalization of AQP4 and GLT-1 has long been studied in brain tissue; however, far less is known about the potential influence that the AQP4/GLT-1 complex may have on AD. Research on the functional interaction of AQP4 and GLT-1 has been demonstrated to be of great significance in the study of AD. Here, we review the interaction of AQP4 and GLT-1 in astrocytes, which might play a pivotal role in the regulation of distinct cellular responses that involve neuroprotection against AD. The association of AQP4 and GLT-1 could greatly supplement previous research regarding neuroprotection against AD. PMID:27057365

  8. Spinal Glutamate Transporters Are Involved in the Development of Electroacupuncture Tolerance

    PubMed Central

    Cui, Luying; Ding, Yi; Zeng, Jie; Feng, Yan; Li, Meng; Ding, Mingxing

    2016-01-01

    Background: Electroacupuncture (EA) tolerance is a gradual decline in EA antinociception because of its repeated or prolonged use. This study aims to explore the role of spinal glutamate transporters (GTs) in EA tolerance (EAT). Methods: Rats were treated with EA once per day for eight consecutive days, and their L4-5 spinal cords were collected at days 0, 2, 4, 6 and 8. The levels of three spinal GTs and their mRNAs were detected with Western blot and pPCR, respectively. Then, riluzole, a positive GT regulator, was administered intrathecally in order to observe its effect on EA analgesia after repeated EA. Results: The expression levels of the spinal GTs increased at days 2 and 4, and gradually decreased as the times of EA increased. At day 8, no difference was observed in the spinal GTs between the sham treatment and the EA treatment. Intrathecal administration of riluzole dose-dependently attenuated the decreased EA analgesia. Conclusion: These results indicated the participation of the spinal GTs in EAT. PMID:26978348

  9. 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

  10. 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

  11. Characterization of the Visceral Antinociceptive Effect of Glial Glutamate Transporter GLT-1 Upregulation by Ceftriaxone

    PubMed Central

    Roman, K.; Yang, M.; Stephens, Robert L.

    2013-01-01

    Recent studies demonstrate that glial glutamate transporter-1 (GLT-1) upregulation attenuates visceral nociception. The present work further characterized the effect of ceftriaxone- (CTX-) mediated GLT-1 upregulation on visceral hyperalgesia. Intrathecal pretreatment with dihydrokainate, a selective GLT-1 antagonist, produced a reversal of the antinociceptive response to bladder distension produced by CTX. The hyperalgesic response to urinary bladder distension caused by intravesicular acrolein was also attenuated by CTX treatment as was the enhanced time spent licking of abdominal area due to intravesicular acrolein. Bladder inflammation via cyclophosphamide injections enhanced the nociceptive to bladder distension; cohorts administered CTX and concomitant cyclophosphamide showed reduced hyperalgesic response. Cyclophosphamide-induced bladder hyperalgesia correlated with a significant 22% increase in GluR1 AMPA receptor subunit expression in the membrane fraction of the lumbosacral spinal cord, which was attenuated by CTX coadministration. Finally, neonatal colon insult-induced hyperalgesia caused by intracolonic mustard oil (2%) administration at P9 and P11 was attenuated by CTX. These studies suggest that GLT-1 upregulation (1) attenuates the hyperalgesia caused by bladder irritation/inflammation or by neonatal colonic insult, (2) acts at a spinal site, and (3) may produce antinociceptive effects by attenuating GluR1 membrane trafficking. These findings support further consideration of this FDA-approved drug to treat chronic pelvic pain syndromes.

  12. 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

  13. Spinal cord injury causes a wide-spread, persistent loss of Kir4.1 and glutamate transporter 1: benefit of 17β-oestradiol treatment

    PubMed Central

    Olsen, Michelle L.; Campbell, Susan C.; McFerrin, Michael B.; Floyd, Candace L.

    2010-01-01

    During neuronal activity astrocytes function to remove extracellular increases in potassium, which are largely mediated by the inwardly-rectifying potassium channel Kir4.1, and to take up excess glutamate via glutamate transporter 1, a glial-specific glutamate transporter. Here we demonstrate that expression of both of these proteins is reduced by nearly 80% following a crush spinal cord injury in adult male rats, 7 days post-injury. This loss extended to spinal segments several millimetres rostral and caudal to the lesion epicentre, and persisted at 4 weeks post-injury. Importantly, we demonstrate that loss of these two proteins is not a direct result of astrocyte loss, as immunohistochemistry at 7 days and western blots at 4 weeks demonstrate a marked up-regulation in glial fibrillary acidic protein expression. Kir4.1 and glutamate transporter 1 expression were partially rescued by post-spinal cord injury administration of physiological levels of 17β-oestradiol (0.08 mg/kg/day) in vivo. Utilizing an in vitro culture system we demonstrate that 17β-oestradiol treatment (50 nM) is sufficient to increase glutamate transporter 1 protein expression in spinal cord astrocytes. This increase in glutamate transporter 1 protein expression was reversed and Kir4.1 expression reduced in the presence of an oestrogen receptor antagonist, Fulvestrant 182 780 suggesting a direct translational regulation of Kir4.1 and glutamate transporter 1 via genomic oestrogen receptors. Using whole-cell patch-clamp recordings in cultured spinal cord astrocytes, we show that changes in protein expression following oestrogen application led to functional changes in Kir4.1 mediated currents. These findings suggest that the neuroprotective benefits previously seen with 17β-oestradiol after spinal cord injury may be in part due to increased Kir4.1 and glutamate transporter 1 expression in astrocytes leading to improved potassium and glutamate homeostasis. PMID:20375134

  14. Spinal cord injury causes a wide-spread, persistent loss of Kir4.1 and glutamate transporter 1: benefit of 17 beta-oestradiol treatment.

    PubMed

    Olsen, Michelle L; Campbell, Susan C; McFerrin, Michael B; Floyd, Candace L; Sontheimer, Harald

    2010-04-01

    During neuronal activity astrocytes function to remove extracellular increases in potassium, which are largely mediated by the inwardly-rectifying potassium channel Kir4.1, and to take up excess glutamate via glutamate transporter 1, a glial-specific glutamate transporter. Here we demonstrate that expression of both of these proteins is reduced by nearly 80% following a crush spinal cord injury in adult male rats, 7 days post-injury. This loss extended to spinal segments several millimetres rostral and caudal to the lesion epicentre, and persisted at 4 weeks post-injury. Importantly, we demonstrate that loss of these two proteins is not a direct result of astrocyte loss, as immunohistochemistry at 7 days and western blots at 4 weeks demonstrate a marked up-regulation in glial fibrillary acidic protein expression. Kir4.1 and glutamate transporter 1 expression were partially rescued by post-spinal cord injury administration of physiological levels of 17beta-oestradiol (0.08 mg/kg/day) in vivo. Utilizing an in vitro culture system we demonstrate that 17beta-oestradiol treatment (50 nM) is sufficient to increase glutamate transporter 1 protein expression in spinal cord astrocytes. This increase in glutamate transporter 1 protein expression was reversed and Kir4.1 expression reduced in the presence of an oestrogen receptor antagonist, Fulvestrant 182,780 suggesting a direct translational regulation of Kir4.1 and glutamate transporter 1 via genomic oestrogen receptors. Using whole-cell patch-clamp recordings in cultured spinal cord astrocytes, we show that changes in protein expression following oestrogen application led to functional changes in Kir4.1 mediated currents. These findings suggest that the neuroprotective benefits previously seen with 17beta-oestradiol after spinal cord injury may be in part due to increased Kir4.1 and glutamate transporter 1 expression in astrocytes leading to improved potassium and glutamate homeostasis. PMID:20375134

  15. Hypothermia protects against oxygen-glucose deprivation-induced neuronal injury by down-regulating the reverse transport of glutamate by astrocytes as mediated by neurons.

    PubMed

    Wang, D; Zhao, Y; Zhang, Y; Zhang, T; Shang, X; Wang, J; Liu, Y; Kong, Q; Sun, B; Mu, L; Liu, X; Wang, G; Li, H

    2013-05-01

    Glutamate is the major mediator of excitotoxic neuronal death following cerebral ischemia. Under severe ischemic conditions, glutamate transporters can functionally reverse to release glutamate, thereby inducing further neuronal injury. Hypothermia has been shown to protect neurons from brain ischemia. However, the mechanism(s) involved remain unclear. Therefore, the aim of this study was to investigate the mechanism(s) mediating glutamate release during brain ischemia-reperfusion injury under hypothermic conditions. Neuron/astrocyte co-cultures were exposed to oxygen-glucose deprivation (OGD) at various temperatures for 2h, and cell viability was assayed 12h after reoxygenation. PI and MAP-2 staining demonstrated that hypothermia significantly decreased neuronal injury. Furthermore, [(3)H]-glutamate uptake assays showed that hypothermia protected rat primary cortical cultures against OGD reoxygenation-induced injury. Protein levels of the astrocytic glutamate transporter, GLT-1, which is primarily responsible for the clearance of extracellular glutamate, were also found to be reduced in a temperature-dependent manner. In contrast, expression of GLT-1 in astrocyte-enriched cultures was found to significantly increase following the addition of neuron-conditioned medium maintained at 37 °C, and to a lesser extent with neuron-conditioned medium at 33 °C. In conclusion, the neuroprotective effects of hypothermia against brain ischemia-reperfusion injury involve down-regulation of astrocytic GLT-1, which mediates the reverse transport of glutamate. Moreover, this process may be regulated by molecules secreted by stressed neurons. PMID:23402854

  16. Membrane topology of the high-affinity L-glutamate transporter (GLAST- 1) of the central nervous system

    PubMed Central

    1996-01-01

    The membrane topology of the high affinity, Na(+)-coupled L-glutamate/L- aspartate transporter (GLAST-1) of the central nervous system has been determined. Truncated GLAST-1 cDNA constructs encoding protein fragments with an increasing number of hydrophobic regions were fused to a cDNA encoding a reporter peptide with two N-glycosylation sites. The respective cRNA chimeras were translated in vitro and in vivo in Xenopus oocytes. Posttranslational N-glycosylation of the two reporter consensus sites monitors the number, size, and orientation of membrane- spanning domains. The results of our experiments suggest a novel 10- transmembrane domain topology of GLAST-1, a representative of the L- glutamate neurotransmitter transporter family, with its NH2 and COOH termini on the cytoplasmic side, six NH2-terminal hydrophobic transmembrane alpha-helices, and four COOH-terminal short hydrophobic domains spanning the bilayer predicted as beta-sheets. PMID:8991097

  17. 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

  18. Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation

    PubMed Central

    Gosselin, Romain-Daniel; Meylan, Patrick; Decosterd, Isabelle

    2013-01-01

    Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release. PMID:24368897

  19. Corticostriatal dysfunction and glutamate transporter 1 (GLT1) in Huntington’s disease: interactions between neurons and astrocytes

    PubMed Central

    Estrada-Sánchez, Ana María; Rebec, George V.

    2012-01-01

    Huntington’s Disease (HD) is a fatally inherited neurodegenerative disorder caused by an expanded glutamine repeat in the N-terminal region of the huntingtin (HTT) protein. The result is a progressively worsening triad of cognitive, emotional, and motor alterations that typically begin in adulthood and end in death 10-20 years later. Autopsy of HD patients indicates massive cell loss in the striatum and its main source of input, the cerebral cortex. Further studies of HD patients and transgenic animal models of HD indicate that corticostriatal neuronal processing is altered long before neuronal death takes place. In fact, altered neuronal function appears to be the primary driver of the HD behavioral phenotype, and dysregulation of glutamate, the excitatory amino acid released by corticostriatal afferents, is believed to play a critical role. Although mutant HTT interferes with the operation of multiple proteins related to glutamate transmission, consistent evidence links the expression of mutant HTT with reduced activity of glutamate transporter 1 (rodent GLT1 or human EAAT2), the astrocytic protein responsible for the bulk of glutamate uptake. Here, we review corticostriatal dysfunction in HD and focus on GLT1 and its expression in astrocytes as a possible therapeutic target. PMID:22905336

  20. 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

  1. 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. PMID:11793340

  2. Differential changes of extracellular aspartate and glutamate in the striatum of domestic chicken evoked by high potassium or distress: an in vivo microdialysis study.

    PubMed

    Zachar, Gergely; Wagner, Zsolt; Tábi, Tamás; Bálint, Eszter; Szökő, Eva; Csillag, András

    2012-08-01

    It has long been proposed that L: -aspartate (Asp) is an excitatory neurotransmitter similar to L: -glutamate (Glu) but with distinct signaling properties. The presence of Asp in excitatory synapses of the medial striatum/nucleus accumbens of domestic chicks suggests that Asp plays a role of neurotransmitter also in the avian brain. Neurotransmitters are released from the presynaptic bouton mostly by Ca(2+) dependent exocytosis. We used in vivo microdialysis to monitor the simultaneous changes of the extracellular levels of Asp and Glu in the medial striatum of young post-hatch domestic chicks. Microdialysis samples were collected from freely moving birds at 5 min intervals and analysed off-line using capillary electrophoresis. Event-related elevations of extracellular Glu and Asp concentrations in response to handling stress and to high KCl (50 mM) were observed. Increase of Glu and Asp on handling stress was 200 and 250 %, whereas on KCl stimulation the values were 300 and 1,000 %, respectively, if stress was applied before high KCl, and 150 and 200 %, respectively, in the absence of stress. In most cases, the amino acids showed correlated changes, Asp concentrations being consistently smaller at resting but exceeding Glu during stimulation. Using Ca(2+) free medium, the KCl triggered elevation of Glu was reduced. When KCl stimulation was combined with tetrodotoxin infusion, there was no significant elevation in Asp or in Glu suggesting that most of the extracellular excitatory amino acids were released by synaptic mechanisms. The results support the suggestion that Asp is co-released with Glu and may play a signaling role (as distinct from that of glutamate) in the striatum of birds. PMID:22547325

  3. The antinociceptive effects of intracerebroventricular administration of Chicago sky blue 6B, a vesicular glutamate transporter inhibitor.

    PubMed

    Yu, Gang; Yi, Shoupu; Wang, Meiliang; Yan, Hui; Yan, Lingdi; Su, Ruibin; Gong, Zehui

    2013-12-01

    Accumulating evidence suggests that vesicular glutamate transporters (VGLUTs), which control the storage and release of glutamate, may play a role in pain processing. Chicago sky blue 6B (CSB6B), which is structurally related to glutamate, is a competitive VGLUT inhibitor without affecting plasma membrane transporters. The present study was designed to investigate the antinociceptive effects of CSB6B in a number of pain models. The hot-plate test was used as an acute thermal pain test. Inflammatory pain was evaluated using acetic acid writhing, formalin, and complete Freund's adjuvant tests. Intracerebroventricular administration of CSB6B did not affect acute thermal pain responses in 50 or 55°C hot plate tests. However, CSB6B attenuated acetic acid-induced writhing in a dose-dependent and time-dependent manner. In addition, CSB6B reduced licking/biting behavior during the second phase, but not during the first phase, following an intraplantar injection of formalin. In the complete Freund's adjuvant test, a significant attenuation of thermal hyperalgesia was also observed in CSB6B-treated mice. At antinociceptive doses, CSB6B did not affect mice spontaneous locomotor activity. The present study shows that pharmacological inhibition of VGLUT activity was sufficient to attenuate experimental inflammatory pain and suggests that regulation of VGLUTs might be a novel therapeutic strategy for the treatment of pain. PMID:24128751

  4. 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.

  5. 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

  6. Submucosal reflexes: distension-evoked ion transport in the guinea pig distal colon.

    PubMed

    Frieling, T; Wood, J D; Cooke, H J

    1992-07-01

    Muscle-stripped segments of distal colon from guinea pigs were mounted in modified flux chambers to determine the effect of distension on mucosal secretion. Ion secretion was monitored as changes in short-circuit current (Isc). Distending forces were pressure gradients established by controlled reduction in liquid volume of the submucosal compartment of the chamber. Volume removal for 10 s or 5 min evoked a monophasic or biphasic increase in Isc, which returned to baseline within 5-20 min. The amplitude of the response correlated with the volume removed and was reduced by bumetanide and Cl-free solutions but not by tetraethylammonium or amiloride. Tetrodotoxin and atropine also suppressed the response. Neither the nicotinic receptor antagonist mecamylamine, the 5-hydroxytryptamine3 (5-HT3) receptor antagonist ICS 205-930, or the prostaglandin synthesis inhibitor piroxicam altered the response. Addition of prostaglandin D2 to the submucosal bath significantly enhanced the response. The results suggest that distension of the colon evokes anion secretion by activation of reflex circuits with cholinergic neurons and muscarinic synapses. Prostaglandins and 5-hydroxytryptamine acting at 5-HT3 receptors appear not to be signal substances in the reflex pathway, which evokes the secretory response to distension. PMID:1636721

  7. 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

  8. Citrin and aralar1 are Ca(2+)-stimulated aspartate/glutamate transporters in mitochondria.

    PubMed

    Palmieri, L; Pardo, B; Lasorsa, F M; del Arco, A; Kobayashi, K; Iijima, M; Runswick, M J; Walker, J E; Saheki, T; Satrústegui, J; Palmieri, F

    2001-09-17

    The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF-hand Ca(2+)-binding motifs in their N-terminal domains. Both proteins and their C-terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H(+). Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca(2+) on the external side of the inner mitochondrial membrane, where the Ca(2+)-binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca(2+) through a mechanism independent of Ca(2+) entry into mitochondria, and suggest a novel mechanism of Ca(2+) regulation of the aspartate/malate shuttle. PMID:11566871

  9. Glutamate presynaptic vesicular transporter and postsynaptic receptor levels correlate with spatial memory status in aging rat models.

    PubMed

    Ménard, Caroline; Quirion, Rémi; Vigneault, Erika; Bouchard, Sylvain; Ferland, Guylaine; El Mestikawy, Salah; Gaudreau, Pierrette

    2015-03-01

    In humans, memory capacities are generally affected with aging, even without any reported neurologic disorders. The mechanisms behind cognitive decline are not well understood. We studied here whether postsynaptic glutamate receptor and presynaptic vesicular glutamate transporters (VGLUTs) levels may change in the course of aging and be related to cognitive abilities using various age-impaired (AI) or age-unimpaired rat strains. Twenty-four-month-old Long-Evans (LE) rats with intact spatial memory maintained postsynaptic ionotropic glutamate receptor levels in the hippocampal-adjacent cortex similar to those of young animals. In contrast, AI rats showed significantly reduced expression of ionotropic glutamate receptor GluR2, NR2A and NR2B subunits. In AI LE rats, VGLUT1 and VGLUT2 levels were increased and negatively correlated with receptor levels as shown by principal component analysis and correlation matrices. We also investigated whether glutamatergic receptors and VGLUT levels were altered in the obesity-resistant LOU/C/Jall (LOU) rat strain which is characterized by intact memory despite aging. No difference was observed between 24-month-old LOU rats and their young counterparts. Taken together, the unaltered spatial memory performance of 24-month-old age-unimpaired LE and LOU rats suggests that intact coordination of the presynaptic and postsynaptic hippocampal-adjacent cortex glutamatergic networks may be important for successful cognitive aging. Accordingly, altered expression of presynaptic and postsynaptic glutamatergic components, such as in AI LE rats, could be considered a marker of age-related cognitive deficits. PMID:25556161

  10. Non-essential roles of cysteine residues in functional expression and redox regulatory pathways for canine glutamate/aspartate transporter based on mutagenic analysis.

    PubMed Central

    Tamahara, Satoshi; Inaba, Mutsumi; Sato, Kota; Matsuki, Naoaki; Hikasa, Yoshiaki; Ono, Ken-Ichiro

    2002-01-01

    A redox regulatory mechanism and a molecular link between oxidative and excitotoxic neurodegeneration have been postulated for high-affinity Na(+)-dependent glutamate transporters. In the present study, mutations were introduced at three cysteine residues in canine glutamate/aspartate transporter (GLAST) to investigate the functional significance of thiol groups in response to oxidation. Cys(-) GLAST, in which all cysteines were replaced by other amino acids, as well as other mutants with disruption of one of three cysteine residues, showed insoluble oligomer formation, which was considered to be due to spontaneous and excessive oxidation as observed in wild-type GLAST. The mutant transporters also showed plasma-membrane localization and glutamate-transport kinetics that were very similar to those of wild-type GLAST. Glutamate-transport activities in COS-7 cells transfected with wild-type and Cys(-) GLAST were inhibited to the same degree when cells were exposed to Hg(2+) and were recovered by the addition of thiol-specific reductant dithiothreitol. These findings suggest that cysteine residues are not critical in functional expression of GLAST and the redox-sensing pathway via glutamate transporters. PMID:12088508

  11. Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody.

    PubMed

    Shashidharan, P; Huntley, G W; Murray, J M; Buku, A; Moran, T; Walsh, M J; Morrison, J H; Plaitakis, A

    1997-10-31

    Neuronal regulation of glutamate homeostasis is mediated by high-affinity sodium-dependent and highly hydrophobic plasma membrane glycoproteins which maintain low levels of glutamate at central synapses. To further elucidate the molecular mechanisms that regulate glutamate metabolism and glutamate flux at central synapses, a monoclonal antibody was produced to a synthetic peptide corresponding to amino acid residues 161-177 of the deduced sequence of the human neuron-specific glutamate transporter III (EAAC1). Immunoblot analysis of human and rat brain total homogenates and isolated synaptosomes from frontal cortex revealed that the antibody immunoreacted with a protein band of apparent Mr approximately 70 kDa. Deglycosylation of immunoprecipitates obtained using the monoclonal antibody yielded a protein with a lower apparent Mr (approximately 65 kDa). These results are consistent with the molecular size of the human EAAC1 predicted from the cloned cDNA. Analysis of the transfected COS-1 cells by immunocytochemistry confirmed that the monoclonal antibody is specific for the neuron-specific glutamate transporter. Immunocytochemical studies of rat cerebral cortex, hippocampus, cerebellum, substantia nigra and spinal cord revealed intense labeling of neuronal somata, dendrites, fine-caliber fibers and puncta. Double-label immunofluorescence using antibody to glial fibrillary acidic protein as a marker for astrocytes demonstrated that astrocytes were not co-labeled for EAAC1. The localization of EAAC1 immunoreactivity in dendrites and particularly in cell somata suggests that this transporter may function in the regulation of other aspects of glutamate metabolism in addition to terminating the action of synaptically released glutamate at central synapses. PMID:9409715

  12. 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

  13. The family of sodium-dependent glutamate transporters: a focus on the GLT-1/EAAT2 subtype.

    PubMed

    Robinson, M B

    1998-12-01

    The acidic amino acids, glutamate and aspartate, are the predominant excitatory neurotransmitters in the mammalian CNS. Under many pathologic conditions, these excitatory amino acids (EAAs) accumulate in the extracellular fluid in CNS and the resultant excessive activation of EAA receptors contributes to brain injury through a process known as 'excitotoxicity'. Unlike many other neurotransmitters, there is no evidence for extracellular metabolism of EAAs, rather, they are cleared by Na+-dependent transport mechanisms. Therefore, this transport process is important for ensuring crisp synaptic signaling as well as limiting the excitotoxic potential of EAAs. With the cloning of five distinct EAA transporters, a variety of tools were developed to characterize individual transporter subtypes, including specific antibodies, expression systems, and probes to delete/knock-down expression of each subtype. These tools are beginning to provide fundamental information that has the potential to impact our understanding of EAA physiology and pathophysiology. For example, biophysical studies of the cloned transporters have led to the observation that some subtypes function as ligand-gated ion channels as well as transporters. With these reagents, it has also been possible to explore the relative contributions of each transporter to the clearance of extracellular EAAs and to begin to examine the regulation of specific transporter subtypes. In this review, an overview of the properties of the transporter subtypes will be presented. The evidence which suggests that the transporter, GLT1/EAAT2, may be sufficient to explain a large percentage of forebrain transport will be critically reviewed. Finally, the studies of regulation of GLT-1 in vitro and in vivo will be described. PMID:10098717

  14. 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

  15. Astrocyte membrane properties are altered in a rat model of developmental cortical malformation but single-cell astrocytic glutamate uptake is robust.

    PubMed

    Hanson, Elizabeth; Danbolt, Niels Christian; Dulla, Chris G

    2016-05-01

    Developmental cortical malformations (DCMs) are linked with severe epilepsy and are caused by both genetic and environmental insults. DCMs include several neurological diseases, such as focal cortical dysplasia, polymicrogyria, schizencephaly, and others. Human studies have implicated astrocyte reactivity and dysfunction in the pathophysiology of DCMs, but their specific role is unknown. As astrocytes powerfully regulate glutamate neurotransmission, and glutamate levels are known to be increased in human epileptic foci, understanding the role of astrocytes in the pathological sequelae of DCMs is extremely important. Additionally, recent studies examining astrocyte glutamate uptake in DCMs have reported conflicting results, adding confusion to the field. In this study we utilized the freeze lesion (FL) model of DCM, which is known to induce reactive astrocytosis and cause significant changes in astrocyte morphology, proliferation, and distribution. Using whole-cell patch clamp recording from astrocytes, we recorded both UV-uncaging and synaptically evoked glutamate transporter currents (TCs), widely accepted assays of functional glutamate transport by astrocytes. With this approach, we set out to test the hypothesis that astrocyte membrane properties and glutamate transport were disrupted in this model of DCM. Though we found that the developmental maturation of astrocyte membrane resistance was disrupted by FL, glutamate uptake by individual astrocytes was robust throughout FL development. Interestingly, using an immunolabeling approach, we observed spatial and developmental differences in excitatory amino acid transporter (EAAT) expression in FL cortex. Spatially specific differences in EAAT2 (GLT-1) and EAAT1 (GLAST) expression suggest that the relative contribution of each EAAT to astrocytic glutamate uptake may be altered in FL cortex. Lastly, we carefully analyzed the amplitudes and onset times of both synaptically- and UV uncaging-evoked TCs. We found that in

  16. 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

  17. 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.

  18. 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

  19. Vesicular glutamate transporter 2 is required for central respiratory rhythm generation but not for locomotor central pattern generation.

    PubMed

    Wallén-Mackenzie, Asa; Gezelius, Henrik; Thoby-Brisson, Muriel; Nygård, Anna; Enjin, Anders; Fujiyama, Fumino; Fortin, Gilles; Kullander, Klas

    2006-11-22

    Glutamatergic excitatory neurotransmission is dependent on glutamate release from presynaptic vesicles loaded by three members of the solute carrier family, Slc17a6-8, which function as vesicular glutamate transporters (VGLUTs). Here, we show that VGLUT2 (Slc17a6) is required for life ex utero. Vglut2 null mutant mice die immediately after birth because of the absence of respiratory behavior. Investigations at embryonic stages revealed that neural circuits in the location of the pre-Bötzinger (PBC) inspiratory rhythm generator failed to become active. However, neurons with bursting pacemaker properties and anatomical integrity of the PBC area were preserved. Vesicles at asymmetric synapses were fewer and malformed in the Vglut2 null mutant hindbrain, probably causing the complete disruption of AMPA/kainate receptor-mediated synaptic activity in mutant PBC cells. The functional deficit results from an inability of PBC neurons to achieve synchronous activation. In contrast to respiratory rhythm generation, the locomotor central pattern generator of Vglut2 null mutant mice displayed normal rhythmic and coordinated activity, suggesting differences in their operating principles. Hence, the present study identifies VGLUT2-mediated signaling as an obligatory component of the developing respiratory rhythm generator. PMID:17122055

  20. Dietary Glutamate Supplementation Ameliorates Mycotoxin-Induced Abnormalities in the Intestinal Structure and Expression of Amino Acid Transporters in Young Pigs

    PubMed Central

    Wu, Miaomiao; Liao, Peng; Deng, Dun; Liu, Gang; Wen, Qingqi; Wang, Yongfei; Qiu, Wei; Liu, Yan; Wu, Xingli; Ren, Wenkai; Tan, Bie; Chen, Minghong; Xiao, Hao; Wu, Li; Li, Tiejun; Nyachoti, Charles M.; Adeola, Olayiwola; Yin, Yulong

    2014-01-01

    The purpose of this study was to investigate the hypothesis that dietary supplementation with glutamic acid has beneficial effects on growth performance, antioxidant system, intestinal morphology, serum amino acid profile and the gene expression of intestinal amino acid transporters in growing swine fed mold-contaminated feed. Fifteen pigs (Landrace×Large White) with a mean body weight (BW) of 55 kg were randomly divided into control group (basal feed), mycotoxin group (contaminated feed) and glutamate group (2% glutamate+contaminated feed). Compared with control group, mold-contaminated feed decreased average daily gain (ADG) and increased feed conversion rate (FCR). Meanwhile, fed mold-contaminated feed impaired anti-oxidative system and intestinal morphology, as well as modified the serum amino acid profile in growing pigs. However, supplementation with glutamate exhibited potential positive effects on growth performance of pigs fed mold-contaminated feed, ameliorated the imbalance antioxidant system and abnormalities of intestinal structure caused by mycotoxins. In addition, dietary glutamate supplementation to some extent restored changed serum amino acid profile caused by mold-contaminated feed. In conclusion, glutamic acid may be act as a nutritional regulating factor to ameliorate the adverse effects induced by mycotoxins. PMID:25405987

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

    PubMed Central

    Li, Huinan; Harlow, Mark L.

    2014-01-01

    Abstract 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. PMID:24744885

  2. 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.

  3. N-Acetylcysteine and Ceftriaxone as Preconditioning Strategies in Focal Brain Ischemia: Influence on Glutamate Transporters Expression.

    PubMed

    Krzyzanowska, Weronika; Pomierny, Bartosz; Budziszewska, Boguslawa; Filip, Malgorzata; Pera, Joanna

    2016-05-01

    Glutamate (Glu) plays a key role in excitotoxicity-related injury in cerebral ischemia. In the brain, Glu homeostasis depends on Glu transporters, including the excitatory amino acid transporters and the cysteine/Glu antiporter (xc-). We hypothesized that drugs acting on Glu transporters, such as ceftriaxone (CEF, 200 mg/kg, i.p.) and N-acetylcysteine (NAC, 150 mg/kg, i.p.), administered repeatedly for 5 days before focal cerebral ischemia in rats and induced by a 90-min middle cerebral artery occlusion (MCAO), may induce brain tolerance to ischemia. We compared the effects of these drugs on brain infarct volume, neurological deficits and the mRNA and protein expression of the Glu transporter-1 (GLT-1) and xc- with the effects of ischemic preconditioning and chemical preconditioning using 3-nitropropionic acid. Administration of CEF and NAC significantly reduced infarct size and neurological deficits caused by a 90-min MCAO. These beneficial effects were accompanied by changes in GLT-1 expression caused by a 90-min MCAO at both the mRNA and protein levels in the frontal cortex, hippocampus, and dorsal striatum. Thus, the results of this study suggest that the regulation of GLT-1 and xc- plays a role in the development of cerebral tolerance to ischemia and that this regulation may be a novel approach in the therapy of brain ischemia. PMID:26861954

  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. Neuroprotective Effects of the Glutamate Transporter Activator (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153) following Traumatic Brain Injury in the Adult Rat.

    PubMed

    Karklin Fontana, Andréia Cristina; Fox, Douglas P; Zoubroulis, Argie; Valente Mortensen, Ole; Raghupathi, Ramesh

    2016-06-01

    Traumatic brain injury (TBI) in humans and in animals leads to an acute and sustained increase in tissue glutamate concentrations within the brain, triggering glutamate-mediated excitotoxicity. Excitatory amino acid transporters (EAATs) are responsible for maintaining extracellular central nervous system glutamate concentrations below neurotoxic levels. Our results demonstrate that as early as 5 min and up to 2 h following brain trauma in brain-injured rats, the activity (Vmax) of EAAT2 in the cortex and the hippocampus was significantly decreased, compared with sham-injured animals. The affinity for glutamate (KM) and the expression of glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST) were not altered by the injury. Administration of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), a GLT-1 activator, beginning immediately after injury and continuing for 24 h, significantly decreased neurodegeneration, loss of microtubule-associated protein 2 and NeuN (+) immunoreactivities, and attenuated calpain activation in both the cortex and the hippocampus at 24 h after the injury; the reduction in neurodegeneration remained evident up to 14 days post-injury. In synaptosomal uptake assays, MS-153 up-regulated GLT-1 activity in the naïve rat brain but did not reverse the reduced activity of GLT-1 in traumatically-injured brains. This study demonstrates that administration of MS-153 in the acute post-traumatic period provides acute and long-term neuroprotection for TBI and suggests that the neuroprotective effects of MS-153 are related to mechanisms other than GLT-1 activation, such as the inhibition of voltage-gated calcium channels. PMID:26200170

  6. Synthetic cathinone MDPV downregulates glutamate transporter subtype I (GLT-1) and produces rewarding and locomotor-activating effects that are reduced by a GLT-1 activator.

    PubMed

    Gregg, Ryan A; Hicks, Callum; Nayak, Sunil U; Tallarida, Christopher S; Nucero, Paul; Smith, Garry R; Reitz, Allen B; Rawls, Scott M

    2016-09-01

    Synthetic cathinones produce dysregulation of monoamine systems, but their effects on the glutamate system and the influence of glutamate on behavioral effects related to cathinone abuse are unknown. A principal regulator of glutamate homeostasis is glutamate transporter subtype 1 (GLT-1), an astrocytic protein that clears glutamate from the extracellular space and influences behavioral effects of established psychostimulants. We hypothesized that repeated administration of the synthetic cathinone, MDPV (3,4-methylenedioxypyrovalerone), would affect GLT-1 expression in the corticolimbic circuit, and that a GLT-1 activator (ceftriaxone, CTX) would reduce rewarding and locomotor-stimulant effects of MDPV in rats. GLT-1 protein expression in the nucleus accumbens (NAcc), but not prefrontal cortex (PFC), was decreased following withdrawal (2, 5 and 10 days) from repeated MDPV treatment, but not immediately after the last MDPV injection. CTX (200 mg/kg) pretreatment did not affect acute locomotor activation produced by MDPV (0.5, 1, 3 mg/kg). However, CTX (200 mg/kg) administered during a 7-day MDPV treatment paradigm attenuated the development of MDPV-induced sensitization of repetitive movements in rats challenged with MDPV following 11 days of drug abstinence. Pretreatment with CTX (200 mg/kg) during a 4-day MDPV (2 mg/kg) conditioned place preference (CPP) paradigm reduced the development of place preference produced by MDPV. The present data demonstrate dysregulation of corticolimbic glutamate transport systems during withdrawal from chronic MDPV exposure, and show that a GLT-1 transporter activator disrupts behavioral effects of MDPV that are related to synthetic cathinone abuse. PMID:27085607

  7. Expression of the activity of cystine/glutamate exchange transporter, system x(c)(-), by xCT and rBAT.

    PubMed

    Wang, Hongyu; Tamba, Michiko; Kimata, Mayumi; Sakamoto, Kazuichi; Bannai, Shiro; Sato, Hideyo

    2003-06-01

    The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells. PMID:12763038

  8. The Amino Acid Transporter JhI-21 Coevolves with Glutamate Receptors, Impacts NMJ Physiology, and Influences Locomotor Activity in Drosophila Larvae.

    PubMed

    Ziegler, Anna B; Augustin, Hrvoje; Clark, Nathan L; Berthelot-Grosjean, Martine; Simonnet, Mégane M; Steinert, Joern R; Geillon, Flore; Manière, Gérard; Featherstone, David E; Grosjean, Yael

    2016-01-01

    Changes in synaptic physiology underlie neuronal network plasticity and behavioral phenomena, which are adjusted during development. The Drosophila larval glutamatergic neuromuscular junction (NMJ) represents a powerful synaptic model to investigate factors impacting these processes. Amino acids such as glutamate have been shown to regulate Drosophila NMJ physiology by modulating the clustering of postsynaptic glutamate receptors and thereby regulating the strength of signal transmission from the motor neuron to the muscle cell. To identify amino acid transporters impacting glutmatergic signal transmission, we used Evolutionary Rate Covariation (ERC), a recently developed bioinformatic tool. Our screen identified ten proteins co-evolving with NMJ glutamate receptors. We selected one candidate transporter, the SLC7 (Solute Carrier) transporter family member JhI-21 (Juvenile hormone Inducible-21), which is expressed in Drosophila larval motor neurons. We show that JhI-21 suppresses postsynaptic muscle glutamate receptor abundance, and that JhI-21 expression in motor neurons regulates larval crawling behavior in a developmental stage-specific manner. PMID:26805723

  9. The Amino Acid Transporter JhI-21 Coevolves with Glutamate Receptors, Impacts NMJ Physiology, and Influences Locomotor Activity in Drosophila Larvae

    PubMed Central

    Ziegler, Anna B.; Augustin, Hrvoje; Clark, Nathan L.; Berthelot-Grosjean, Martine; Simonnet, Mégane M.; Steinert, Joern R.; Geillon, Flore; Manière, Gérard; Featherstone, David E.; Grosjean, Yael

    2016-01-01

    Changes in synaptic physiology underlie neuronal network plasticity and behavioral phenomena, which are adjusted during development. The Drosophila larval glutamatergic neuromuscular junction (NMJ) represents a powerful synaptic model to investigate factors impacting these processes. Amino acids such as glutamate have been shown to regulate Drosophila NMJ physiology by modulating the clustering of postsynaptic glutamate receptors and thereby regulating the strength of signal transmission from the motor neuron to the muscle cell. To identify amino acid transporters impacting glutmatergic signal transmission, we used Evolutionary Rate Covariation (ERC), a recently developed bioinformatic tool. Our screen identified ten proteins co-evolving with NMJ glutamate receptors. We selected one candidate transporter, the SLC7 (Solute Carrier) transporter family member JhI-21 (Juvenile hormone Inducible-21), which is expressed in Drosophila larval motor neurons. We show that JhI-21 suppresses postsynaptic muscle glutamate receptor abundance, and that JhI-21 expression in motor neurons regulates larval crawling behavior in a developmental stage-specific manner. PMID:26805723

  10. Glutamate dysregulation in the trigeminal ganglion: a novel mechanism for peripheral sensitization of the craniofacial region.

    PubMed

    Laursen, J C; Cairns, B E; Dong, X D; Kumar, U; Somvanshi, R K; Arendt-Nielsen, L; Gazerani, P

    2014-01-01

    In the trigeminal ganglion (TG), satellite glial cells (SGCs) form a functional unit with neurons. It has been proposed that SGCs participate in regulating extracellular glutamate levels and that dysfunction of this SGC capacity can impact nociceptive transmission in craniofacial pain conditions. This study investigated whether SGCs release glutamate and whether elevation of TG glutamate concentration alters response properties of trigeminal afferent fibers. Immunohistochemistry was used to assess glutamate content and the expression of excitatory amino acid transporter (EAAT)1 and EAAT2 in TG sections. SGCs contained glutamate and expressed EAAT1 and EAAT2. Potassium chloride (10 mM) was used to evoke glutamate release from cultured rat SGCs treated with the EAAT1/2 inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)ben zoyl]amino]phenyl]methoxy]-L-aspartic acid (TFB-TBOA) or control. Treatment with TFB-TBOA (1 and 10 μM) significantly reduced the glutamate concentration from 10.6 ± 1.1 to 5.8 ± 1.4 μM and 3.0 ± 0.8 μM, respectively (p<0.05). Electrophysiology experiments were conducted in anaesthetized rats to determine the effect of intraganglionic injections of glutamate on the response properties of ganglion neurons that innervated either the temporalis or masseter muscle. Intraganglionic injection of glutamate (500 mM, 3 μl) evoked afferent discharge and significantly reduced muscle afferent mechanical threshold. Glutamate-evoked discharge was attenuated bythe N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovalerate (APV) and increased by TFB-TBOA, whereas mechanical sensitization was only sensitive to APV. Antidromic invasion of muscle afferent fibers by electrical stimulation of the caudal brainstem (10 Hz) or local anesthesia of the brainstem with lidocaine did not alter glutamate-induced mechanical sensitization. These findings provide a novel mechanism whereby dysfunctional trigeminal SGCs could contribute to cranial muscle tenderness in

  11. Inhibitory effects of (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) on the astrocytic sodium responses to glutamate.

    PubMed

    Bozzo, Luigi; Chatton, Jean-Yves

    2010-02-26

    Astrocytes are responsible for the majority of the clearance of extracellular glutamate released during neuronal activity. dl-threo-beta-benzyloxyaspartate (TBOA) is extensively used as inhibitor of glutamate transport activity, but suffers from relatively low affinity for the transporter. Here, we characterized the effects of (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), a recently developed inhibitor of the glutamate transporter on mouse cortical astrocytes in primary culture. The glial Na(+)-glutamate transport system is very efficient and its activation by glutamate causes rapid intracellular Na(+) concentration (Na(+)(i)) changes that enable real time monitoring of transporter activity. Na(+)(i) was monitored by fluorescence microscopy in single astrocytes using the fluorescent Na(+)-sensitive probe sodium-binding benzofuran isophtalate. When applied alone, TFB-TBOA, at a concentration of 1 microM, caused small alterations of Na(+)(i). TFB-TBOA inhibited the Na(+)(i) response evoked by 200 microM glutamate in a concentration-dependent manner with IC(50) value of 43+/-9 nM, as measured on the amplitude of the Na(+)(i) response. The maximum inhibition of glutamate-evoked Na(+)(i) increase by TFB-TBOA was >80%, but was only partly reversible. The residual response persisted in the presence of the AMPA/kainate receptor antagonist CNQX. TFB-TBOA also efficiently inhibited Na(+)(i) elevations caused by the application of d-aspartate, a transporter substrate that does not activate non-NMDA ionotropic receptors. TFB-TBOA was found not to influence the membrane properties of cultured cortical neurons recorded in whole-cell patch clamp. Thus, TFB-TBOA, with its high potency and its apparent lack of neuronal effects, appears to be one of the most useful pharmacological tools available so far for studying glial glutamate transporters. PMID:20026319

  12. 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

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:26085636

  14. 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.

  15. Vesicular glutamate transporter VGLUT1 has a role in hippocampal long-term potentiation and spatial reversal learning.

    PubMed

    Balschun, Detlef; Moechars, Diederik; Callaerts-Vegh, Zsuzsanna; Vermaercke, Ben; Van Acker, Nathalie; Andries, Luc; D'Hooge, Rudi

    2010-03-01

    Vesicular glutamate transporters 1 and 2 (VGLUT1, VGLUT2) show largely complementary distribution in the mature rodent brain and tend to segregate to synapses with different physiological properties. In the hippocampus, VGLUT1 is the dominate subtype in adult animals, whereas VGLUT2 is transiently expressed during early postnatal development. We generated and characterized VGLUT1 knockout mice in order to examine the functional contribution of this transporter to hippocampal synaptic plasticity and hippocampus-dependent spatial learning. Because complete deletion of VGLUT1 resulted in postnatal lethality, we used heterozygous animals for analysis. Here, we report that deletion of VGLUT1 resulted in impaired hippocampal long-term potentiation (LTP) in the CA1 region in vitro. In contrast, heterozygous VGLUT2 mice that were investigated for comparison did not show any changes in LTP. The reduced ability of VGLUT1-deficient mice to express LTP was accompanied by a specific deficit in spatial reversal learning in the water maze. Our data suggest a functional role of VGLUT1 in forms of hippocampal synaptic plasticity that are required to adapt and modify acquired spatial maps to external stimuli and changes. PMID:19574394

  16. Transcription Factor Nrf1 Negatively Regulates the Cystine/Glutamate Transporter and Lipid-Metabolizing Enzymes

    PubMed Central

    Tsujita, Tadayuki; Peirce, Vivian; Baird, Liam; Matsuyama, Yuka; Takaku, Misaki; Walsh, Shawn V.; Griffin, Julian L.; Uruno, Akira

    2014-01-01

    Liver-specific Nrf1 (NF-E2-p45-related factor 1) knockout mice develop nonalcoholic steatohepatitis. To identify postnatal mechanisms responsible for this phenotype, we generated an inducible liver-specific Nrf1 knockout mouse line using animals harboring an Nrf1flox allele and a rat CYP1A1-Cre transgene (Nrf1flox/flox::CYP1A1-Cre mice). Administration of 3-methylcholanthrene (3-MC) to these mice (Nrf1flox/flox::CYP1A1-Cre+3MC mice) resulted in loss of hepatic Nrf1 expression. The livers of mice lacking Nrf1 accumulated lipid, and the hepatic fatty acid (FA) composition in such animals differed significantly from that in the Nrf1flox/flox::CYP1A1-Cre control. This change was provoked by upregulation of several FA metabolism genes. Unexpectedly, we also found that the level of glutathione was increased dramatically in livers of Nrf1flox/flox::CYP1A1-Cre+3MC mice. While expression of glutathione biosynthetic enzymes was unchanged, xCT, a component of the cystine/glutamate antiporter system xc−, was significantly upregulated in livers of Nrf1flox/flox::CYP1A1-Cre+3MC mice, suggesting that Nrf1 normally suppresses xCT. Thus, stress-inducible expression of xCT is a two-step process: under homeostatic conditions, Nrf1 effectively suppresses nonspecific transactivation of xCT, but when cells encounter severe oxidative/electrophilic stress, Nrf1 is displaced from an antioxidant response element (ARE) in the gene promoter while Nrf2 is recruited to the ARE. Thus, Nrf1 controls both the FA and the cystine/cysteine content of hepatocytes by participating in an elaborate regulatory network. PMID:25092871

  17. 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

  18. 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

  19. Glutamic acid as anticancer agent: An overview

    PubMed Central

    Dutta, Satyajit; Ray, Supratim; Nagarajan, K.

    2013-01-01

    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. PMID:24227952

  20. Motor Neuron-Specific Overexpression of the Presynaptic Choline Transporter: Impact on Motor Endurance and Evoked Muscle Activity

    PubMed Central

    Lund, David; Ruggiero, Alicia M.; Ferguson, Shawn M.; Wright, Jane; English, Brett A.; Reisz, Peter A.; Whitaker, Sarah M.; Peltier, Amanda C.; Blakely, Randy D.

    2010-01-01

    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 hr. 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 hrs, 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. PMID:20888396

  1. SV2A and SV2C are not vesicular Ca2+ transporters but control glucose-evoked granule recruitment.

    PubMed

    Iezzi, Mariella; Theander, Sten; Janz, Roger; Loze, Chantal; Wollheim, Claes B

    2005-12-01

    Synaptic vesicle protein 2 (SV2) is expressed in neuroendocrine cells as three homologous isoforms, SV2A, SV2B and SV2C. Ca2+-dependent function in exocytosis has been attributed to SV2A and SV2B, without elucidation of the mechanism. The role of SV2C has not yet been addressed. Here we characterize the three SV2 isoforms and define their involvement in regulated insulin secretion. SV2A and SV2C are associated with insulin-containing granules and synaptic-like-microvesicles (SLM) in INS-1E insulinoma and primary beta-cells, whereas SV2B is only present on SLM. Neither overexpression nor isoform-specific silencing of SV2A or SV2C by RNA interference modifies depolarization-triggered cytosolic [Ca2+] rises or secretory granule [Ca2+], measured with a VAMP-2 aequorin chimera. This strongly argues against any Ca2+ transport function of SV2. Moreover, up- or downregulation of these isoforms has no influence on K+-induced insulin release suggesting that SV2 does not affect the Ca2+-dependent step(s) of exocytosis. By contrast, glucose-elicited secretion is inhibited during the sustained rather than the early phase, placing the action of SV2 on the recruitment of granules from the reserve pool to the plasma membrane. This conclusion is reinforced by capacitance measurements in glucose-stimulated SV2C-deficient cells. Like capacitance, evoked and basal hormone release are attenuated more by silencing of SV2C compared with SV2A. This indicates only partial redundancy and highlights a key role for SV2C in the secretory process. PMID:16306227

  2. 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. PMID:26483543

  3. KETAMINE ALTERS RAT FLASH EVOKED POTENTIALS (JOURNAL VERSION)

    EPA Science Inventory

    Discovering the neurotransmitters involved in the generation of flash evoked potentials (FEPs) would enhance the use of FEPs in screening for and assessment of neurological damage. Recent evidence suggests that the excitatory amino acids, glutamate and aspartate, may be transmitt...

  4. 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.

  5. The vesicular glutamate transporter-1 upstream promoter and first intron each support glutamatergic-specific expression in rat postrhinal cortex

    PubMed Central

    Zhang, Guo-rong; Li, Xu; Cao, Haiyan; Zhao, Hua; Geller, Alfred I.

    2011-01-01

    Multiple applications of direct gene transfer into neurons require restricting expression to glutamatergic neurons, or specific subclasses of glutamatergic neurons. Thus, it is desirable to develop and analyze promoters that support glutamatergic-specific expression. The three vesicular glutamate transporters (VGLUTs) are found in different populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported on a plasmid (amplicon) Herpes Simplex Virus vector that contains a VGLUT1 promoter. This vector supports long-term expression in VGLUT1-containing glutamatergic neurons in rat postrhinal (POR) cortex, but does not support expression in VGLUT2-containing glutamatergic neurons in the ventral medial hypothalamus. This VGLUT1 promoter contains both the VGLUT1 upstream promoter and the VGLUT1 first intron. In this study, we begin to isolate and analyze the glutamatergic-specific regulatory elements in this VGLUT1 promoter. We show that the VGLUT1 upstream promoter and first intron each support glutamatergic-specific expression. We isolated a small, basal VGLUT1 promoter that does not support glutamatergic-specific expression. Next, we fused either the VGLUT1 upstream promoter or the first intron to this basal promoter. The VGLUT1 upstream promoter or the first intron, fused to the basal promoter, each supported glutamatergic-specific expression in POR cortex. PMID:21172319

  6. The ATRA-dependent overexpression of the glutamate transporter EAAC1 requires RARbeta induction.

    PubMed

    Bianchi, Massimiliano G; Gazzola, Gian C; Cagnin, Silvia; Kagechika, Hiroyuki; Bussolati, Ovidio

    2009-09-01

    The mechanisms underlying trafficking and membrane targeting of EAAC1, the rodent counterpart of the human EAAT3 carrier for anionic amino acids, are well characterized. In contrast, much less is known on the regulation of Slc1a1, the gene that encodes for the transporter. We have recently found that all-trans retinoic acid (ATRA) stimulates EAAC1 expression and anionic amino acid transport in C6 rat glioma cells. We report here that the ATRA effect on EAAC1 activity was inhibited by the specific RAR antagonist LE540 and mimicked by Am80, a RAR agonist, but not by the RXR agonist HX630. Moreover, the ATRA-dependent induction of Slc1a1 mRNA required the synthesis of a protein intermediate and was not associated with changes in the messenger half-life. ATRA treatment induced the expression of both Rarb mRNA and RARbeta protein several hours before the induction of Slc1a1, while the mRNA for RFX1, a transcription factor recently involved in Slc1a1 transcription, was unchanged. In addition, Rarb silencing markedly inhibited the ATRA-dependent increase of both Rarb and Slc1a1 mRNAs. We conclude that in C6 glioma cells the induction of Slc1a1 by ATRA requires the synthesis of RARbeta, suggesting that the receptor is involved in the regulation of the transporter gene. PMID:19450544

  7. Roles of the NMDA Receptor and EAAC1 Transporter in the Modulation of Extracellular Glutamate by Low and High Affinity AMPA Receptors in the Cerebellum in Vivo: Differential Alteration in Chronic Hyperammonemia.

    PubMed

    Cabrera-Pastor, Andrea; Taoro, Lucas; Llansola, Marta; Felipo, Vicente

    2015-12-16

    The roles of high- and low-affinity AMPA receptors in modulating extracellular glutamate in the cerebellum remain unclear. Altered glutamatergic neurotransmission is involved in neurological alterations in hyperammonemia, which differently affects high- and low-affinity AMPA receptors. The aims were to assess by in vivo microdialysis (a) the effects of high- and low-affinity AMPA receptor activation on extracellular glutamate in the cerebellum; (b) whether chronic hyperammonemia alters extracellular glutamate modulation by high- and/or low-affinity AMPA receptors; and (c) the contribution of NMDA receptors and EAAC1 transporter to AMPA-induced changes in extracellular glutamate. In control rats, high affinity receptor activation does not affect extracellular glutamate but increases glutamate if NMDA receptors are blocked. Low affinity AMPA receptor activation increases transiently extracellular glutamate followed by reduction below basal levels and return to basal values. The reduction is associated with transient increased membrane expression of EAAC1 and is prevented by blocking NMDA receptors. Blocking NMDA receptors with MK-801 induces a transient increase in extracellular glutamate which is associated with reduced membrane expression of EAAC1 followed by increased membrane expression of the glutamate transporter GLT-1. Chronic hyperammonemia does not affect responses to activation of low affinity AMPA receptors. Activation of high affinity AMPA receptors increases extracellular glutamate in hyperammonemic rats by an NMDA receptor-dependent mechanism. In conclusion, these results show that there is a tightly controlled interplay between AMPA and NMDA receptors and an EAAC1 transporter in controlling extracellular glutamate. Hyperammonemia alters high- but not low-affinity AMPA receptors. PMID:26428532

  8. 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

  9. 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

  10. 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

  11. Adenosine Monophosphate-activated Protein Kinase Regulates Interleukin-1β Expression and Glial Glutamate Transporter Function in Rodents with Neuropathic Pain

    PubMed Central

    Maixner, Dylan W.; Yan, Xisheng; Gao, Mei; Yadav, Ruchi; Weng, Han-Rong

    2015-01-01

    Background Neuroinflammation and dysfunctional glial glutamate transporters (GTs) in the spinal dorsal horn (SDH) are implicated in the genesis of neuropathic pain. We determined if adenosine monophosphate-activated protein kinase (AMPK) in the SDH regulates these processes in rodents with neuropathic pain. Methods Hind paw withdrawal responses to radiant heat and mechanical stimuli were used to assess nociceptive behaviors. Spinal markers related to neuroinflammation and glial GTs were determined by Western blotting. AMPK activities were manipulated pharmacologically and genetically. Regulation of glial GTs was determined by measuring protein expression and activities of glial GTs. Results AMPK activities were reduced in the SDH of rats (n = 5) with thermal hyperalgesia induced by nerve injury, which were accompanied with the activation of astrocytes, increased production of interleukin-1beta and activities of glycogen synthase kinase 3β, and suppressed protein expression of glial glutamate transporter-1. Thermal hyperalgesia was reversed by spinal activation of AMPK in neuropathic rats (n = 10), and induced by inhibiting spinal AMPK in naïve rats (n = 7 to 8). Spinal AMPKα knockdown (n = 6) and AMPKα1 conditional knockout (n = 6) induced thermal hyperalgesia and mechanical allodynia. These genetic alterations mimicked the changes of molecular markers induced by nerve injury. Pharmacological activation of AMPK enhanced glial GT activity in mice with neuropathic pain (n = 8) and attenuated glial glutamate transporter-1 internalization induced by interleukin-1β (n = 4). Conclusion These findings suggest enhancing spinal AMPK activities could be an effective approach for the treatment of neuropathic pain. PMID:25710409

  12. 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 Central

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

    2009-01-01

    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 ∼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 ∼90 % glutamatergic neuron-specific expression. The GAD67 promoter supported ∼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 a glutamatergic or GABAergic

  13. Beneficial effect of magnesium lithospermate B on cerebral ischemia-reperfusion injury in rats involves the regulation of miR-107/glutamate transporter 1 pathway.

    PubMed

    Yang, Zhong-Bao; Luo, Xiu-Ju; Ren, Kai-Di; Peng, Jing-Jie; Tan, Bin; Liu, Bin; Lou, Zheng; Xiong, Xiao-Ming; Zhang, Xiao-Jie; Ren, Xian; Peng, Jun

    2015-11-01

    Recent studies uncovered that glutamate accumulation following cerebral ischemia-reperfusion (I/R) was related to the dysfunction of miR-107/glutamate transporter-1(GLT-1) pathway and magnesium lithospermate B (MLB) possesses the pharmacological activity of anti-excitotoxicity. This study aims to explore whether MLB is able to protect rat brain from excitatory neurotoxicity during I/R by modulating miR-107/GLT-1 pathway. Rats were subjected to 2h of cerebral ischemia following by 24h of reperfusion to establish an I/R injury model, which showed an increase in neurological deficit score, infarct volume and cellular apoptosis concomitant with glutamate accumulation, miR-107 elevation and GLT-1 down-regulation. Administration of MLB reduced I/R-induced cerebral injury accompanied by a reverse in glutamate accumulation, miR-107 and GLT-1 expression. Next, we examined the association of MLB with miR-107/GLT-1 pathway in a nerve cell hypoxia/reoxygenation (H/R) injury model. H/R treatment increased the nerve cells apoptosis concomitant with glutamate accumulation and miR-107 elevation, and suppressed GLT-1 expression, mimicking our in vivo findings. All these effects were reversed in the presence of MLB, confirming a strong correlation between MLB and miR-107/GLT-1 pathway. Based on these observations, we conclude that MLB is able to protect the rat brain from excitatory neurotoxicity during I/R through the regulation of miR-107/GLT-1 pathway. PMID:26420356

  14. 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

  15. 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

  16. Glutamate-induced sensitization of rat masseter muscle fibers.

    PubMed

    Cairns, B E; Gambarota, G; Svensson, P; Arendt-Nielsen, L; Berde, C B

    2002-01-01

    In rats, intradermal or intraarticular injection of glutamate or selective excitatory amino acid receptor agonists acting at peripheral excitatory amino acid receptors can decrease the intensity of mechanical stimulation required to evoke nocifensive behaviors, an indication of hyperalgesia. Since excitatory amino acid receptors have been found on the terminal ends of cutaneous primary afferent fibers, it has been suggested that increased tissue glutamate levels may have a direct sensitizing effect on primary afferent fibers, in particular skin nociceptors. However, less is known about the effects of glutamate on deep tissue afferent fibers. In the present study, a series of experiments were undertaken to investigate the effect of intramuscular injection of glutamate on the excitability and mechanical threshold of masseter muscle afferent fibers in anesthetized rats of both sexes. Injection of 1.0 M, but not 0.1 M glutamate evoked masseter muscle afferent activity that was significantly greater than that evoked by isotonic saline. The mechanical threshold of masseter muscle afferent fibers, which was assessed with a Von Frey hair, was reduced by approximately 50% for a period of 30 min after injection of 1.0 M glutamate, but was unaffected by injections of 0.1 M glutamate or isotonic saline. Injection of 25% dextrose, which has the same osmotic strength as 1.0 M glutamate, did not evoke significant activity in or decrease the mechanical threshold of masseter muscle afferent fibers. Magnetic resonance imaging experiments confirmed that injection of 25% dextrose and 1.0 M glutamate produced similar edema volumes in the masseter muscle tissue. Co-injection of 0.1 M kynurenate, an excitatory amino acid receptor antagonist, and 1.0 M glutamate attenuated glutamate-evoked afferent activity and prevented glutamate-induced mechanical sensitization. When male and female rats were compared, no difference in the baseline mechanical threshold or in the magnitude of glutamate

  17. 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

  18. Distribution of Vesicular Glutamate Transporter 2 (VGluT2) in the Primary Visual Cortex of the Macaque and Human

    PubMed Central

    Garcia-Marin, Virginia; Ahmed, Tunazzina H.; Afzal, Yasmeen C.; Hawken, Michael J.

    2014-01-01

    The majority of thalamic terminals in V1 arise from lateral geniculate nucleus (LGN) afferents. Thalamic afferent terminals are preferentially labeled by an isoform of the vesicular glutamate transporter, VGluT2. The goal of our study was to determine the distribution of VGluT2-ir puncta in macaque and human visual cortex. First, we investigated the distribution of VGluT2-ir puncta in all layers of macaque monkey primary visual cortex (V1), and found a very close correspondence between the known distribution of LGN afferents from previous studies and the distribution of VGluT2-immunoreactive (-ir) puncta. There was also a close correspondence between cytochrome oxidase density and VGluT2-ir puncta distribution. After validating the correspondence in macaque, we made a comparative study in human V1. In many aspects, the distribution of VGluT2-ir puncta in human was qualitatively similar to that of the macaque: high densities in layer 4C, patches of VGluT2-ir puncta in the supragranular layer (2/3), lower but clear distribution in layers 1 and 6, and very few puncta in layers 5 and 4B. However, there were also important differences between macaques and humans. In layer 4A of human, there was a sparse distribution of VGluT2-ir puncta, whereas in macaque, there was a dense distribution with the characteristic honeycomb organization. The results suggest important changes in the pattern of cortical VGluT2 immunostaining that may be related to evolutionary differences in the cortical organization of LGN afferents between Old World monkeys and humans. PMID:22684983

  19. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability. PMID

  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. 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. PMID:26259098

  3. 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

  4. Glutamate in peripheral organs: Biology and pharmacology.

    PubMed

    Du, Jie; Li, Xiao-Hui; Li, Yuan-Jian

    2016-08-01

    Glutamate is a versatile molecule existing in both the central nervous system and peripheral organs. Previous studies have mainly focussed on the biological effect of glutamate in the brain. Recently, abundant evidence has demonstrated that glutamate also participates in the regulation of physiopathological functions in peripheral tissues, including the lung, kidney, liver, heart, stomach and immune system, where the glutamate/glutamate receptor/glutamate transporter system plays an important role in the pathogenesis of certain diseases, such as myocardial ischaemia/reperfusion injury and acute gastric mucosa injury. All these findings provide new insight into the biology and pharmacology of glutamate and suggest a potential therapeutic role of glutamate in non-neurological diseases. PMID:27164423

  5. Synthesis and preliminary pharmacological evaluation of novel derivatives of L-β-threo-benzylaspartate as inhibitors of the neuronal glutamate transporter EAAT-3

    PubMed Central

    Mavencamp, Terri L.; Rhoderick, Joseph F.; Bridges, Richard J.; Esslinger, C. Sean

    2008-01-01

    A series of β-benzylaspartate derivatives were prepared from N-trityl-L-aspartate dimethyl ester and evaluated as inhibitors of neuronal glutamate transporter EAAT3. The result of the structure-activity studies suggest that the position occupied by the aromatic ring of β-benzylaspartate within the binding site of EAAT3 may be different from that occupied by comparable groups in previously identified inhibitors, such as L-threo-benzyloxy aspartate (TBOA). Further, halogen substitutions at the 3-postition of the aromatic ring of β-benzylaspartate can increase the potency with which the analogues inhibit EAAT3. PMID:18650095

  6. Effect of glutamate transporter EAAT2 gene variants and gray matter deficits on working memory in schizophrenia.

    PubMed

    Poletti, S; Radaelli, D; Bosia, M; Buonocore, M; Pirovano, A; Lorenzi, C; Cavallaro, R; Smeraldi, E; Benedetti, F

    2014-05-01

    Glutamate is the major excitatory neurotransmitter in the brain, with up to 40% of all synapses being glutamatergic. An altered glutamatergic transmission could play a critical role in working memory deficts observed in schizophrenia and could underline progressive changes such as grey matter loss throughout the brain. The aim of the study was to investigate if gray matter volume and working memory could be modulated by a genetic polymorphism related to glutamatergic function. Fifty schizophrenia patients underwent magnetic resonance and working memory testing outside of the scanner and were genotyped for rs4354668 EAAT2 polymorphism. Carriers of the G allele had lower gray matter volumes than T/T homozygote and worse working memory performance. Poor working memory performance was associated with gray matter reduction. Differences between the three genotypes are more relevant among patients showing poor performance at the 2-back task. Since glutamate abnormalities are known to be involved in excitotoxic processes, the decrease in cortical thickness observed in schizophrenia patients could be linked to an excess of extracellular glutamate. The differential effect of EAAT2 observed between good and poor performers suggests that the effect of EEAT2 on gray matter might reveal in the presence of a pathological process affecting gray matter. PMID:24076156

  7. 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

  8. A dominant-negative variant of SNAP-23 decreases the cell surface expression of the neuronal glutamate transporter EAAC1 by slowing constitutive delivery.

    PubMed

    Fournier, Keith M; Robinson, Michael B

    2006-01-01

    A family of high-affinity transporters controls the extracellular concentration of glutamate in the brain, ensuring appropriate excitatory signaling and preventing excitotoxicity. There is evidence that one of the neuronal glutamate transporters, EAAC1, is rapidly recycled on and off the plasma membrane with a half-life of no more than 5-7 min in both C6 glioma cells and cortical neurons. Syntaxin 1A has been implicated in the trafficking of several neurotransmitter transporters and in the regulation of EAAC1, but it has not been determined if this SNARE protein is required for EAAC1 trafficking. Expression of two different sets of SNARE proteins was examined in C6 glioma with Western blotting. These cells did not express syntaxin 1A, vesicle-associated membrane protein-1 (VAMP1), or synaptosomal-associated protein of 25 kDa (SNAP-25), but did express a family of SNARE proteins that has been implicated in glucose transporter trafficking, including syntaxin 4, vesicle-associated membrane protein-2 (VAMP2), and synaptosomal-associated protein of 23 kDa (SNAP-23). cDNAs encoding variants of SNAP-23 were co-transfected with Myc-tagged EAAC1 to determine if SNAP-23 function was required for maintenance of EAAC1 surface expression. Expression of a dominant-negative variant of SNAP-23 that lacks a domain required for SNARE complex assembly decreased the fraction of EAAC1 found on the cell surface and decreased total EAAC1 expression, while two control constructs had no effect. The dominant-negative variant of SNAP-23 also slowed the rate of EAAC1 delivery to the plasma membrane. These data strongly suggest that syntaxin 1A is not required for EAAC1 trafficking and provide evidence that SNAP-23 is required for constitutive recycling of EAAC1. PMID:16516346

  9. Marked synergism between mutant SOD1 and glutamate transport inhibition in the induction of motor neuronal degeneration in spinal cord slice cultures.

    PubMed

    Yin, Hong Z; Weiss, John H

    2012-04-11

    Loss of astrocytic glutamate transport capacity in ALS spinal cord supports an excitotoxic contribution to motor neuron (MN) damage in the disease, and dominant gain of function mutations in Cu/Zn superoxide dismutase (SOD1) cause certain familial forms of ALS. We have used organotypic slice cultures from wild type and G93A SOD1 mutant rat spinal cords to examine interactions between excitotoxicity and the presence of mutant SOD1 in the induction of MN degeneration. Slice cultures were prepared from 1 week old pups, and after an additional week in vitro, some were exposed to either a low level (30 μM) of the glutamate uptake inhibitor, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) for 3 weeks, or a higher level (50 μM) for 48 h, followed by histochemical labeling to assess MN injury. In wild type animals these exposures caused relatively little MN degeneration. Similarly, little MN degeneration was seen in slices from SOD1 mutant animals that were not exposed to PDC. However, addition of PDC to SOD1 mutant slices resulted in substantial MN injury, which was markedly attenuated by a Ca2+ permeable AMPA-type (Ca-AMPA) glutamate channel blocker, or by a nitric oxide synthase antagonist. These observations illustrate the utility of the organotypic culture model for the investigation of intracellular interactions underlying MN degeneration in ALS, and support the hypothesis that activation of Ca-AMPA channels on MNs provides a metabolic burden that synergizes with deleterious effects of mutant SOD1 in the induction of MN injury. PMID:22370146

  10. 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

  11. 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

  12. Expression of the vesicular glutamate transporters-1 and -2 in adult mouse dorsal root ganglia and spinal cord and their regulation by nerve injury.

    PubMed

    Brumovsky, P; Watanabe, M; Hökfelt, T

    2007-06-29

    The expression of two vesicular glutamate transporters (VGLUTs), VGLUT1 and VGLUT2, was studied with immunohistochemistry in lumbar dorsal root ganglia (DRGs), the lumbar spinal cord and the skin of the adult mouse. About 12% and 65% of the total number of DRG neuron profiles (NPs) expressed VGLUT1 and VGLUT2, respectively. VGLUT1-immunoreactive (IR) NPs were usually medium- to large-sized, in contrast to a majority of small- or medium-sized VGLUT2-IR NPs. Most VGLUT1-IR NPs did not coexpress calcitonin gene-related peptide (CGRP) or bound isolectin B4 (IB4). In contrast, approximately 31% and approximately 42% of the VGLUT2-IR DRG NPs were also CGRP-IR or bound IB4, respectively. Conversely, virtually all CGRP-IR and IB4-binding NPs coexpressed VGLUT2. Moderate colocalization between VGLUT1 and VGLUT2 was also observed. Sciatic nerve transection induced a decrease in the overall number of VGLUT1- and VGLUT2-IR NPs (both ipsi- and contralaterally) and, in addition, a parallel, unilateral increase of VGLUT2-like immunoreactivity (LI) in a subpopulation of mostly small NPs. In the dorsal horn of the spinal cord, strong VGLUT1-LI was detected, particularly in deep dorsal horn layers and in the ventral horns. VGLUT2-LI was abundant throughout the gray spinal matter, 'radiating' into/from the white matter. A unilateral dorsal rhizotomy reduced VGLUT1-LI, while apparently leaving unaffected the VGLUT2-LI. Transport through axons for both VGLUTs was confirmed by their accumulation after compression of the sciatic nerve or dorsal roots. In the hind paw skin, abundant VGLUT2-IR nerve fibers were observed, sometimes associated with Merkel cells. Lower numbers of VGLUT1-IR fibers were also detected in the skin. Some VGLUT1-IR and VGLUT2-IR fibers were associated with hair follicles. Based on these data and those by Morris et al. [Morris JL, Konig P, Shimizu T, Jobling P, Gibbins IL (2005) Most peptide-containing sensory neurons lack proteins for exocytotic release and

  13. 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

    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

  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. 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

  16. [Reduction in hypoxia-derived neuroinflammation and dysfunctional glutamate transporters by minocycline may restore hypoxia-injured cognition of neonatal rat].

    PubMed

    Li, Hong-Chun; Xiao, Jie; Huang, Yi-Long; Li, Long-Jun; Jiang, Hong; Huang, Li-Xuan; Yang, Ting; Yang, Ling; Li, Fan

    2016-04-25

    The aim of the present study was to investigate the effects of minocycline on cognitive functions in neonatal rat after hypoxia exposure and the underlying mechanism. A model of hypoxic brain damage (HBD) was developed by exposing postnatal 1 day (P1) rats to systemic hypoxia. The rats were intraperitoneally injected with normal saline (Hy group) or minocycline (Hy + M group) 2 h after hypoxia exposure. Some other P1 rats that were not subjected to systemic hypoxia were used as normal control (NG group). The Y-maze test was used to evaluate learning and memory ability on postnatal day 30. Inflammatory mediators (Iba-1, IL-1β, TNF-α and TGF-β1), glutamate transporters (EAAT1 and EAAT2), total Tau and phosphorylated Tau (phosphorylation sites: Tyr18, Thr205, Thr231, Ser396 and Ser404) protein expressions in the hippocampus were detected by Western blot 7 d after hypoxic exposure. The results showed that hypoxia induced learning and memory impairments of the neonatal rats, and minocycline administration could reverse the effects of hypoxia. The protein expression levels of Iba-1, IL-1β, TNF-α, EAAT2 and Tau phosphorylated at T231 were increased, but the total Tau expression was decreased in the hippocampus of the rats from Hy group 7 d after hypoxia exposure. In the hypoxia-treated rats, minocycline down-regulated Iba-1, IL-1β, TNF-α and EAAT2 protein expressions significantly, but did not affect total Tau and phosphorylated Tau protein expressions. Our results suggest that minocycline can prevent cognitive deficits of rats with hypoxia exposure, and the underlying mechanism may involve the inhibition of neuroinflammation and dysfunctional glutamate transporters but not the regulation of the Tau hyperphosphorylation. PMID:27108901

  17. Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3*

    PubMed Central

    Slugoski, Melissa D.; Smith, Kyla M.; Ng, Amy M. L.; Yao, Sylvia Y. M.; Karpinski, Edward; Cass, Carol E.; Baldwin, Stephen A.; Young, James D.

    2009-01-01

    Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na+ and H+ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na+/nucleoside and H+/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H+ dependence (all mutants), shift in Na+:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na+-specific hCNT1, uncoupled Na+ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine. PMID:19380587

  18. Partial Loss of the Glutamate Transporter GLT-1 Alters Brain Akt and Insulin Signaling in a Mouse Model of Alzheimer’s Disease

    PubMed Central

    Meeker, Kole D.; Meabon, James S.; Cook, David G.

    2016-01-01

    The glutamate transporter GLT-1 (also called EAAT2 in humans) plays a critical role in regulating extracellular glutamate levels in the central nervous system (CNS). In Alzheimer’s disease (AD),EAAT2 loss is associated with neuropathology and cognitive impairment. In keeping with this, we have reported that partial GLT-1 loss (GLT-1+/−) causes early-occurring cognitive deficits in mice harboring familial AD AβPPswe/PS1ΔE9 mutations. GLT-1 plays important roles in several molecular pathways that regulate brain metabolism, including Akt and insulin signaling in astrocytes. Significantly, AD pathogenesis also involves chronic Akt activation and reduced insulin signaling in the CNS. In this report we tested the hypothesis that GLT-1 heterozygosity (which reduces GLT-1 to levels that are comparable to losses in AD patients) in AβPPswe/PS1ΔE9 mice would induce sustained activation of Akt and disturb components of the CNS insulin signaling cascade. We found that partial GLT-1 loss chronically increased Akt activation (reflected by increased phosphorylation at serine 473), impaired insulin signaling (reflected by decreased IRβ phosphorylation of tyrosines 1150/1151 and increased IRS-1 phosphorylation at serines 632/635 –denoted as 636/639 in humans), and reduced insulin degrading enzyme (IDE) activity in brains of mice expressing familial AβPPswe/PS1ΔE9 AD mutations. GLT-1 loss also caused an apparent compensatory increase in IDE activity in the liver, an organ that has been shown to regulate peripheral amyloid-β levels and expresses GLT-1. Taken together, these findings demonstrate that partial GLT-1 loss can cause insulin/Akt signaling abnormalities that are in keeping with those observed in AD. PMID:25589729

  19. 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

  20. Inhibitory effect of taurine on veratridine-evoked D-[3H]aspartate release from murine corticostriatal slices: involvement of chloride channels and mitochondria.

    PubMed

    Molchanova, Svetlana M; Oja, Simo S; Saransaari, Pirjo

    2007-01-26

    We have previously shown that the inhibitory neuromodulator taurine attenuates the release of preloaded D-[3H]aspartate from murine corticostriatal slices evoked by ischemic conditions or by application of the sodium channel agonist veratridine. The release of D-[3H]aspartate (a non-metabolized analog of glutamate) was used as an index of glutamate release. The aim of the present study was to reveal the molecular mechanisms responsible for this inhibitory effect of taurine. It was shown that 10 mM taurine suppresses D-[3H]aspartate release evoked by 0.1 mM veratridine, but does not affect the high-K+ -(50 mM) or ouabain- (0.1 mM) evoked release. Taurine had no effect in Ca2+ -free medium when the synaptic exocytosis of D-[3H]aspartate was inhibited. Nor did it suppress the release from slices preloaded with the competitive glutamate uptake blocker DL-threo-beta-hydroxyaspartate (THBA), which inhibits D-[3H]aspartate release mediated by the reverse action of glutamate transporters. Omission of Cl- from the incubation medium reduced the effect of taurine, signifying the involvement of a Cl- channel. The glycine receptor antagonist strychnine and the GABA(A) receptor antagonist bicuculline did not block the taurine effect, although picrotoxin, a less specific blocker of agonist-gated chloride channels, completely prevented the effect of taurine on veratridine-induced D-[3H]aspartate release. The respiratory chain blocker rotenone or mitochondrial protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in combination with the mitochondrial ATPase inhibitor oligomycin, which inhibits the mitochondrial Ca2+ uniporter, also reduced the effect of taurine. The results obtained in the present study show that taurine acts specifically on the release of preloaded D-[3H]aspartate evoked by veratridine, but not on that evoked by other depolarizing agents, and affects the release mediated both by synaptic exocytosis and the reverse action of glutamate transporter. Taurine

  1. 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...

  2. Disorders of glutamate metabolism.

    PubMed

    Kelly, A; Stanley, C A

    2001-01-01

    The significant role the amino acid glutamate assumes in a number of fundamental metabolic pathways is becoming better understood. As a central junction for interchange of amino nitrogen, glutamate facilitates both amino acid synthesis and degradation. In the liver, glutamate is the terminus for release of ammonia from amino acids, and the intrahepatic concentration of glutamate modulates the rate of ammonia detoxification into urea. In pancreatic beta-cells, oxidation of glutamate mediates amino acid-stimulated insulin secretion. In the central nervous system, glutamate serves as an excitatory neurotransmittor. Glutamate is also the precursor of the inhibitory neurotransmittor GABA, as well as glutamine, a potential mediator of hyperammonemic neurotoxicity. The recent identification of a novel form of congenital hyperinsulinism associated with asymptomatic hyperammonemia assigns glutamate oxidation by glutamate dehydrogenase a more important role than previously recognized in beta-cell insulin secretion and hepatic and CNS ammonia detoxification. Disruptions of glutamate metabolism have been implicated in other clinical disorders, such as pyridoxine-dependent seizures, confirming the importance of intact glutamate metabolism. This article will review glutamate metabolism and clinical disorders associated with disrupted glutamate metabolism. PMID:11754524

  3. Vesicular Glutamate (VGluT), GABA (VGAT), and Acetylcholine (VAChT) Transporters in Basal Forebrain Axon Terminals Innervating the Lateral Hypothalamus

    PubMed Central

    HENNY, PABLO; JONES, BARBARA E.

    2008-01-01

    The basal forebrain (BF) is known to play important roles in cortical activation and sleep, which are likely mediated by chemically differentiated cell groups including cholinergic, γ-aminobutyric acid (GABA)ergic and other unidentified neurons. One important target of these cells is the lateral hypothalamus (LH), which is critical for arousal and the maintenance of wakefulness. To determine whether chemically specific BF neurons provide an innervation to the LH, we employed anterograde transport of 10,000 MW biotinylated dextran amine (BDA) together with immunohistochemical staining of the vesicular transporter proteins (VTPs) for glutamate (VGluT1, -2, and -3), GABA (VGAT), or acetylcholine (ACh, VAChT). In addition, we applied triple staining for the postsynaptic proteins (PSPs), PSD-95 with VGluT or Gephyrin (Geph) with VGAT, to examine whether the BDA-labeled varicosities may form excitatory or inhibitory synapses in the LH. Axons originating from BDA-labeled neurons in the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) descended within the medial forebrain bundle and extended collateral varicose fibers to contact LH neurons. In the LH, the BDA-labeled varicosities were immunopositive (+) for VAChT (~10%), VGluT2 (~25%), or VGAT (~50%), revealing an important influence of newly identified glutamatergic together with GABAergic BF inputs. Moreover, in confocal microscopy, VGluT2+ and VGAT+ terminals were apposed to PSD-95+ and Geph+ profiles respectively, indicating that they formed synaptic contacts with LH neurons. The important inputs from glutamatergic and GABAergic BF cells could thus regulate LH neurons in an opposing manner to stimulate vs. suppress cortical activation and behavioral arousal reciprocally. PMID:16572456

  4. 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.

  5. Scent-evoked nostalgia.

    PubMed

    Reid, Chelsea A; Green, Jeffrey D; Wildschut, Tim; Sedikides, Constantine

    2015-01-01

    Can scents evoke nostalgia; what might be the psychological implications of such an evocation? Participants sampled 12 scents and rated the extent to which each scent was familiar, arousing and autobiographically relevant, as well as the extent to which each scent elicited nostalgia. Participants who were high (compared to low) in nostalgia proneness reported more scent-evoked nostalgia, and scents elicited greater nostalgia to the extent that they were arousing, familiar and autobiographically relevant. Scent-evoked nostalgia predicted higher levels of positive affect, self-esteem, self-continuity, optimism, social connectedness and meaning in life. In addition, scent-evoked nostalgia was characterised by more positive emotions than either non-nostalgic autobiographical memories or non-nostalgic non-autobiographical memories. Finally, scent-evoked nostalgia predicted in-the-moment feelings of personal (general or object-specific) nostalgia. The findings represent a foray into understanding the triggers and affective signature of scent-evoked nostalgia. PMID:24456210

  6. Glutamate. Its applications in food and contribution to health.

    PubMed

    Jinap, S; Hajeb, P

    2010-08-01

    This article reviews application of glutamate in food and its benefits and role as one of the common food ingredients used. Monosodium glutamate is one of the most abundant naturally occurring amino acids which frequently added as a flavor enhancer. It produced a unique taste that cannot be provided by other basic taste (saltiness, sourness, sweetness and bitterness), referred to as a fifth taste (umami). Glutamate serves some functions in the body as well, serving as an energy source for certain tissues and as a substrate for glutathione synthesis. Glutamate has the potential to enhance food intake in older individuals and dietary free glutamate evoked a visceral sensation from the stomach, intestine and portal vein. Small quantities of glutamate used in combination with a reduced amount of table salt during food preparation allow for far less salt to be used during and after cooking. Because glutamate is one of the most intensely studied food ingredients in the food supply and has been found safe, the Joint Expert Committee on Food Additives of the United Nations Food and Agriculture Organization and World Health Organization placed it in the safest category for food additives. Despite a widespread belief that glutamate can elicit asthma, migraine headache and Chinese Restaurant Syndrome (CRS), there are no consistent clinical data to support this claim. In addition, findings from the literature indicate that there is no consistent evidence to suggest that individuals may be uniquely sensitive to glutamate. PMID:20470841

  7. Aberrant Rab11-Dependent Trafficking of the Neuronal Glutamate Transporter EAAC1 Causes Oxidative Stress and Cell Death in Huntington's Disease

    PubMed Central

    Valencia, Antonio; Sapp, Ellen; Masso, Nicholas; Alexander, Jonathan; Reeves, Patrick; Kegel, Kimberly B.; Aronin, Neil

    2010-01-01

    Oxidative stress contributes to neurodegeneration in Huntington's disease (HD). However, the origins of oxidative stress in HD remain unclear. Studies in HD transgenic models suggest involvement of mitochondrial dysfunction, which would lead to overproduction of reactive oxygen species (ROS). Impaired mitochondria complexes occur in late stages of HD but not in presymptomatic or early-stage HD patients. Thus, other mechanisms may account for the earliest source of oxidative stress caused by endogenous mutant huntingtin. Here, we report that decreased levels of a major intracellular antioxidant glutathione coincide with accumulation of ROS in primary HD neurons prepared from embryos of HD knock-in mice (HD140Q/140Q), which have human huntingtin exon 1 with 140 CAG repeats inserted into the endogenous mouse huntingtin gene. Uptake of extracellular cysteine through the glutamate/cysteine transporter EAAC1 is required for de novo synthesis of glutathione in neurons. We found that, compared with wild-type neurons, HD neurons had lower cell surface levels of EAAC1 and were deficient in taking up cysteine. Constitutive trafficking of EAAC1 from recycling endosomes relies on Rab11 activity, which is defective in the brain of HD140Q/140Q mice. Enhancement of Rab11 activity by expression of a dominant-active Rab11 mutant in primary HD neurons ameliorated the deficit in cysteine uptake, increased levels of intracellular glutathione, normalized clearance of ROS, and improved neuronal survival. Our data support a novel mechanism for oxidative stress in HD: Rab11 dysfunction slows trafficking of EAAC1 to the cell surface and impairs cysteine uptake, thereby leading to deficient synthesis of glutathione. PMID:20357106

  8. Opioid-glutamate interactions in rat locus coeruleus neurons.

    PubMed

    Oleskevich, S; Clements, J D; Williams, J T

    1993-09-01

    1. The effect of mu-opioids on the glutamate response was investigated in rat locus coeruleus (LC) neurons by intracellular recording in the brain slice preparation. Glutamate responses were evoked by bath application of selective glutamate agonists, glutamate iontophoresis, and stimulation of excitatory afferents. 2. The mu-opioid agonist D-Ala2-MePhe4-Gly-ol5-enkephalin (DAMGO; 1 microM) potentiated the response to bath application of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid by 91 and 142%, respectively, in slices cut in the horizontal plane. The mechanism of action of this effect was investigated under conditions that limited the DAMGO-induced hyperpolarization and improved the space clamp of the neuron through 1) addition of barium, 2) increase in extracellular potassium concentration, 3) sectioning of the LC in the coronal plane, and 4) addition of carbenoxolone. Each experimental manipulation decreased the DAMGO outward current and reduced the mu-opioid potentiation of the glutamate response. The results suggest that the mu-opioid-mediated potentiation of the glutamate response is dependent on membrane hyperpolarization. 3. Neither forskolin nor the phorbol ester 4b-phorbol 12,13-dibutyrate (PDBu) altered the glutamate-mediated inward currents. The potentiation of the glutamate response by DAMGO was not affected by PDBu. 4. The mu-opioids DAMGO and [met]5enkephalin (10 microM) did not significantly affect the NMDA receptor-mediated depolarization (mean 14%) evoked by local application of glutamate but inhibited the NMDA receptor-mediated synaptic potential (mean 25%).(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7693886

  9. Glutamate receptor ligands as anxiolytics.

    PubMed

    Chojnacka-Wójcik, E; Kłodzinska, A; Pilc, A

    2001-08-01

    The glutamatergic system has received considerable attention over recent years as a potential target for anxiolytic drugs. In spite of the pronounced anxiolytic-like effects of competitive and non-competitive antagonists of NMDA receptors in animal models of anxiety, these substances can not be regarded as potential anxiolytic drugs, mainly due to their side-effect profiles (eg, ataxia, myorelaxation, impairment of learning and memory processes and psychotomimetic effects). Antagonists and partial agonists of the glycine, receptor inhibit function of the NMDA receptor complex and evoke in animals an anxiolytic-like response. Although data concerning anti-anxiety-like effects of glycine, receptor antagonists are not very promising, studies are underway to develop new, brain-penetrating agents devoid of side effects. Further developments are necessary to more fully elucidate the possible involvement of AMPA/kainate receptors in anxiety. The recent discovery of metabotropic glutamate receptors, which modulate the function of the glutamatergic system, offers new hope for discovery of a new generation of anxiolytics. MPEP, a highly selective, brain penetrable, noncompetitive mGlu5 receptor antagonist, evokes anxiolytic-like effects in several animal models of anxiety, remaining remarkably free of side effects. LY-354740, a selective brain-penetrable group II mGlu receptor agonist, evokes marked anxiolytic-like effects in animal models of anxiety. LY-354740 causes mild sedation in mice, does not disturb motor coordination and has no potential to cause dependence. Therefore mGlu receptor ligands may become the anxiolytics of the future, free from the side effects characteristic of benzodiazepines. PMID:11892923

  10. Human vestibular evoked responses.

    PubMed

    Muñoz-Gamboa, C; Jiménez-Cruz, J

    1994-01-01

    The results of an experimental series dedicated to the acquisition of human vestibular evoked responses are presented. In these series, manual stimulation is applied to a normal group of subjects with rotational acceleration impulses. Every stimulus is large in magnitude and very short in duration, producing small head movements of only a few degrees through a specially designed head immobilization helmet. Results correspond to middle latency vestibular evoked responses. PMID:7968862

  11. Silicon Wafer-Based Platinum Microelectrode Array Biosensor for Near Real-Time Measurement of Glutamate in Vivo

    PubMed Central

    Wassum, Kate M.; Tolosa, Vanessa M.; Wang, Jianjun; Walker, Eric; Monbouquette, Harold G.; Maidment, Nigel T.

    2008-01-01

    Using Micro-Electro-Mechanical-Systems (MEMS) technologies, we have developed silicon wafer-based platinum microelectrode arrays (MEAs) modified with glutamate oxidase (GluOx) for electroenzymatic detection of glutamate in vivo. These MEAs were designed to have optimal spatial resolution for in vivo recordings. Selective detection of glutamate in the presence of the electroactive interferents, dopamine and ascorbic acid, was attained by deposition of polypyrrole and Nafion. The sensors responded to glutamate with a limit of detection under 1μM and a sub-1-second response time in solution. In addition to extensive in vitro characterization, the utility of these MEA glutamate biosensors was also established in vivo. In the anesthetized rat, these MEA glutamate biosensors were used for detection of cortically-evoked glutamate release in the ventral striatum. The MEA biosensors also were applied to the detection of stress-induced glutamate release in the dorsal striatum of the freely-moving rat. PMID:19543440

  12. 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

  13. WAY208466 inhibits glutamate release at hippocampal nerve terminals.

    PubMed

    Wang, Hue Yu; Lu, Cheng Wei; Lin, Tzu Yu; Kuo, Jinn Rung; Wang, Su Jane

    2016-06-15

    Evidence suggests that the glutamatergic system plays a crucial role in the pathophysiology and treatment of depression. This study investigates the effect of WAY208466, a 5-HT6 receptor agonist exhibiting an antidepressant effect, on glutamate release from rat hippocampal nerve terminals (synaptosomes). WAY208466 inhibited the Ca(2+)-dependent release of glutamate that was evoked by exposing the synaptosomes to the potassium channel blocker 4-aminopyridine, and the selective 5-HT6 receptor antagonist SB258585 blocked this phenomenon. The WAY208466-mediated inhibition of glutamate release was associated with a reduction of 4-aminopyridine-induced increase in the cytosolic free Ca(2+) concentration ([Ca(2+)]C) mediated via Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels. WAY208466 did not alter the resting synaptosomal membrane potential or 4-aminopyridine-mediated depolarization; thus, the inhibition of the Ca(2+) influx could not be attributed to the decrease in synaptosomal excitability caused by 5-HT6 receptor activation. Furthermore, the effect of WAY208466 on 4-aminopyridine-evoked glutamate release was prevented by a Gi/Go-protein inhibitor pertussis toxin, adenylate cyclase inhibitor SQ22536, and a protein kinase A inhibitor H89. These results suggest that WAY208466 acts at the 5-HT6 receptors present in the hippocampal nerve terminals to suppress the Gi/Go-protein-coupled adenylate cyclase/protein kinase A cascade, which subsequently reduces the Ca(2+) influx via N- and P/Q-type Ca(2+) channels to inhibit the evoked glutamate release. This finding implicated a potential therapeutic role of 5-HT6 receptor agonist in the treatment of depression and other neurological diseases associated with glutamate excitotoxicity. PMID:27068148

  14. Chronic at-level thermal hyperalgesia following rat cervical contusion spinal cord injury is accompanied by neuronal and astrocyte activation and loss of the astrocyte glutamate transporter, GLT1, in superficial dorsal horn.

    PubMed

    Putatunda, Rajarshi; Hala, Tamara J; Chin, Jeannie; Lepore, Angelo C

    2014-09-18

    Neuropathic pain is a form of pathological nociception that occurs in a significant portion of traumatic spinal cord injury (SCI) patients, resulting in debilitating and often long-term physical and psychological burdens. While many peripheral and central mechanisms have been implicated in neuropathic pain, central sensitization of dorsal horn spinothalamic tract (STT) neurons is a major underlying substrate. Furthermore, dysregulation of extracellular glutamate homeostasis and chronic astrocyte activation play important underlying roles in persistent hyperexcitability of these superficial dorsal horn neurons. To date, central sensitization and astrocyte changes have not been characterized in cervical SCI-induced neuropathic pain models, despite the fact that a major portion of SCI patients suffer contusion trauma to cervical spinal cord. In this study, we have characterized 2 rat models of unilateral cervical contusion SCI that behaviorally result in chronic persistence of thermal hyperalgesia in the ipsilateral forepaw. In addition, we find that STT neurons are chronically activated in both models when compared to laminectomy-only uninjured rats. Finally, persistent astrocyte activation and significantly reduced expression of the major CNS glutamate transporter, GLT1, in superficial dorsal horn astrocytes are associated with both excitability changes in STT neurons and the neuropathic pain behavioral phenotype. In conclusion, we have characterized clinically-relevant rodent models of cervical contusion-induced neuropathic pain that result in chronic activation of both STT neurons and astrocytes, as well as compromise in astrocyte glutamate transporter expression. These models can be used as important tools to further study mechanisms underlying neuropathic pain post-SCI and to test potential therapeutic interventions. PMID:24833066

  15. Exciting Times for Pancreatic Islets: Glutamate Signaling in Endocrine Cells.

    PubMed

    Otter, Silke; Lammert, Eckhard

    2016-03-01

    Glutamate represents a key excitatory neurotransmitter in the central nervous system, and also modulates the function and viability of endocrine cells in pancreatic islets. In insulin-secreting beta cells, glutamate acts as an intracellular messenger, and its transport into secretory granules promotes glucose- and incretin-stimulated insulin secretion. Mitochondrial degradation of glutamate also contributes to insulin release when glutamate dehydrogenase is allosterically activated. It also signals extracellularly via glutamate receptors (AMPA and NMDA receptors) to modulate glucagon, insulin and somatostatin secretion, and islet cell survival. Its degradation products, GABA and γ-hydroxybutyrate, are released and also influence islet cell behavior. Thus, islet glutamate receptors, such as the NMDA receptors, might serve as possible drug targets to develop new medications for adjunct treatment of diabetes. PMID:26740469

  16. SOMATOSENSORY EVOKED POTENTIALS

    EPA Science Inventory

    Somatosensory evoked potentials (SEPs) have been used by neuroscientists for many years. The versatility of the method is attested to be the differing purposes to which it has been applied. Initially, SEPs were used to uncover basic principles of sensory processing. A casual glan...

  17. Metabotropic glutamate receptors depress vagal and aortic baroreceptor signal transmission in the NTS.

    PubMed

    Liu, Z; Chen, C Y; Bonham, A C

    1998-11-01

    We sought to determine whether metabotropic glutamate receptors contribute to frequency-dependent depression of vagal and aortic baroreceptor signal transmission in the nucleus of the solitary tract (NTS) in vivo. In alpha-chloralose-anesthetized rabbits, we determined the number of extracellular action potentials synaptically evoked by low (1 Hz)- or high-frequency vagal (3-20 Hz) or aortic depressor nerve (ADN) (6-80 Hz) stimulation and postsynaptically evoked by the ionotropic glutamate receptor agonist alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). The metabotropic glutamate receptor agonist (2S,1'S, 2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) attenuated NTS responses monosynaptically evoked by 1-Hz vagus stimulation by 34% (n = 25; P = 0.011), while augmenting AMPA-evoked responses by 64% (n = 17; P = 0.026). The metabotropic glutamate receptor antagonist alpha-methyl-4-phosphonophenylglycine (MPPG) did not affect NTS responses to low-frequency vagal stimulation (n = 11) or AMPA (n = 10) but augmented responses to high-frequency stimulation by 50% (n = 25; P = 0.0001). MPPG also augmented NTS responses to high-frequency ADN stimulation by 35% (n = 9; P = 0.048) but did not affect responses to low-frequency stimulation (n = 9) or AMPA (n = 7). The results suggest that metabotropic glutamate receptors, presumably at presynaptic sites, contribute to frequency-dependent depression of vagal and aortic baroreceptor signal transmission in NTS. PMID:9815076

  18. Glutamate release from astrocytic gliosomes under physiological and pathological conditions.

    PubMed

    Milanese, Marco; Bonifacino, Tiziana; Zappettini, Simona; Usai, Cesare; Tacchetti, Carlo; Nobile, Mario; Bonanno, Giambattista

    2009-01-01

    Glial subcellular particles (gliosomes) have been purified from rat cerebral cortex or mouse spinal cord and investigated for their ability to release glutamate. Confocal microscopy showed that gliosomes are enriched with glia-specific proteins, such as GFAP and S-100 but not neuronal proteins, such as PSD-95, MAP-2, and beta-tubulin III. Furthermore, gliosomes exhibit labeling neither for integrin-alphaM nor for myelin basic protein, specific for microglia and oligodendrocytes, respectively. The gliosomal fraction contains proteins of the exocytotic machinery coexisting with GFAP. Consistent with ultrastructural analysis, several nonclustered vesicles are present in the gliosome cytoplasm. Finally, gliosomes represent functional organelles that actively export glutamate when subjected to releasing stimuli, such as ionomycin, high KCl, veratrine, 4-aminopyridine, AMPA, or ATP by mechanisms involving extracellular Ca2+, Ca2+ release from intracellular stores as well as reversal of glutamate transporters. In addition, gliosomes can release glutamate also by a mechanism involving heterologous transporter activation (heterotransporters) located on glutamate-releasing and glutamate transporter-expressing (homotransporters) gliosomes. This glutamate release involves reversal of glutamate transporters and anion channel opening, but not exocytosis. Both the exocytotic and the heterotransporter-mediated glutamate release were more abundant in gliosomes prepared from the spinal cord of transgenic mice, model of amyotrophic lateral sclerosis, than in controls; suggesting the involvement of astrocytic glutamate release in the excitotoxicity proposed as a cause of motor neuron degeneration. The results support the view that gliosomes may represent a viable preparation that allows to study mechanisms of astrocytic transmitter release and its regulation in healthy animals and in animal models of brain diseases. PMID:19607977

  19. 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

  20. 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

  1. Astrocyte/neuron ratio and its importance on glutamate toxicity: an in vitro voltammetric study.

    PubMed

    Hacimuftuoglu, Ahmet; Tatar, Abdulgani; Cetin, Damla; Taspinar, Numan; Saruhan, Fatih; Okkay, Ufuk; Turkez, Hasan; Unal, Deniz; Stephens, Robert Louis; Suleyman, Halis

    2016-08-01

    The purpose of this study was to clarify the relationship between neuron cells and astrocyte cells in regulating glutamate toxicity on the 10th and 20th day in vitro. A mixed primary culture system from newborn rats that contain cerebral cortex neurons cells was employed to investigate the glutamate toxicity. All cultures were incubated with various glutamate concentrations, then viability tests and histological analyses were performed. The activities of glutamate transporters were determined by using in vitro voltammetry technique. Viable cell number was decreased significantly on the 10th day at 10(-7) M and at 10(-6) M glutamate applications, however, viable cell number was not decreased at 20th day. Astrocyte number was increased nearly six times on the 20th day as compared to the 10th day. The peak point of glutamate reuptake capacity was about 2 × 10(-4) M on the 10th day and 10(-3) M on the 20th day. According to our results, we suggested that astrocyte age was important to maintain neuronal survival against glutamate toxicity. Thus, we revealed activation or a trigger point of glutamate transporters on astrocytes due to time since more glutamate was taken up by astrocytes when glutamate transporters on the astrocyte were triggered with high exogenous glutamate concentrations. In conclusion, the present investigation is the first voltammetric study on the reuptake parameters of glutamate in vitro. PMID:26438331

  2. 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

  3. 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

  4. 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

  5. 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

  6. Laser-scanning astrocyte mapping reveals increased glutamate-responsive domain size and disrupted maturation of glutamate uptake following neonatal cortical freeze-lesion

    PubMed Central

    Armbruster, Moritz; Hampton, David; Yang, Yongjie; Dulla, Chris G.

    2014-01-01

    Astrocytic uptake of glutamate shapes extracellular neurotransmitter dynamics, receptor activation, and synaptogenesis. During development, glutamate transport becomes more robust. How neonatal brain insult affects the functional maturation of glutamate transport remains unanswered. Neonatal brain insult can lead to developmental delays, cognitive losses, and epilepsy; the disruption of glutamate transport is known to cause changes in synaptogenesis, receptor activation, and seizure. Using the neonatal freeze-lesion (FL) model, we have investigated how insult affects the maturation of astrocytic glutamate transport. As lesioning occurs on the day of birth, a time when astrocytes are still functionally immature, this model is ideal for identifying changes in astrocyte maturation following insult. Reactive astrocytosis, astrocyte proliferation, and in vitro hyperexcitability are known to occur in this model. To probe astrocyte glutamate transport with better spatial precision we have developed a novel technique, Laser Scanning Astrocyte Mapping (LSAM), which combines glutamate transport current (TC) recording from astrocytes with laser scanning glutamate photolysis. LSAM allows us to identify the area from which a single astrocyte can transport glutamate and to quantify spatial heterogeneity in the rate of glutamate clearance kinetics within that domain. Using LSAM, we report that cortical astrocytes have an increased glutamate-responsive area following FL and that TCs have faster decay times in distal, as compared to proximal processes. Furthermore, the developmental shift from GLAST- to GLT-1-dominated clearance is disrupted following FL. These findings introduce a novel method to probe astrocyte glutamate uptake and show that neonatal cortical FL disrupts the functional maturation of cortical astrocytes. PMID:25249939

  7. Overexpression of the astrocyte glutamate transporter GLT1 exacerbates phrenic motor neuron degeneration, diaphragm compromise, and forelimb motor dysfunction following cervical contusion spinal cord injury.

    PubMed

    Li, Ke; Nicaise, Charles; Sannie, Daniel; Hala, Tamara J; Javed, Elham; Parker, Jessica L; Putatunda, Rajarshi; Regan, Kathleen A; Suain, Valérie; Brion, Jean-Pierre; Rhoderick, Fred; Wright, Megan C; Poulsen, David J; Lepore, Angelo C

    2014-05-28

    A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for ≥6 weeks. We used intraspinal delivery of adeno-associated virus type 8 (AAV8)-Gfa2 vector to rat cervical spinal cord ventral horn for targeting focal astrocyte GLT1 overexpression in areas of PhMN loss. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in transduction primarily of GFAP(+) astrocytes that persisted for ≥6 weeks postinjury, as well as increased intraspinal GLT1 protein expression. Surprisingly, we found that astrocyte-targeted GLT1 overexpression increased lesion size, PhMN loss, phrenic nerve axonal degeneration, and diaphragm neuromuscular junction denervation, and resulted in reduced functional diaphragm innervation as assessed by phrenic nerve-diaphragm compound muscle action potential recordings. These results demonstrate that GLT1 overexpression via intraspinal AAV-Gfa2-GLT1 delivery exacerbates neuronal damage and increases respiratory impairment following cervical SCI. PMID:24872566

  8. Taurine attenuates D-[3H]aspartate release evoked by depolarization in ischemic corticostriatal slices.

    PubMed

    Molchanova, Svetlana M; Oja, Simo S; Saransaari, Pirjo

    2006-07-12

    Taurine is thought to be protective in ischemia due to its neuroinhibitory effects. The present aim was to assess the ability of taurine to attenuate glutamate release evoked by ischemia and to determine which component of this release is affected. The release of preloaded D-[(3)H]aspartate (a non-metabolized analog of glutamate) from superfused murine corticostriatal slices was used as index of glutamate release. Preincubation of corticostriatal slices with 10 mM taurine reduced the D-[(3)H]aspartate release evoked by either chemical ischemia (0.5 mM NaCN in glucose-free medium) or oxygen-glucose deprivation. The taurine uptake inhibitor guanidinoethanesulfonate (5 mM), the glycine receptor antagonist strychnine (0.1 mM) and the GABA(A) receptor antagonist bicuculline (0.1 mM) did not block the taurine effect. To determine which component of ischemia-induced glutamate release is affected by taurine, three pathways of this release were pharmacologically modeled. Unlabeled D-aspartate (0.5 mM) and hypo-osmotic medium (NaCl reduced by 50 mM) evoked D-[(3)H]aspartate release via homoexchange and hypo-osmotic release pathways, respectively. Taurine did not influence these pathways. However, it suppressed the synaptic release of D-[(3)H]aspartate evoked by the voltage-gated sodium channel opener veratridine (0.1 mM). Taurine thus reduces glutamate release under ischemic conditions by affecting the depolarization-evoked component. PMID:16781687

  9. 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

  10. Importance of glutamate-generating metabolic pathways for memory consolidation in chicks.

    PubMed

    Gibbs, Marie E; Hertz, Leif

    2005-07-15

    Glutamatergic and noradrenergic stimulation is essential for formation of memory of single-trial discriminative avoidance of colored beads in the 1-day-old chick. Transmitter glutamate is released soon after training and again before memory consolidation 30 min after training. Memory consolidation is abolished by posttraining injection of iodoacetate, which inhibits glycolysis and thus not only energy metabolism but also pyruvate carboxylase-dependent glucose conversion to glutamate, needed for consolidation; a similar effect is evoked by the antagonists propranolol acting at beta(2)-adrenoceptors or SR59230A acting at beta(3)-adrenoceptors. This paper shows that the effect of these inhibitors can be overcome by central injection of glutamine, providing an alternate source of transmitter glutamate and compensating for the inhibition of glycolysis by iodoacetate or the blockade of adrenergic stimulation of glycogenolysis by propranolol or of glucose uptake by SR59230A. Conversely, inhibition of memory consolidation by methionine sulfoximine (MSO), an inhibitor of glutamine synthetase and thus of the glutamate-glutamine cycle, essential for neuronal reaccumulation of previously released transmitter glutamate, could be challenged by noradrenaline, stimulating glucose uptake and glycogenolysis and providing glutamate synthesis from glucose to compensate for the lack of return of previously released glutamate. Also, administration of either glutamine or noradrenaline could prevent the spontaneous decay of labile memory 30 min after training on a weakened stimulus, suggesting that direct supply of glutamate from glucose may secure sufficient supplies of transmitter glutamate for release prior to memory consolidation at 30 min. PMID:15929064

  11. Cystine/glutamate antiporter blockage induces myelin degeneration.

    PubMed

    Soria, Federico N; Zabala, Alazne; Pampliega, Olatz; Palomino, Aitor; Miguelez, Cristina; Ugedo, Luisa; Sato, Hideyo; Matute, Carlos; Domercq, María

    2016-08-01

    The cystine/glutamate antiporter is a membrane transport system responsible for the uptake of extracellular cystine and release of intracellular glutamate. It is the major source of cystine in most cells, and a key regulator of extrasynaptic glutamate in the CNS. Because cystine is the limiting factor in the biosynthesis of glutathione, and glutamate is the most abundant neurotransmitter, the cystine/glutamate antiporter is a central player both in antioxidant defense and glutamatergic signaling, two events critical to brain function. However, distribution of cystine/glutamate antiporter in CNS has not been well characterized. Here, we analyzed expression of the catalytic subunit of the cystine/glutamate antiporter, xCT, by immunohistochemistry in histological sections of the forebrain and spinal cord. We detected labeling in neurons, oligodendrocytes, microglia, and oligodendrocyte precursor cells, but not in GFAP(+) astrocytes. In addition, we examined xCT expression and function by qPCR and cystine uptake in primary rat cultures of CNS, detecting higher levels of antiporter expression in neurons and oligodendrocytes. Chronic inhibition of cystine/glutamate antiporter caused high toxicity to cultured oligodendrocytes. In accordance, chronic blockage of cystine/glutamate antiporter as well as glutathione depletion caused myelin disruption in organotypic cerebellar slices. Finally, mice chronically treated with sulfasalazine, a cystine/glutamate antiporter inhibitor, showed a reduction in the levels of myelin and an increase in the myelinated fiber g-ratio. Together, these results reveal that cystine/glutamate antiporter is expressed in oligodendrocytes, where it is a key factor to the maintenance of cell homeostasis. GLIA 2016. GLIA 2016;64:1381-1395. PMID:27247047

  12. Glycine release is regulated by metabotropic glutamate receptors sensitive to mGluR2/3 ligands and activated by N-acetylaspartylglutamate (NAAG).

    PubMed

    Romei, Cristina; Raiteri, Maurizio; Raiteri, Luca

    2013-03-01

    The presence of metabotropic glutamate receptors (mGluRs) of group II modulating glycine exocytosis from glycinergic nerve endings of mouse spinal cord was investigated. Purified synaptosomes were selectively prelabeled with [(3)H]glycine through the neuronal transporter GlyT2 and subsequently depolarized by superfusion with 12 mM KCl. The selective mGluR2/3 agonist LY379268 inhibited the K(+)-evoked overflow of [(3)H]glycine in a concentration-dependent manner (EC(50) about 0.2 nM). The effect of LY379268 was prevented by the selective mGluR2/3 antagonist LY341495 (IC(50) about 1 nM). N-acetylaspartylglutamate (NAAG) inhibited [(3)H]glycine overflow with extraordinary potency (EC(50) about 50 fmol). In contrast, glutamate was ineffective up to 0.1 nM, excluding that glutamate contamination of commercial NAAG samples is responsible for the reported activity of NAAG at mGluR3. LY341495 antagonized the NAAG inhibition of [(3)H]glycine release. The effect of a combination of maximally effective concentrations of LY379268 and NAAG exhibited no additivity. The non-hydrolysable NAAG analogue N-acetylaspartyl-β-linked glutamate (β-NAAG) antagonized NAAG and LY379268. In conclusion, our results show that glycinergic nerve endings in spinal cord are endowed with group II mGluRs mediating inhibition of glycine exocytosis. NAAG can activate these presynaptic receptors with extremely high affinity and with characteristics compatible with the reported mGluR3 pharmacology. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. PMID:22659408

  13. 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.

  14. 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.

  15. 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. PMID:23990041

  16. Dietary Glutamate: Interactions With the Enteric Nervous System

    PubMed Central

    Wang, Guo-Du; Wang, Xi-Yu; Xia, Yun

    2014-01-01

    Background/Aims Digestion of dietary protein elevates intraluminal concentrations of glutamate in the small intestine, some of which gain access to the enteric nervous system (ENS). Glutamate, in the central nervous system (CNS), is an excitatory neurotransmitter. A dogma that glutamatergic neurophysiology in the ENS recapitulates CNS glutamatergic function persists. We reassessed the premise that glutamatergic signaling in the ENS recapitulates its neurotransmitter role in the CNS. Methods Pharmacological analysis of actions of receptor agonists and antagonists in concert with immunohistochemical localization of glutamate transporters and receptors was used. Analysis focused on intracellularly-recorded electrical and synaptic behavior of ENS neurons, on stimulation of mucosal secretion by secretomotor neurons in the submucosal plexus and on muscle contractile behavior mediated by musculomotor neurons in the myenteric plexus. Results Immunoreactivity for glutamate was expressed in ENS neurons. ENS neurons expressed immunoreactivity for the EAAC-1 glutamate transporter. Neither L-glutamate nor glutamatergic receptor agonists had excitatory actions on ENS neurons. Metabotropic glutamatergic receptor agonists did not directly stimulate neurogenic mucosal chloride secretion. Neither L-glutamate nor the metabotropic glutamatergic receptor agonist, aminocyclopentane-1,3-dicarboxylic acid (ACPD), changed the mean amplitude of spontaneously occurring contractions in circular or longitudinal strips of intestinal wall from either guinea pig or human small intestinal preparations. Conclusions Early discoveries, for excitatory glutamatergic neurotransmission in the CNS, inspired enthusiasm that investigation in the ENS would yield discoveries recapitulating the CNS glutamatergic story. We found this not to be the case. PMID:24466444

  17. 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.

  18. Prefrontal glutamate correlates of methamphetamine sensitization and preference.

    PubMed

    Lominac, Kevin D; Quadir, Sema G; Barrett, Hannah M; McKenna, Courtney L; Schwartz, Lisa M; Ruiz, Paige N; Wroten, Melissa G; Campbell, Rianne R; Miller, Bailey W; Holloway, John J; Travis, Katherine O; Rajasekar, Ganesh; Maliniak, Dan; Thompson, Andrew B; Urman, Lawrence E; Kippin, Tod E; Phillips, Tamara J; Szumlinski, Karen K

    2016-03-01

    Methamphetamine (MA) is a widely misused, highly addictive psychostimulant that elicits pronounced deficits in neurocognitive function related to hypo-functioning of the prefrontal cortex (PFC). Our understanding of how repeated MA impacts excitatory glutamatergic transmission within the PFC is limited, as is information about the relationship between PFC glutamate and addiction vulnerability/resiliency. In vivo microdialysis and immunoblotting studies characterized the effects of MA (ten injections of 2 mg/kg, i.p.) upon extracellular glutamate in C57BL/6J mice and upon glutamate receptor and transporter expression, within the medial PFC. Glutamatergic correlates of both genetic and idiopathic variance in MA preference/intake were determined through studies of high vs. low MA-drinking selectively bred mouse lines (MAHDR vs. MALDR, respectively) and inbred C57BL/6J mice exhibiting spontaneously divergent place-conditioning phenotypes. Repeated MA sensitized drug-induced glutamate release and lowered indices of N-methyl-d-aspartate receptor expression in C57BL/6J mice, but did not alter basal extracellular glutamate content or total protein expression of Homer proteins, or metabotropic or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors. Elevated basal glutamate, blunted MA-induced glutamate release and ERK activation, as well as reduced protein expression of mGlu2/3 and Homer2a/b were all correlated biochemical traits of selection for high vs. low MA drinking, and Homer2a/b levels were inversely correlated with the motivational valence of MA in C57BL/6J mice. These data provide novel evidence that repeated, low-dose MA is sufficient to perturb pre- and post-synaptic aspects of glutamate transmission within the medial PFC and that glutamate anomalies within this region may contribute to both genetic and idiopathic variance in MA addiction vulnerability/resiliency. PMID:26742098

  19. Ammonia Mediates Methamphetamine-Induced Increases in Glutamate and Excitotoxicity

    PubMed Central

    Halpin, Laura E; Northrop, Nicole A; Yamamoto, Bryan K

    2014-01-01

    Ammonia has been identified to have a significant role in the long-term damage to dopamine and serotonin terminals produced by methamphetamine (METH), but how ammonia contributes to this damage is unknown. Experiments were conducted to identify whether increases in brain ammonia affect METH-induced increases in glutamate and subsequent excitotoxicity. Increases in striatal glutamate were measured using in vivo microdialysis. To examine the role of ammonia in mediating changes in extracellular glutamate after METH exposure, lactulose was used to decrease plasma and brain ammonia. Lactulose is a non-absorbable disaccharide, which alters the intestinal lumen through multiple mechanisms that lead to the increased peripheral excretion of ammonia. METH caused a significant increase in extracellular glutamate that was prevented by lactulose. Lactulose had no effect on METH-induced hyperthermia. To determine if ammonia contributed to excitotoxicity, the effect of METH and lactulose treatment on calpain-mediated spectrin proteolysis was measured. METH significantly increased calpain-specific spectrin breakdown products, and this increase was prevented with lactulose treatment. To examine if ammonia-induced increases in extracellular glutamate were mediated by excitatory amino-acid transporters, the reverse dialysis of ammonia, the glutamate transporter inhibitor, DL-threo-β-benzyloxyaspartic acid (TBOA), or the combination of the two directly into the striatum of awake, freely moving rats was conducted. TBOA blocked the increases in extracellular glutamate produced by the reverse dialysis of ammonia. These findings demonstrate that ammonia mediates METH-induced increases in extracellular glutamate through an excitatory amino-acid transporter to cause excitotoxicity. PMID:24165886

  20. 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

  1. 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

  2. Metabotropic Glutamate Receptors

    PubMed Central

    Dillon, James; Franks, Christopher J.; Murray, Caitriona; Edwards, Richard J.; Calahorro, Fernando; Ishihara, Takeshi; Katsura, Isao; Holden-Dye, Lindy; O'Connor, Vincent

    2015-01-01

    Glutamatergic neurotransmission is evolutionarily conserved across animal phyla. A major class of glutamate receptors consists of the metabotropic glutamate receptors (mGluRs). In C. elegans, three mGluR genes, mgl-1, mgl-2, and mgl-3, are organized into three subgroups, similar to their mammalian counterparts. Cellular reporters identified expression of the mgls in the nervous system of C. elegans and overlapping expression in the pharyngeal microcircuit that controls pharyngeal muscle activity and feeding behavior. The overlapping expression of mgls within this circuit allowed the investigation of receptor signaling per se and in the context of receptor interactions within a neural network that regulates feeding. We utilized the pharmacological manipulation of neuronally regulated pumping of the pharyngeal muscle in the wild-type and mutants to investigate MGL function. This defined a net mgl-1-dependent inhibition of pharyngeal pumping that is modulated by mgl-3 excitation. Optogenetic activation of the pharyngeal glutamatergic inputs combined with electrophysiological recordings from the isolated pharyngeal preparations provided further evidence for a presynaptic mgl-1-dependent regulation of pharyngeal activity. Analysis of mgl-1, mgl-2, and mgl-3 mutant feeding behavior in the intact organism after acute food removal identified a significant role for mgl-1 in the regulation of an adaptive feeding response. Our data describe the molecular and cellular organization of mgl-1, mgl-2, and mgl-3. Pharmacological analysis identified that, in these paradigms, mgl-1 and mgl-3, but not mgl-2, can modulate the pharyngeal microcircuit. Behavioral analysis identified mgl-1 as a significant determinant of the glutamate-dependent modulation of feeding, further highlighting the significance of mGluRs in complex C. elegans behavior. PMID:25869139

  3. 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

  4. 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. PMID:26221781

  5. 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

  6. Increased glutamate-stimulated release of dopamine in substantia nigra of a rat model for attention-deficit/hyperactivity disorder--lack of effect of methylphenidate.

    PubMed

    Warton, Fleur L; Howells, Fleur M; Russell, Vivienne A

    2009-12-01

    Attention-deficit/hyperactivity disorder (ADHD) is a behavioural disorder that has been associated with dysfunction of the dopaminergic system. Abnormal dopamine function could be the result of a primary defect in dopamine neurons (neuronal firing, dopamine transporter, synthesis, receptor function) or an indirect result of impaired glutamate and/or noradrenergic regulation of dopamine neurons. There is considerable evidence to suggest that dopamine release is impaired at mesolimbic and nigrostriatal dopaminergic terminals. However, it is not known whether dysregulation occurs at the level of the cell bodies in the ventral tegmental area of the midbrain (VTA) and substantia nigra (SN). An in vitro superfusion technique was used to measure dopamine release in a widely used model of ADHD, the spontaneously hypertensive rat (SHR), and its normotensive Wistar-Kyoto (WKY) control. At approximately 30 days of age, rats were analysed for behavioural differences in the open field in response to acute treatment with methylphenidate (0.5 to 2 mg/kg in condensed milk, oral self-administration). In addition, rats were treated chronically with methylphenidate (2 mg/kg, oral self-administration, twice daily for 14 days from postnatal day 21 to 34) before the VTA and the SN were analysed for glutamate-stimulated and depolarization-evoked release of dopamine in these areas. In support of its use as an animal model for ADHD, SHR were more active in the open field and displayed less anxiety-like behaviour than WKY. Neither strain showed any effect of treatment with methylphenidate. A significant difference was observed in glutamate-stimulated release of dopamine in the SN of SHR and WKY, with SHR releasing more dopamine, consistent with the hypothesis of altered glutamate regulation of dopamine neurons in SHR. PMID:19821016

  7. Modulation of the intracellular calcium concentration in photoreceptor terminals by a presynaptic metabotropic glutamate receptor

    PubMed Central

    Koulen, Peter; Kuhn, Rainer; Wässle, Heinz; Brandstätter, Johann Helmut

    1999-01-01

    Fast excitatory neurotransmission in the central nervous system is mediated through glutamate acting on ionotropic glutamate receptors. However, glutamate acting on metabotropic glutamate receptors (mGluRs) can also exert an inhibitory action. Here, we report by immunocytochemistry and physiology, to our knowledge, the first glutamate receptor to be found in terminals of photoreceptors in the mammalian retina—the group III metabotropic glutamate receptor mGluR8. Glutamate is the transmitter of photoreceptors, and thus mGluR8 functions as an autoreceptor. Activation of mGluR8 by the group III mGluR agonists l-2-amino-4-phosphonobutyrate and l-serine-O-phosphate, or by glutamate itself, evokes a decrease in the intracellular calcium ion concentration ([Ca2+]i) in isolated photoreceptors. This effect is blocked by the group III mGluR antagonists (RS)-α-methyl-4-phosphonophenylglycine and (RS)-α-methylserine-O-phosphate. Agonists for other classes of glutamate receptors—n-methyl-d-aspartic acid, quisqualic acid, kainic acid, or (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid—have no effect on the [Ca2+]i in isolated photoreceptors. The down-regulation of the [Ca2+]i in photoreceptors by mGluR8 provides evidence for an inhibitory feedback loop at the photoreceptor synapse in the mammalian retina. This negative feedback may be a mechanism for the fine adjustment of the light-regulated release of glutamate from photoreceptors and may serve as a safety device against excitotoxic levels of release at this tonic synapse. Such a mechanism may provide a model for feedback inhibition in other parts of the central nervous system. PMID:10449793

  8. Mystixin-7 Peptide Protects Ionotropic Glutamatergic Mechanisms against Glutamate-Induced Excitotoxicity In Vitro

    PubMed Central

    2016-01-01

    Hyperactivation of the N-methyl-D-aspartic acid type glutamate receptors (NMDARs) causes glutamate excitotoxicity, a process potentially important for many neurological diseases. This study aims to investigate protective effects of the synthetic corticotrophin-releasing factor-like peptide, mystixin-7 (MTX), on model glutamate-induced excitotoxicity in vitro. The technique online monitoring of electrophysiological parameters (excitatory glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPAR) and NMDAR-dependent postsynaptic mechanisms) in the olfactory cortex slices was used. Application of L-glutamate in toxic concentration (20 mM) on slices evoked hyperactivation of NMDARs and weaker activation of the AMPARs. Upon further action agonist, the excessive activation of glutamate receptors was replaced by their irreversible blockade. Pretreatment of the slices using MTX in different concentrations (50 and 100 mg/mL) protected both NMDARs and AMPARs from glutamate-induced damage. An enzymatic treatment of MTX reduced hyperactivation of both NMDARs and AMPARs. The present study demonstrated that MTX minipeptide protected the functioning of both NMDARs and AMPARs against glutamate-induced damage. The MTX peptide is a prospective candidate for elaborated medication in treatment of neurological diseases. PMID:27504123

  9. Riluzole rescues alterations in rapid glutamate transients in the hippocampus of rTg4510 mice.

    PubMed

    Hunsberger, Holly C; Hickman, James E; Reed, Miranda N

    2016-06-01

    Those at risk for Alzheimer's disease (AD) often exhibit hippocampal hyperexcitability in the years preceding diagnosis. Our previous work with the rTg(TauP301L)4510 tau mouse model of AD suggests that this increase in hyperexcitability is likely mediated by an increase in depolarization-evoked glutamate release and a decrease in glutamate uptake, alterations of which correlate with learning and memory deficits. Treatment with riluzole restored glutamate regulation and rescued memory deficits in the TauP301L model. Here, we used enzyme-based ceramic microelectrode array technology to measure real-time phasic glutamate release and uptake events in the hippocampal subregions of TauP301L mice. For the first time, we demonstrate that perturbations in glutamate transients (rapid, spontaneous bursts of glutamate) exist in a tau mouse model of AD mouse model and that riluzole mitigates these alterations. These results help to inform our understanding of how glutamate signaling is altered in the disease process and also suggest that riluzole may serve as a clinically applicable therapeutic approach in AD. PMID:26744018

  10. Chemoenzymatic Synthesis of ortho-, meta-, and para-Substituted Derivatives of l-threo-3-Benzyloxyaspartate, An Important Glutamate Transporter Blocker

    PubMed Central

    de Villiers, Jandré; de Villiers, Marianne; Geertsema, Edzard M; Raj, Hans; Poelarends, Gerrit J

    2015-01-01

    A simple, three-step chemoenzymatic synthesis of l-threo-3-benzyloxyaspartate (l-TBOA), as well as l-TBOA derivatives with F, CF3, and CH3 substituents at the aromatic ring, starting from dimethyl acetylenedicarboxylate was investigated. These chiral amino acids, which are extremely difficult to prepare by chemical synthesis, form an important class of inhibitors of excitatory amino acid transporters involved in the regulation of glutamatergic neurotransmission. In addition, a new chemical procedure for the synthesis of racemic mixtures of TBOA and its derivatives was explored. These chemically prepared racemates are valuable reference compounds in chiral-phase HPLC to establish the enantiopurities of the corresponding chemoenzymatically prepared amino acids. PMID:26251674

  11. Ionotropic glutamate receptors in the external lateral parabrachial nucleus participate in processing cardiac sympathoexcitatory reflexes

    PubMed Central

    Guo, Zhi-Ling; Longhurst, John C.

    2012-01-01

    Stimulation of cardiac sympathetic afferents during myocardial ischemia with metabolites such as bradykinin (BK) evokes sympathoexcitatory reflex responses and activates neurons in the external lateral parabrachial nucleus (elPBN). The present study tested the hypothesis that this region in the pons processes sympathoexcitatory cardiac reflexes through an ionotropic glutamate receptor mechanism. The ischemic metabolite BK (0.1–1 μg) was injected into the pericardial space of anesthetized and bilaterally vagotomized or intact cats. Hemodynamic and renal sympathetic nerve activity (RSNA) responses to repeated administration of BK before and after unilateral 50-nl microinjections of kynurenic acid (Kyn; 25 mM), 2-amino-5-phosphonopentanoic acid (AP5; 25 mM), and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzol(F)quinoxaline (NBQX; 10 mM) into the elPBN were recorded. Intrapericardial BK evoked significant increases in mean arterial pressure (MAP) and RSNA in seven vagotomized cats. After blockade of glutamate receptors with the nonselective glutamate receptor antagonist Kyn, the BK-evoked reflex increases in MAP (50 ± 6 vs. 29 ± 2 mmHg) and RSNA (59 ± 8.6 vs. 29 ± 4.7%, before vs. after) were significantly attenuated. The BK-evoked responses returned to pre-Kyn levels 85 min after the application of Kyn. Similarly, BK-evoked reflex responses were reversibly attenuated by blockade of glutamate N-methyl-d-aspartate (NMDA) receptors with AP5 (n = 5) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors with NBQX (n = 5). In contrast, we observed that the repetitive administration of BK evoked consistent reflex responses including MAP and RSNA before and after microinjection of 50 nl of the artificial cerebrospinal fluid vehicle into the elPBN in five animals. Microinjection of glutamate receptor antagonists into regions outside the elPBN did not alter BK-induced reflex responses. Microinjection of Kyn into the elPBN reversibly attenuated BK

  12. Pivotal Enzyme in Glutamate Metabolism of Poly-γ-Glutamate-Producing Microbes

    PubMed Central

    Ashiuchi, Makoto; Yamamoto, Takashi; Kamei, Tohru

    2013-01-01

    The extremely halophilic archaeon Natrialba aegyptiaca secretes the L-homo type of poly-γ-glutamate (PGA) as an extremolyte. We examined the enzymes involved in glutamate metabolism and verified the presence of L-glutamate dehydrogenases, L-aspartate aminotransferase, and L-glutamate synthase. However, neither glutamate racemase nor D-amino acid aminotransferase activity was detected, suggesting the absence of sources of D-glutamate. In contrast, D-glutamate-rich PGA producers mostly possess such intracellular sources of D-glutamate. The results of our present study indicate that the D-glutamate-anabolic enzyme “glutamate racemase” is pivotal in the biosynthesis of PGA. PMID:25371338

  13. Riluzole rescues glutamate alterations, cognitive deficits, and tau pathology associated with P301L tau expression.

    PubMed

    Hunsberger, Holly C; Weitzner, Daniel S; Rudy, Carolyn C; Hickman, James E; Libell, Eric M; Speer, Rebecca R; Gerhardt, Greg A; Reed, Miranda N

    2015-10-01

    Hyperexcitability of the hippocampus is a commonly observed phenomenon in the years preceding a diagnosis of Alzheimer's disease (AD). Our previous work suggests a dysregulation in glutamate neurotransmission may mediate this hyperexcitability, and glutamate dysregulation correlates with cognitive deficits in the rTg(TauP301L)4510 mouse model of AD. To determine whether improving glutamate regulation would attenuate cognitive deficits and AD-related pathology, TauP301L mice were treated with riluzole (~ 12.5 mg/kg/day p.o.), an FDA-approved drug for amyotrophic lateral sclerosis that lowers extracellular glutamate levels. Riluzole-treated TauP301L mice exhibited improved performance in the water radial arm maze and the Morris water maze, associated with a decrease in glutamate release and an increase in glutamate uptake in the dentate gyrus, cornu ammonis 3 (CA3), and cornu ammonis 1 (CA1) regions of the hippocampus. Riluzole also attenuated the TauP301L-mediated increase in hippocampal vesicular glutamate transporter 1, which packages glutamate into vesicles and influences glutamate release; and the TauP301L-mediated decrease in hippocampal glutamate transporter 1, the major transporter responsible for removing glutamate from the extracellular space. The TauP301L-mediated reduction in PSD-95 expression, a marker of excitatory synapses in the hippocampus, was also rescued by riluzole. Riluzole treatment reduced total levels of tau, as well as the pathological phosphorylation and conformational changes in tau associated with the P301L mutation. These findings open new opportunities for the development of clinically applicable therapeutic approaches to regulate glutamate in vulnerable circuits for those at risk for the development of AD. PMID:26146790

  14. 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

  15. Glutamate transporter homolog-based model predicts that anion-π interaction is the mechanism for the voltage-dependent response of prestin.

    PubMed

    Lovas, Sándor; He, David Z Z; Liu, Huizhan; Tang, Jie; Pecka, Jason L; Hatfield, Marcus P D; Beisel, Kirk W

    2015-10-01

    Prestin is the motor protein of cochlear outer hair cells. Its unique capability to perform direct, rapid, and reciprocal electromechanical conversion depends on membrane potential and interaction with intracellular anions. How prestin senses the voltage change and interacts with anions are still unknown. Our three-dimensional model of prestin using molecular dynamics simulations predicts that prestin contains eight transmembrane-spanning segments and two helical re-entry loops and that tyrosyl residues are the structural specialization of the molecule for the unique function of prestin. Using site-directed mutagenesis and electrophysiological techniques, we confirmed that residues Tyr(367), Tyr(486), Tyr(501), and Tyr(508) contribute to anion binding, interacting with intracellular anions through novel anion-π interactions. Such weak interactions, sensitive to voltage and mechanical stimulation, confer prestin with a unique capability to perform electromechanical and mechanoelectric conversions with exquisite sensitivity. This novel mechanism is completely different from all known mechanisms seen in ion channels, transporters, and motor proteins. PMID:26283790

  16. 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

  17. cAMP Response Element-binding Protein (CREB) and Nuclear Factor κB Mediate the Tamoxifen-induced Up-regulation of Glutamate Transporter 1 (GLT-1) in Rat Astrocytes*

    PubMed Central

    Karki, Pratap; Webb, Anton; Smith, Keisha; Lee, Kyuwon; Son, Deok-Soo; Aschner, Michael; Lee, Eunsook

    2013-01-01

    Tamoxifen (TX), a selective estrogen receptor modulator, exerts antagonistic effects on breast tissue and is used to treat breast cancer. Recent evidence also suggests that it may act as an agonist in brain tissue. We reported previously that TX enhanced the expression and function of glutamate transporter 1 (GLT-1) in rat astrocytes, an effect that was mediated by TGF-α. To gain further insight into the mechanisms that mediate TX-induced up-regulation of GLT-1 (EAAT2 in humans), we investigated its effect on GLT-1 at the transcriptional level. TX phosphorylated the cAMP response element-binding protein (CREB) and recruited CREB to the GLT-1 promoter consensus site. The effect of TX on astrocytic GLT-1 was attenuated by the inhibition of PKA, the upstream activator of the CREB pathway. In addition, the effect of TX on GLT-1 promoter activity was abolished by the inhibition of the NF-κB pathway. Furthermore, TX recruited the NF-κB subunits p65 and p50 to the NF-κB binding domain of the GLT-1 promoter. Mutation of NF-κB (triple, −583/-282/-251) or CRE (-308) sites on the GLT-1 promoter led to significant repression of the promoter activity, but neither mutant completely abolished the TX-induced GLT-1 promoter activity. Mutation of both the NF-κB (-583/-282/-251) and CRE (-308) sites led to a complete abrogation of the effect of TX on GLT-1 promoter activity. Taken together, our findings establish that TX regulates GLT-1 via the CREB and NF-κB pathways. PMID:23955341

  18. 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

  19. Alcohol-seeking behavior is associated with increased glutamate transmission in basolateral amygdala and nucleus accumbens as measured by glutamate-oxidase coated biosensors

    PubMed Central

    Gass, Justin T.; Sinclair, Courtney M.; Cleva, Richard M.; Widholm, John J.; Olive, M. Foster

    2010-01-01

    Relapse is one of the most problematic aspects in the treatment of alcoholism and is often triggered by alcohol-associated environmental cues. Evidence indicates that glutamate neurotransmission plays a critical role in cue-induced relapse-like behavior, as inhibition of glutamate neurotransmission can prevent reinstatement of alcohol-seeking behavior. However, few studies have examined specific changes in extracellular glutamate levels in discrete brain regions produced by exposure to alcohol-associated cues. The purpose of this study was to use glutamate oxidase (GluOx)-coated biosensors to monitor changes in extracellular glutamate in specific brain regions during cue-induced reinstatement of alcohol-seeking behavior. Male Wistar rats were implanted with indwelling jugular vein catheters and intracerebral guide cannula aimed at the basolateral amygdala (BLA) or nucleus accumbens (NAc) core, and then trained to self-administer alcohol intravenously. A separate group of animals was trained to self-administer food pellets. Each reinforcer was accompanied by the presentation of a light/tone stimulus. Following stabilization of responding for alcohol or food reinforcement and subsequent extinction training, animals were implanted with precalibrated biosensors and then underwent a 1 hr cue-induced reinstatement testing period. As determined by GluOx-coated biosensors, extracellular levels of glutamate were increased in the BLA and NAc core during cue-induced reinstatement of alcohol-seeking behavior. The cumulative change in extracellular glutamate in both regions was significantly greater for cue-induced reinstatement of alcohol-seeking behavior versus that of food-seeking behavior. These results indicate that increases in glutamate transmission in the BLA and NAc core may be a neurochemical substrate of cue-evoked alcohol-seeking behavior. PMID:21054692

  20. 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

  1. 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

  2. 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. PMID:26414427

  3. Genotoxicity of monosodium glutamate.

    PubMed

    Ataseven, Nazmiye; Yüzbaşıoğlu, Deniz; Keskin, Ayten Çelebi; Ünal, Fatma

    2016-05-01

    Monosodium glutamate (MSG) is one of the most widely used flavor enhancers throughout the world. The aim of this study is to investigate the genotoxic potential of MSG by using chromosome aberrations (CAs), sister-chromatid exchanges (SCEs), cytokinesis-blocked micronucleus (CBMN), and random amplified polymorphic DNA-polimerase chain reaction (RAPD-PCR) in cultured human lymphocytes and alkaline comet assays in isolated human lymphocytes, which were incubated with six concentrations (250, 500, 1000, 2000, 4000 and 8000 μg/mL) of MSG. The result of this study indicated that MSG significantly and dose dependently increased the frequencies of CAs, SCE and MN in all treatments and times, compared with control. However, the replication (RI) and nuclear division indices (NDI) were not affected. In this paper, in vitro genotoxic effects of the MSG was also investigated on human peripheral lymphocytes by analysing the RAPD-PCR with arbitrary 10-mer primers. The changes occurring in RAPD profiles after MSG treatment include increase or decrease in band intensity and gain or loss of bands. In the comet assay, this additive caused DNA damage at all concentrations in isolated human lymphocytes after 1-h in vitro exposure. Our results demonstrate that MSG is genotoxic to the human peripheral blood lymphocytes in vitro. PMID:26929995

  4. Presynaptic kainate receptor facilitation of glutamate release involves protein kinase A in the rat hippocampus

    PubMed Central

    Rodríguez-Moreno, Antonio; Sihra, Talvinder S

    2004-01-01

    We have explored the mechanisms involved in the facilitation of glutamate release mediated by the activation of kainate receptors in the rat hippocampus using isolated nerve terminal (synaptosome) and slice preparations. In hippocampal nerve terminals, kainate (KA) produced an increase of glutamate release at concentrations of agonist ranging from 10 to 1000 μm. In hippocampal slices, KA at low nanomolar concentrations (20–50 nm) also produced an increase of evoked excitatory postsynaptic currents (eEPSCs) at mossy fibre–CA3 synapses. In both, synaptosomes and slices, the effect of KA was antagonized by CNQX, and persisted after pretreatment with a cocktail of antagonists for other receptors whose activation could potentially have produced facilitation of release. These data indicate that the facilitation of glutamate release observed is mediated by the activation of presynaptic glutamate receptors of the kainate type. Mechanistically, the observed effects of KA appear to be the same in synaptosomal and slice preparations. Thus, the effect of KA on glutamate release and mossy fibre–CA3 synaptic transmission was occluded by the stimulation of adenylyl cyclase by forskolin and suppressed by the inhibition of protein kinase A by H-89 or Rp-Br-cAMP. We conclude that kainate receptors present at presynaptic terminals in the rat hippocampus mediate the facilitation of glutamate release through a mechanism involving the activation of an adenylyl cyclase–second messenger cAMP–protein kinase A signalling cascade. PMID:15107475

  5. 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

  6. 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. PMID:26911692

  7. Mechanisms for maintaining extracellular glutamate levels in the anoxic turtle striatum.

    PubMed

    Milton, Sarah L; Thompson, John W; Lutz, Peter L

    2002-05-01

    The turtle Trachemys scripta is one of a limited group of vertebrates that can withstand hours to days without oxygen. One facet of anoxic survival is the turtle's ability to maintain basal extracellular glutamate levels, whereas in most vertebrates, anoxia triggers massive excitotoxic glutamate release. We investigated glutamate release and reuptake in the anoxic turtle and the effects of adenosine and ATP-sensitive potassium (K(ATP)) channels on glutamate homeostasis. Striatal extracellular glutamate was measured in anesthetized T. scripta by microdialysis in normoxia and over 2-h anoxia. Glutamate release is decreased by 44% in the early anoxic turtle; this anoxia-induced decrease in glutamate release was prevented when K(ATP) channels and adenosine receptors were blocked simultaneously but not when either mechanism was blocked individually. We hypothesize that the continued release and reuptake of glutamate during anoxia help maintain neuronal tone and aid in the recovery of a functional neuronal network after long periods of anoxia, whereas activation of adenosine and/or K(ATP) conserves energy by reducing glutamate release and lowering transport costs. PMID:11959671

  8. 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

  9. The glutamate hypothesis of schizophrenia: evidence from human brain tissue studies

    PubMed Central

    Hu, Wei; MacDonald, Matthew L.; Elswick, Daniel E.; Sweet, Robert A.

    2014-01-01

    A number of studies have indicated that antagonists of the N-methyl-d-aspartate (NMDA) subtypes of glutamate receptors can cause schizophrenia-like symptoms in healthy individuals and exacerbate symptoms in individuals with schizophrenia. These findings have led to the glutamate hypothesis of schizophrenia. Here we review the evidence for this hypothesis in postmortem studies of brain tissue from individuals affected by schizophrenia, summarizing studies of glutamate neuron morphology, of expression of glutamate receptors and transporters, and of the synthesizing and metabolizing enzymes for glutamate and its co-agonists. We found consistent evidence of morphological alterations of dendrites of glutamatergic neurons in the cerebral cortex of subjects with schizophrenia and of reduced levels of the axon bouton marker synaptophysin. There were no consistent alterations of mRNA expression of glutamate receptors, although there has been limited study of the corresponding proteins. Studies of the glutamate metabolic pathway have been limited, although there is some evidence that excitatory amino acid transporter-2, glutamine synthetase, and glutaminase have altered expression in schizophrenia. Future studies would benefit from additional direct examination of glutamatergic proteins. Further advances, such as selective testing of synaptic microdomains, cortical layers, and neuronal subtypes, may also be required to elucidate the nature of glutamate signaling impairments in schizophrenia. PMID:25315318

  10. 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.

  11. Metabotropic glutamate receptors depress glutamate-mediated synaptic input to rat midbrain dopamine neurones in vitro.

    PubMed

    Wigmore, M A; Lacey, M G

    1998-02-01

    1. Glutamate (AMPA receptor-mediated) excitatory postsynaptic potentials (e.p.s.ps.), evoked by electrical stimulation rostral to the recording site, were examined by intracellular microelectrode recording from dopamine neurones in parasagittal slices of rat ventral midbrain. 2. The e.p.s.p. was depressed by the group III metabotropic glutamate (mGlu) receptor agonist L-2-amino-4-phosphonobutyric acid (L-AP4; 0.01-30 microM) by up to 60% with an EC50 of 0.82 microM. The depression induced by L-AP4 (3 microM) was reversed by the group III preferring mGlu receptor antagonist, alpha-methyl-4-phosphonophenylglycine (MPPG; 250 microM). 3. The group I and II mGlu agonist, 1S,3R-aminocyclopentanedicarboxylic acid (ACPD; 3-30 microM) also depressed the e.p.s.p. in a concentration-dependent manner. The effect of ACPD (10 microM) was reversed by (+)-alpha-methyl-4-carboxyphenylglycine (MCPG; 1 mM; 4 cells). This effect of ACPD was also partially antagonized (by 50.3+/-15.7%, 4 cells) by MPPG (250 microM). 4. The selective agonist at group I mGlu receptors, dihydroxyphenylglycine (DHPG; 100 microM), decreased e.p.s.p. amplitude by 27.1+/-8.2% (7 cells), as did the group II mGlu receptor-selective agonist (1S,1R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; 1 microM) by 26.7+/-4.3% (5 cells). 5. DHPG (10-100 microM) caused a depolarization of the recorded cell, as did ACPD (3-30 microM), whereas no such postsynaptic effect of either L-AP4 or DCG-IV was observed. 6. These results provide evidence for the presence of presynaptic inhibitory metabotropic glutamate autoreceptors from the mGlu receptor groups II and III on descending glutamatergic inputs to midbrain dopamine neurones. Group I mGlu receptors mediate a postsynaptic depolarization, and can also depress glutamatergic transmission, but may not necessarily be localized presynaptically. These sites represent novel drug targets for treatment of schizophrenia and movement disorders of basal ganglia origin. PMID

  12. Metabotropic glutamate receptors depress glutamate-mediated synaptic input to rat midbrain dopamine neurones in vitro

    PubMed Central

    Wigmore, Mark A; Lacey, Michael G

    1998-01-01

    Glutamate (AMPA receptor-mediated) excitatory postsynaptic potentials (e.p.s.ps.), evoked by electrical stimulation rostral to the recording site, were examined by intracellular microelectrode recording from dopamine neurones in parasagittal slices of rat ventral midbrain. The e.p.s.p. was depressed by the group III metabotropic glutamate (mGlu) receptor agonist L-2-amino-4-phosphonobutyric acid (L-AP4; 0.01–30 μM) by up to 60% with an EC50 of 0.82 μM. The depression induced by L-AP4 (3 μM) was reversed by the group III preferring mGlu receptor antagonist, α-methyl-4-phosphonophenylglycine (MPPG; 250 μM). The group I and II mGlu agonist, 1S,3R-aminocyclopentanedicarboxylic acid (ACPD; 3–30 μM) also depressed the e.p.s.p. in a concentration-dependent manner. The effect of ACPD (10 μM) was reversed by (+)-α-methyl-4-carboxyphenylglycine (MCPG; 1 mM; 4 cells). This effect of ACPD was also partially antagonized (by 50.3±15.7%, 4 cells) by MPPG (250 μM). The selective agonist at group I mGlu receptors, dihydroxyphenylglycine (DHPG; 100 μM), decreased e.p.s.p. amplitude by 27.1±8.2% (7 cells), as did the group II mGlu receptor-selective agonist (1S,1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; 1 μM) by 26.7±4.3% (5 cells). DHPG (10–100 μM) caused a depolarization of the recorded cell, as did ACPD (3–30 μM), whereas no such postsynaptic effect of either L-AP4 or DCG-IV was observed. These results provide evidence for the presence of presynaptic inhibitory metabotropic glutamate autoreceptors from the mGlu receptor groups II and III on descending glutamatergic inputs to midbrain dopamine neurones. Group I mGlu receptors mediate a postsynaptic depolarization, and can also depress glutamatergic transmission, but may not necessarily be localized presynaptically. These sites represent novel drug targets for treatment of schizophrenia and movement disorders of basal ganglia origin. PMID:9517386

  13. Long-term NMDAR antagonism correlates reduced astrocytic glutamate uptake with anxiety-like phenotype

    PubMed Central

    Zimmer, Eduardo R.; Torrez, Vitor R.; Kalinine, Eduardo; Augustin, Marina C.; Zenki, Kamila C.; Almeida, Roberto F.; Hansel, Gisele; Muller, Alexandre P.; Souza, Diogo O.; Machado-Vieira, Rodrigo; Portela, Luis V.

    2015-01-01

    The role of glutamate N-methyl-D-aspartate receptor (NMDAR) hypofunction has been extensively studied in schizophrenia; however, less is known about its role in anxiety disorders. Recently, it was demonstrated that astrocytic GLT-1 blockade leads to an anxiety-like phenotype. Although astrocytes are capable of modulating NMDAR activity through glutamate uptake transporters, the relationship between astrocytic glutamate uptake and the development of an anxiety phenotype remains poorly explored. Here, we aimed to investigative whether long-term antagonism of NMDAR impacts anxiety-related behaviors and astrocytic glutamate uptake. Memantine, an NMDAR antagonist, was administered daily for 24 days to healthy adult CF-1 mice by oral gavage at doses of 5, 10, or 20 mg/kg. The mice were submitted to a sequential battery of behavioral tests (open field, light–dark box and elevated plus-maze tests). We then evaluated glutamate uptake activity and the immunocontents of glutamate transporters in the frontoparietal cortex and hippocampus. Our results demonstrated that long-term administration of memantine induces anxiety-like behavior in mice in the light–dark box and elevated plus-maze paradigms. Additionally, the administration of memantine decreased glutamate uptake activity in both the frontoparietal cortex and hippocampus without altering the immunocontent of either GLT-1 or GLAST. Remarkably, the memantine-induced reduction in glutamate uptake was correlated with enhancement of an anxiety-like phenotype. In conclusion, long-term NMDAR antagonism with memantine induces anxiety-like behavior that is associated with reduced glutamate uptake activity but that is not dependent on GLT-1 or GLAST protein expression. Our study suggests that NMDAR and glutamate uptake hypofunction may contribute to the development of conditions that fall within the category of anxiety disorders. PMID:26089779

  14. 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

  15. 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....

  16. 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....

  17. 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....

  18. 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....

  19. 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...

  20. Glutamate-induced long-term potentiation of the frequency of miniature synaptic currents in cultured hippocampal neurons

    NASA Astrophysics Data System (ADS)

    Malgaroli, Antonio; Tsien, Richard W.

    1992-05-01

    Glutamate application at synapses between hippocampal neurons in culture produces long-term potentiation of the frequency of spontaneous miniature synaptic currents, together with long-term potentiation of evoked synaptic currents. The mini frequency potentiation is initiated postsynaptically and requires activity of NMDA receptors. Although the frequency of unitary quanta! responses increases strongly, their amplitude remains little changed with potentiation. Tests of postsynaptic responsiveness rule out recruitment of latent glutamate receptor clusters. Thus, postsynaptic induction can lead to enhancement of presynaptic transmitter release. The sustained potentiation of mini frequency is expressed even in the absence of Ca2+ entry into presynaptic terminals.

  1. 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

  2. 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. PMID:26471418

  3. Reactive oxygen species induced by presynaptic glutamate receptor activation is involved in [(3)H]GABA release from rat brain cortical nerve terminals.

    PubMed

    Tarasenko, A; Krupko, O; Himmelreich, N

    2012-12-01

    We investigated the production of reactive oxygen species (ROS) as a response to presynaptic glutamate receptor activation, and the role of ROS in neurotransmitter (GABA) release. Experiments were performed with rat brain cortical synaptosomes using glutamate, NMDA and kainate as agonists of glutamate receptors. ROS production was evaluated with the fluorogenic compound dichlorodihydrofluorescein diacetate (H(2)DCF-DA), and GABA release was studied using synaptosomes loaded with [(3)H]GABA. All agonists were found to stimulate ROS production, and specific antagonists of NMDA and kainate/AMPA receptors, dizocilpine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-done (CNQX), significantly inhibited the ROS increase. Spontaneous as well as agonist-evoked ROS production was effectively attenuated by diphenyleneiodonium (DPI), a commonly used potent inhibitor of NADPH oxidase activity, that suggests a high contribution of NADPH-oxidase to this process. The replacement of glucose with pyruvate or the simultaneous presence of both substrates in the medium led to the decrease in spontaneous and NMDA-evoked ROS production, but to the increase in ROS production induced by kainate. Scavenging of agonist-evoked ROS production by a potent antioxidant N-acetylcysteine was tightly correlated with the inhibition of agonist-evoked GABA release. Together, these findings show that the activation of presynaptic glutamate receptors induces an increase in ROS production, and there is a tight correlation between ROS production and GABA secretion. The pivotal role of kainate/AMPA receptors in ROS production is under discussion. PMID:22864357

  4. 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. PMID:26719090

  5. L-glutamate may be the fast excitatory transmitter of Aplysia sensory neurons.

    PubMed Central

    Dale, N; Kandel, E R

    1993-01-01

    Although modulation of synaptic transmission between Aplysia mechanosensory and motor neurons has been an important model for processes thought to underlie simple forms of learning and memory, the nature of the fast excitatory transmitter utilized by the sensory neurons has remained obscure. To identify the sensory neuron transmitter, we first examined the detailed properties of the synaptic response evoked in motor neurons cocultured with pleural sensory neurons. The excitatory postsynaptic current had a nonlinear current-voltage relation with a reversal potential between 0 and 10 mV and a plateau region between -40 and -70 mV. When the concentration of Mg2+ in the artificial sea water was lowered to 5 mM, the current-voltage relation of the excitatory postsynaptic current became linear, suggesting that Mg2+ blocks the postsynaptic receptor in a voltage-dependent manner. After screening a variety of small molecules, we found that L-glutamate could mimic the actions of the sensory neuron transmitter: responses to L-glutamate also had a reversal potential between 0 and 10 mV and a nonlinear current-voltage relation that could be made linear by lowering external Mg2+. To demonstrate further similarity of action between L-glutamate and the endogenous transmitter, we utilized four antagonists (kynurenate, 6,7-dinitroquinoxaline-2,3-dione, D-aspartate, and D-glutamate) to block in a dose-dependent manner the actions of L-glutamate and the natural transmitter. We therefore suggest that the sensory neurons use a glutamate-like transmitter and favor L-glutamate itself, because no other naturally occurring amino acid that we have studied has had similar actions. As the postsynaptic receptor for the sensory neuron transmitter is weakly blocked in a voltage-dependent manner by Mg2+, the excitatory receptors innervated by the Aplysia sensory neuron may represent a distant precursor of the vertebrate N-methyl-D-aspartate receptor. PMID:8102205

  6. The dose makes the poison: from glutamate-mediated neurogenesis to neuronal atrophy and depression.

    PubMed

    Rubio-Casillas, Alberto; Fernández-Guasti, Alonso

    2016-08-01

    Experimental evidence has demonstrated that glutamate is an essential factor for neurogenesis, whereas another line of research postulates that excessive glutamatergic neurotransmission is associated with the pathogenesis of depression. The present review shows that such paradox can be explained within the framework of hormesis, defined as biphasic dose responses. Low glutamate levels activate adaptive stress responses that include proteins that protect neurons against more severe stress. Conversely, abnormally high levels of glutamate, resulting from increased release and/or decreased removal, cause neuronal atrophy and depression. The dysregulation of the glutamatergic transmission in depression could be underlined by several factors including a decreased inhibition (γ-aminobutyric acid or serotonin) or an increased excitation (primarily within the glutamatergic system). Experimental evidence shows that the activation of N-methyl-D-aspartate receptor (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR) can exert two opposite effects on neurogenesis and neuron survival depending on the synaptic or extrasynaptic concentration. Chronic stress, which usually underlies experimental and clinical depression, enhances glutamate release. This overactivates NMDA receptors (NMDAR) and consequently impairs AMPAR activity. Various studies show that treatment with antidepressants decreases plasma glutamate levels in depressed individuals and regulates glutamate receptors by reducing NMDAR function by decreasing the expression of its subunits and by potentiating AMPAR-mediated transmission. Additionally, it has been shown that chronic treatment with antidepressants having divergent mechanisms of action (including tricyclics, selective serotonin reuptake inhibitors, and ketamine) markedly reduced depolarization-evoked glutamate release in the hippocampus. These data, taken together, suggest that the glutamatergic system could be a final

  7. Modeling Electrically Evoked Otoacoustic Emissions

    NASA Astrophysics Data System (ADS)

    Grosh, K.; Deo, N.; Parthasarathi, A. A.; Nuttall, A. L.; Zheng, J. F.; Ren, T. Y.

    2003-02-01

    Electrical evoked otoacoustic emissions (EEOAE) are used to investigate in vivo cochlear electromechanical function. Round window electrical stimulation gives rise to a broad frequency EEOAE response, from 100 Hz or below to 40 kHz in guinea pigs. Placing bipolar electrodes very close to the basilar membrane (in the scala vestibuli and scala tympani) gives rise to a much narrower frequency range of EEOAE, limited to around 20 kHz when the electrodes are placed near the 18 kHz best frequency place. Model predictions using a three dimensional fluid model in conjunction with a simple model for outer hair cell (OHC) activity are used to interpret the experimental results. The model is solved using a 2.5D finite-element formulation. Predictions show that the high-frequency limit of the excitation is determined by the spatial extent of the current stimulus (also called the current spread). The global peaks in the EEOAE spectra are interpreted as constructive interference between electrically evoked backward traveling waves and forward traveling waves reflected from the stapes. Steady-state response predictions of the model are presented.

  8. 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

  9. 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

  10. Dexmedetomidine infusion and somatosensory evoked potentials.

    PubMed

    Bloom, M; Beric, A; Bekker, A

    2001-10-01

    Intraoperative neurophysiologic monitoring requires information on the effects of anesthetic drugs because these drugs can directly alter evoked potentials, thus interfering with monitoring. We report on our evaluation of the effect of the recently introduced alpha2-adrenergic agonist, dexmedetomidine, on the somatosensory evoked potentials in two patients undergoing cervico-occipital fusion. Our results suggest that, although dexmedetomidine can affect the later cortical peaks of somatosensory evoked potentials (SSEPs), consistent and reproducible potentials can be recorded. PMID:11733664

  11. CCL5-glutamate interaction in central nervous system: Early and acute presynaptic defects in EAE mice.

    PubMed

    Di Prisco, Silvia; Merega, Elisa; Milanese, Marco; Summa, Maria; Casazza, Simona; Raffaghello, Lizzia; Pistoia, Vito; Uccelli, Antonio; Pittaluga, Anna

    2013-12-01

    We investigated the CCL5-glutamate interaction in the cortex and in the spinal cord from mice with Experimental Autoimmune Encephalomyelitis (EAE) at 13 and 21/30 days post immunization (d.p.i.), representing the onset and the peak of the disease, respectively. An early reduction of the KCl-evoked glutamate release was observed in cortical terminals from EAE mice at 13 d.p.i., persisting until 21/30 d.p.i. A concomitant reduction of the depolarization-evoked cyclic adenosine monophosphate (cAMP), but not of the inositol 1,4,5-trisphosphate (IP3) cortical production also occurred at 13 d.p.i, that still was detectable at the acute stage of disease (21 dp.i.). Inasmuch, the CCL5-mediated inhibition of glutamate exocytosis observed in control mice turned to facilitation in EAE mouse cortex at 13 d.p.i., then becoming undetectable at 21/30 d.p.i. Differently, glutamate exocytosis, as well as IP3 and cAMP productions were unaltered in spinal cord synaptosomes from EAE mice at 13 d.p.i., but significantly increased at 21/30 d.p.i., while the presynaptic CCL5-mediated facilitation of glutamate exocytosis observed in control mice remained unchanged. In both CNS regions, the presynaptic defects were parallelled by increased CCL5 availability. Inasmuch, the presynaptic defects so far described in EAE mice were reminiscent of the effects acute CCL5 exerts in control conditions. Based on these observations we propose that increased CCL5 bioavailability could have a role in determining the abovedescribed impaired presynaptic impairments in both CNS regions. These presynaptic defects could be relevant to the onset of early cognitive impairments and acute neuroinflammation and demyelinating processes observed in multiple sclerosis patients. PMID:23958452

  12. 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.

  13. 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.

  14. Some Operational Characteristics of Glycine Release in Rat Retina: The Role of Reverse Mode Operation of Glycine Transporter Type-1 (GlyT-1) in Ischemic Conditions.

    PubMed

    Hanuska, Adrienn; Szénási, Gábor; Albert, Mihaly; Koles, Laszlo; Varga, Agoston; Szabo, Andras; Matyus, Peter; Harsing, Laszlo G

    2016-02-01

    Rat posterior eyecups containing the retina were prepared, loaded with [(3)H]glycine and superfused in order to determine its release originated from glycinergic amacrine cells and/or glial cells. Deprivation of oxygen and glucose from the Krebs-bicarbonate buffer used for superfusion evoked a marked increase of [(3)H]glycine release, an effect that was found to be external Ca(2+)-independent. Whereas oxygen and glucose deprivation increased [(3)H]glycine release, its uptake was reduced suggesting that energy deficiency shifts glycine transporter type-1 operation from normal to reverse mode. The increased release of [(3)H]glycine evoked by oxygen and glucose deprivation was suspended by addition of the non-competitive glycine transporter type-1 inhibitor NFPS and the competitive inhibitor ACPPB further suggesting the involvement of this transporter in the mediation of [(3)H]glycine release. Oxygen and glucose deprivation also evoked [(3)H]glutamate release from rat retina and the concomitantly occurring release of the NMDA receptor agonist glutamate and the coagonist glycine makes NMDA receptor pathological overstimulation possible in hypoxic conditions. [(3)H]Glutamate release was suspended by addition of the excitatory amino acid transporter inhibitor TBOA. Sarcosine, a substrate inhibitor of glycine transporter type-1, also increased [(3)H]glycine release probably by heteroexchange shifting transporter operation into reverse mode. This effect of sarcosine was also external Ca(2+)-independent and could be suspended by NFPS. Energy deficiency in retina induced by ouabain, an inhibitor of the Na(+)-K(+)-dependent ATPase, and by rotenone, a mitochondrial complex I inhibitor added with the glycolytic inhibitor 2-deoxy-D-glucose, led to increase of retinal [(3)H]glycine efflux. These effects of ouabain and rotenone/2-deoxy-D-glucose could also be blocked by NFPS pointed to the preferential reverse mode operation of glycine transporter type-1 as a consequence of

  15. Abnormal glutamate release in aged BTBR mouse model of autism

    PubMed Central

    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. PMID:26617779

  16. 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.

  17. 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.

  18. USE OF SENSORY EVOKED POTENTIALS IN TOXICOLOGY

    EPA Science Inventory

    The rationale for studying sensory systems as an integral part of neurotoxicological examinations is presented. The role of evoked potentials in assessing brain dysfunction in general and sensory systems in particular is also presented. Four types of sensory evoked potentials (br...

  19. Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus

    PubMed Central

    Wall, Mark J; Dale, Nicholas

    2013-01-01

    The neuromodulator adenosine plays an important role in many physiological and pathological processes within the mammalian CNS. However, the precise mechanisms of how the concentration of extracellular adenosine increases following neural activity remain contentious. Here we have used microelectrode biosensors to directly measure adenosine release induced by focal stimulation in stratum radiatum of area CA1 in mouse hippocampal slices. Adenosine release was both action potential and Ca2+ dependent and could be evoked with low stimulation frequencies and small numbers of stimuli. Adenosine release required the activation of ionotropic glutamate receptors and could be evoked by local application of glutamate receptor agonists. Approximately 40% of stimulated-adenosine release occurred by translocation of adenosine via equilibrative nucleoside transporters (ENTs). This component of release persisted in the presence of the gliotoxin fluoroacetate and thus results from the direct release of adenosine from neurons. A reduction of adenosine release in the presence of NTPDase blockers, in slices from CD73−/− and dn-SNARE mice, provides evidence that a component of adenosine release arises from the extracellular metabolism of ATP released from astrocytes. This component of release appeared to have slower kinetics than the direct ENT-mediated release of adenosine. These data suggest that activity-dependent adenosine release is surprisingly complex and, in the hippocampus, arises from at least two distinct mechanisms with different cellular sources. PMID:23713028

  20. [The disposing techniques of evoked potentials].

    PubMed

    Liu, H G; Zhou, L; Gu, J; Jing, D Z

    2000-11-01

    This paper is to bring forward the new disposing techniques of evoked potentials which include four aspect techniques of the averaging, the recording, digital sampling and filters about the averaging, evoked potential amplitude, evoked potential latency, evoked potential recording, and evoked potential generations. The technique of the averaging including signal filtering and a periodic averaging, can enhance EP dedection. The commercial EP machines also plot changes in latency between serial EP studies in order to detect trends in peak latency. The modern digital EP recording device consists of sensory stimator, recording amplifiers with analog filters, an analog-to-digital converter, a digital signal averager, and a display and storage system. A sample-and-hold function is one of the recent developments which used EP collectors that provide simultaneous recording with multiple channels employing different time and voltage scales and sampling rates. The EP data may be further processed following A-D conversion by digital filters. PMID:12583248

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Increase of extracellular glutamate concentration increases its oxidation and diminishes glucose oxidation in isolated mouse hippocampus: reversible by TFB-TBOA.

    PubMed

    Torres, Felipe Vasconcelos; Hansen, Fernanda; Locks-Coelho, Lucas Doridio

    2013-08-01

    Glutamate concentration at the synaptic level must be kept low in order to prevent excitotoxicity. Astrocytes play a key role in brain energetics, and also astrocytic glutamate transporters are responsible for the vast majority of glutamate uptake in CNS. Experiments with primary astrocytic cultures suggest that increased influx of glutamate cotransported with sodium at astrocytes favors its flux to the tricarboxylic acid cycle instead of the glutamate-glutamine cycle. Although metabolic coupling can be considered an emergent field of research with important recent discoveries, some basic aspects of glutamate metabolism still have not been characterized in brain tissue. Therefore, the aim of this study was to investigate whether the presence of extracellular glutamate is able to modulate the use of glutamate and glucose as energetic substrates. For this purpose, isolated hippocampi of mice were incubated with radiolabeled substrates, and CO2 radioactivity and extracellular lactate were measured. Our results point to a diminished oxidation of glucose with increasing extracellular glutamate concentration, glutamate presumably being the fuel, and might suggest that oxidation of glutamate could buffer excitotoxic conditions by high glutamate concentrations. In addition, these findings were reversed when glutamate uptake by astrocytes was impaired by the presence of (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-L-aspartic acid (TFB-TBOA). Taken together, our findings argue against the lactate shuttle theory, because glutamate did not cause any detectable increase in extracellular lactate content (or, presumably, in glycolysis), because the glutamate is being used as fuel instead of going to glutamine and back to neurons. PMID:23359514

  8. Feedback-induced glutamate spillover enhances negative feedback from horizontal cells to cones

    PubMed Central

    Vroman, Rozan; Kamermans, Maarten

    2015-01-01

    Key points In the retina, horizontal cells feed back negatively to cone photoreceptors. Glutamate released from cones can spill over to neighbouring cones. Here we show that cone glutamate release induced by negative feedback can also spill over to neighbouring cones. This glutamate activates the glutamate transporter-associated chloride current in these neighbouring cones, which leads to a change in their membrane potential and thus modulates their output. In this way, feedback-induced glutamate spillover enhances negative feedback from horizontal cells to cones, thus forming an additional feedback pathway. This effect will be particularly prominent in cones that are strongly hyperpolarized by light. Abstract Inhibition in the outer retina functions via an unusual mechanism. When horizontal cells hyperpolarize the activation potential of the Ca2+ current of cones shifts to more negative potentials. The underlying mechanism consists of an ephaptic component and a Panx1/ATP-mediated component. Here we identified a third feedback component, which remains active outside the operating range of the Ca2+ current. We show that the glutamate transporters of cones can be activated by glutamate released from their neighbours. This pathway can be triggered by negative feedback from horizontal cells to cones, thus providing an additional feedback pathway. This additional pathway is mediated by a Cl− current, can be blocked by either removing the gradient of K+ or by adding the glutamate transporter blocker TBOA, or low concentrations of Zn2+. These features point to a glutamate transporter-associated Cl− current. The pathway has a delay of 4.7 ± 1.7 ms. The effectiveness of this pathway in modulating the cone output depends on the equilibrium potential of Cl− (ECl) and the membrane potential of the cone. Because estimates of ECl show that it is around the dark resting membrane potential of cones, the activation of the glutamate transporter-associated Cl− current

  9. Characterization of electrically evoked field potentials in the medial prefrontal cortex and orbitofrontal cortex of the rat: modulation by monoamines

    PubMed Central

    Wallace, Joanne; Jackson, Rosanna K; Shotton, Tanya L; Munjal, Ishaana; McQuade, Richard; Gartside, Sarah E

    2015-01-01

    Medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC) play critical roles in cognition and behavioural control. Glutamatergic, GABAergic, and monoaminergic dysfunction in the prefrontal cortex has been hypothesised to underlie symptoms in neuropsychiatric disorders. Here we characterised electrically-evoked field potentials in the mPFC and OFC. Electrical stimulation evoked field potentials in layer V/VI of the mPFC and layer V of the OFC. The earliest component (approximately 2 ms latency) was insensitive to glutamate receptor blockade and was presumed to be presynaptic. Later components were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX (20 μM) and were assumed to reflect monosynaptic (latency 4-6 ms) and polysynaptic activity (latency 6-40 ms) mediated by glutamate via AMPA/kainate receptor. In the mPFC, but not the OFC, the monosynaptic component was also partly blocked by 2-amino-5-phosphonopentanoic acid (AP-5 (50-100 μM) indicating the involvement of NMDA receptors. Bicuculline (3-10 μM) enhanced the monosynaptic component suggesting electrically-evoked and/or glutamate induced GABA release inhibits the monosynaptic component via GABAA receptor activation. There were complex effects of bicuculline on polysynaptic components. In the mPFC both the mono- and polysynaptic components were attenuated by 5-HT (10-100 μM) and NA (30 and 60 μM) and the monosynaptic component was attenuated by DA (100 μM). In the OFC the mono-and polysynaptic components were also attenuated by 5-HT (100 μM), NA (10-100 μM) but DA (10-100 μM) had no effect. We propose that these pharmacologically characterised electrically-evoked field potentials in the mPFC and OFC are useful models for the study of prefrontal cortical physiology and pathophysiology. PMID:23932190

  10. Presynaptic c-Jun N-terminal Kinase 2 regulates NMDA receptor-dependent glutamate release

    PubMed Central

    Nisticò, Robert; Florenzano, Fulvio; Mango, Dalila; Ferraina, Caterina; Grilli, Massimo; Di Prisco, Silvia; Nobili, Annalisa; Saccucci, Stefania; D'Amelio, Marcello; Morbin, Michela; Marchi, Mario; Mercuri, Nicola B.; Davis, Roger J.; Pittaluga, Anna; Feligioni, Marco

    2015-01-01

    Activation of c-Jun N-terminal kinase (JNK) signaling pathway is a critical step for neuronal death occurring in several neurological conditions. JNKs can be activated via receptor tyrosine kinases, cytokine receptors, G-protein coupled receptors and ligand-gated ion channels, including the NMDA glutamate receptors. While JNK has been generally associated with postsynaptic NMDA receptors, its presynaptic role remains largely unexplored. Here, by means of biochemical, morphological and functional approaches, we demonstrate that JNK and its scaffold protein JIP1 are also expressed at the presynaptic level and that the NMDA-evoked glutamate release is controlled by presynaptic JNK-JIP1 interaction. Moreover, using knockout mice for single JNK isoforms, we proved that JNK2 is the essential isoform in mediating this presynaptic event. Overall the present findings unveil a novel JNK2 localization and function, which is likely to play a role in different physiological and pathological conditions. PMID:25762148

  11. 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

  12. 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

  13. 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

  14. Peritumoural glutamate correlates with post-operative seizures in supratentorial gliomas.

    PubMed

    Neal, Andrew; Yuen, Tanya; Bjorksten, Andrew R; Kwan, Patrick; O'Brien, Terence J; Morokoff, Andrew

    2016-09-01

    To examine the impact of glutamate on post-operative seizures and survival in a cohort of patients with grade II to IV supratentorial glioma. A retrospective analysis was performed on 216 patients who underwent surgery for supratentorial gliomas. Primary explanatory variables were peritumoural and/or tumoural glutamate concentrations, glutamate transporter expression (EAAT2 and SXC). Univariate and multivariate survival analysis was performed with primary outcomes of time to first post-operative seizure and overall survival. Subgroup analysis was performed in patients with de novo glioblastomas who received adjuvant chemoradiotherapy. 47 (21.8 %), 34 (15.8 %) and 135 (62.5 %) WHO grade II, III and IV gliomas respectively were followed for a median of 15.8 months. Following multivariate analysis, there was a non-significant association between higher peritumoural glutamate concentrations and time to first post-operative seizure (HR 2.07, CI 0.98-4.37, p = 0.06). In subgroup analysis of 81 glioblastoma patients who received adjunct chemoradiotherapy, peritumoural glutamate concentration was significantly associated with time to first post-operative seizure (HR 3.10, CI 1.20-7.97, p = 0.02). In both the overall cohort and subgroup analysis no glutamate cycle biomarkers were predictive of overall survival. Increased concentrations of peritumoural glutamate were significantly associated with shorter periods of post-operative seizure freedom in patients with de novo glioblastomas treated with adjuvant chemoradiotherapy. No glutamate cycle biomarkers were predictive of overall survival. These results suggest that therapies targeting glutamate may be beneficial in tumour associated epilepsy. PMID:27311724

  15. 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

  16. 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

  17. External QX-314 inhibits evoked cranial primary afferent synaptic transmission independent of TRPV1.

    PubMed

    Hofmann, Mackenzie E; Largent-Milnes, Tally M; Fawley, Jessica A; Andresen, Michael C

    2014-12-01

    The cell-impermeant lidocaine derivative QX-314 blocks sodium channels via intracellular mechanisms. In somatosensory nociceptive neurons, open transient receptor potential vanilloid type 1 (TRPV1) receptors provide a transmembrane passageway for QX-314 to produce long-lasting analgesia. Many cranial primary afferents express TRPV1 at synapses on neurons in the nucleus of the solitary tract and caudal trigeminal nucleus (Vc). Here, we investigated whether QX-314 interrupts neurotransmission from primary afferents in rat brain-stem slices. Shocks to the solitary tract (ST) activated highly synchronous evoked excitatory postsynaptic currents (ST-EPSCs). Application of 300 μM QX-314 increased the ST-EPSC latency from TRPV1+ ST afferents, but, surprisingly, it had similar actions at TRPV1- ST afferents. Continued exposure to QX-314 blocked evoked ST-EPSCs at both afferent types. Neither the time to onset of latency changes nor the time to ST-EPSC failure differed between responses for TRPV1+ and TRPV1- inputs. Likewise, the TRPV1 antagonist capsazepine failed to prevent the actions of QX-314. Whereas QX-314 blocked ST-evoked release, the frequency and amplitude of spontaneous EPSCs remained unaltered. In neurons exposed to QX-314, intracellular current injection evoked action potentials suggesting a presynaptic site of action. QX-314 acted similarly at Vc neurons to increase latency and block EPSCs evoked from trigeminal tract afferents. Our results demonstrate that QX-314 blocked nerve conduction in cranial primary afferents without interrupting the glutamate release mechanism or generation of postsynaptic action potentials. The TRPV1 independence suggests that QX-314 either acted extracellularly or more likely entered these axons through an undetermined pathway common to all cranial primary afferents. PMID:25185814

  18. External QX-314 inhibits evoked cranial primary afferent synaptic transmission independent of TRPV1

    PubMed Central

    Largent-Milnes, Tally M.; Fawley, Jessica A.; Andresen, Michael C.

    2014-01-01

    The cell-impermeant lidocaine derivative QX-314 blocks sodium channels via intracellular mechanisms. In somatosensory nociceptive neurons, open transient receptor potential vanilloid type 1 (TRPV1) receptors provide a transmembrane passageway for QX-314 to produce long-lasting analgesia. Many cranial primary afferents express TRPV1 at synapses on neurons in the nucleus of the solitary tract and caudal trigeminal nucleus (Vc). Here, we investigated whether QX-314 interrupts neurotransmission from primary afferents in rat brain-stem slices. Shocks to the solitary tract (ST) activated highly synchronous evoked excitatory postsynaptic currents (ST-EPSCs). Application of 300 μM QX-314 increased the ST-EPSC latency from TRPV1+ ST afferents, but, surprisingly, it had similar actions at TRPV1− ST afferents. Continued exposure to QX-314 blocked evoked ST-EPSCs at both afferent types. Neither the time to onset of latency changes nor the time to ST-EPSC failure differed between responses for TRPV1+ and TRPV1− inputs. Likewise, the TRPV1 antagonist capsazepine failed to prevent the actions of QX-314. Whereas QX-314 blocked ST-evoked release, the frequency and amplitude of spontaneous EPSCs remained unaltered. In neurons exposed to QX-314, intracellular current injection evoked action potentials suggesting a presynaptic site of action. QX-314 acted similarly at Vc neurons to increase latency and block EPSCs evoked from trigeminal tract afferents. Our results demonstrate that QX-314 blocked nerve conduction in cranial primary afferents without interrupting the glutamate release mechanism or generation of postsynaptic action potentials. The TRPV1 independence suggests that QX-314 either acted extracellularly or more likely entered these axons through an undetermined pathway common to all cranial primary afferents. PMID:25185814

  19. Role of Na,K-ATPase α1 and α2 Isoforms in the Support of Astrocyte Glutamate Uptake

    PubMed Central

    Illarionava, Nina B.; Brismar, Hjalmar; Aperia, Anita; Gunnarson, Eli

    2014-01-01

    Glutamate released during neuronal activity is cleared from the synaptic space via the astrocytic glutamate/Na+ co-transporters. This transport is driven by the transmembrane Na+ gradient mediated by Na,K-ATPase. Astrocytes express two isoforms of the catalytic Na,K-ATPase α subunits; the ubiquitously expressed α1 subunit and the α2 subunit that has a more specific expression profile. In the brain α2 is predominantly expressed in astrocytes. The isoforms differ with regard to Na+ affinity, which is lower for α2. The relative roles of the α1 and α2 isoforms in astrocytes are not well understood. Here we present evidence that the presence of the α2 isoform may contribute to a more efficient restoration of glutamate triggered increases in intracellular sodium concentration [Na+]i. Studies were performed on primary astrocytes derived from E17 rat striatum expressing Na,K-ATPase α1 and α2 and the glutamate/Na+ co-transporter GLAST. Selective inhibition of α2 resulted in a modest increase of [Na+]i accompanied by a disproportionately large decrease in uptake of aspartate, an indicator of glutamate uptake. To compare the capacity of α1 and α2 to handle increases in [Na+]i triggered by glutamate, primary astrocytes overexpressing either α1 or α2 were used. Exposure to glutamate 200 µM caused a significantly larger increase in [Na+]i in α1 than in α2 overexpressing cells, and as a consequence restoration of [Na+]i, after glutamate exposure was discontinued, took longer time in α1 than in α2 overexpressing cells. Both α1 and α2 interacted with astrocyte glutamate/Na+ co-transporters via the 1st intracellular loop. PMID:24901986

  20. 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

  1. Glutamate Racemase Mutants of Bacillus anthracis

    PubMed Central

    Oh, So-Young; Richter, Stefan G.; Missiakas, Dominique M.

    2015-01-01

    ABSTRACT d-Glutamate is an essential component of bacterial peptidoglycan and a building block of the poly-γ-d-glutamic acid (PDGA) capsule of Bacillus anthracis, the causative agent of anthrax. Earlier work suggested that two glutamate racemases, encoded by racE1 and racE2, are each essential for growth of B. anthracis, supplying d-glutamic acid for the synthesis of peptidoglycan and PDGA capsule. Earlier work could not explain, however, why two enzymes that catalyze the same reaction may be needed for bacterial growth. Here, we report that deletion of racE1 or racE2 did not prevent growth of B. anthracis Sterne (pXO1+ pXO2−), the noncapsulating vaccine strain, or of B. anthracis Ames (pXO1+ pXO2+), a fully virulent, capsulating isolate. While mutants with deletions in racE1 and racE2 were not viable, racE2 deletion delayed vegetative growth of B. anthracis following spore germination and caused aberrant cell shapes, phenotypes that were partially restored by exogenous d-glutamate. Deletion of racE1 or racE2 from B. anthracis Ames did not affect the production or stereochemical composition of the PDGA capsule. A model is presented whereby B. anthracis, similar to Bacillus subtilis, utilizes two functionally redundant racemase enzymes to synthesize d-glutamic acid for peptidoglycan synthesis. IMPORTANCE Glutamate racemases, enzymes that convert l-glutamate to d-glutamate, are targeted for antibiotic development. Glutamate racemase inhibitors may be useful for the treatment of bacterial infections such as anthrax, where the causative agent, B. anthracis, requires d-glutamate for the synthesis of peptidoglycan and poly-γ-d-glutamic acid (PDGA) capsule. Here we show that B. anthracis possesses two glutamate racemase genes that can be deleted without abolishing either bacterial growth or PDGA synthesis. These data indicate that drug candidates must inhibit both glutamate racemases, RacE1 and RacE2, in order to block B. anthracis growth and achieve therapeutic

  2. 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

  3. Wireless Instantaneous Neurotransmitter Concentration System–based amperometric detection of dopamine, adenosine, and glutamate for intraoperative neurochemical monitoring

    PubMed Central

    Agnesi, Filippo; Tye, Susannah J.; Bledsoe, Jonathan M.; Griessenauer, Christoph J.; Kimble, Christopher J.; Sieck, Gary C.; Bennet, Kevin E.; Garris, Paul A.; Blaha, Charles D.; Lee, Kendall H.

    2009-01-01

    Object In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time. Methods The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme-linked electrode to measure glutamate; and 3) a multiple enzyme-linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal

  4. Glutamate uptake block triggers deadly rhythmic bursting of neonatal rat hypoglossal motoneurons

    PubMed Central

    Sharifullina, Elina; Nistri, Andrea

    2006-01-01

    In the brain the extracellular concentration of glutamate is controlled by glial transporters that restrict the neurotransmitter action to synaptic sites and avoid excitotoxicity. Impaired transport of glutamate occurs in many cases of amyotrophic lateral sclerosis, a devastating motoneuron disease. Motoneurons of the brainstem nucleus hypoglossus are among the most vulnerable, giving early symptoms like slurred speech and dysphagia. However, the direct consequences of extracellular glutamate build-up, due to uptake block, on synaptic transmission and survival of hypoglossal motoneurons remain unclear and have been studied using the neonatal rat brainstem slice preparation as a model. Patch clamp recording from hypoglossal motoneurons showed that, in about one-third of these cells, inhibition of glutamate transport with the selective blocker dl-threo-β-benzyloxyaspartate (TBOA; 50 μ m) unexpectedly led to the emergence of rhythmic bursting consisting of inward currents of long duration with superimposed fast oscillations and synaptic events. Synaptic inhibition block facilitated bursting. Bursts had a reversal potential near 0 mV, and were blocked by tetrodotoxin, the gap junction blocker carbenoxolone, or antagonists of AMPA, NMDA or mGluR1 glutamate receptors. Intracellular Ca2+ imaging showed bursts as synchronous discharges among motoneurons. Synergy of activation of distinct classes of glutamate receptor plus gap junctions were therefore essential for bursting. Ablating the lateral reticular formation preserved bursting, suggesting independence from propagated network activity within the brainstem. TBOA significantly increased the number of dead motoneurons, an effect prevented by the same agents that suppressed bursting. Bursting thus represents a novel hallmark of motoneuron dysfunction triggered by glutamate uptake block. PMID:16455692

  5. 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

  6. 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

  7. 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

  8. The effect of N-acetyl-aspartyl-glutamate and N-acetyl-aspartate on white matter oligodendrocytes.

    PubMed

    Kolodziejczyk, Karolina; Hamilton, Nicola B; Wade, Anna; Káradóttir, Ragnhildur; Attwell, David

    2009-06-01

    Elevations of the levels of N-acetyl-aspartyl-glutamate (NAAG) and N-acetyl-aspartate (NAA) are associated with myelin loss in the leucodystrophies Canavan's disease and Pelizaeus-Merzbacher-like disease. NAAG and NAA can activate and antagonize neuronal N-methyl-D-aspartate (NMDA) receptors, and also act on group II metabotropic glutamate receptors. Oligodendrocytes and their precursors have recently been shown to express NMDA receptors, and activation of these receptors in ischaemia leads to the death of oligodendrocyte precursors and the loss of myelin. This raises the possibility that the failure to develop myelin, or demyelination, occurring in the leucodystrophies could reflect an action of NAAG or NAA on oligodendrocyte NMDA receptors. However, since the putative subunit composition of NMDA receptors on oligodendrocytes differs from that of neuronal NMDA receptors, the effects of NAAG and NAA on them are unknown. We show that NAAG, but not NAA, evokes an inward membrane current in cerebellar white matter oligodendrocytes, which is reduced by NMDA receptor block (but not by block of metabotropic glutamate receptors). The size of the current evoked by NAAG, relative to that evoked by NMDA, was much smaller in oligodendrocytes than in neurons, and NAAG induced a rise in [Ca(2+)](i) in neurons but not in oligodendrocytes. These differences in the effect of NAAG on oligodendrocytes and neurons may reflect the aforementioned difference in receptor subunit composition. In addition, as a major part of the response in oligodendrocytes was blocked by tetrodotoxin (TTX), much of the NAAG-evoked current in oligodendrocytes is a secondary consequence of activating neuronal NMDA receptors. Six hours exposure to 1 mM NAAG did not lead to the death of cells in the white matter. We conclude that an action of NAAG on oligodendrocyte NMDA receptors is unlikely to be a major contributor to white matter damage in the leucodystrophies. PMID:19383832

  9. 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

  10. Glutamate-gated Chloride Channels*

    PubMed Central

    Wolstenholme, Adrian J.

    2012-01-01

    Glutamate-gated chloride channels (GluCls) are found only in protostome invertebrate phyla but are closely related to mammalian glycine receptors. They have a number of roles in these animals, controlling locomotion and feeding and mediating sensory inputs into behavior. In nematodes and arthropods, they are targeted by the macrocyclic lactone family of anthelmintics and pesticides, making the GluCls of considerable medical and economic importance. Recently, the three-dimensional structure of a GluCl was solved, the first for any eukaryotic ligand-gated anion channel, revealing a macrocyclic lactone-binding site between the channel domains of adjacent subunits. This minireview will highlight some unique features of the GluCls and illustrate their contribution to our knowledge of the entire Cys loop ligand-gated ion channel superfamily. PMID:23038250

  11. L-glutamic acid: a neurotransmitter candidate for cone photoreceptors in human and rat retinas.

    PubMed Central

    Brandon, C; Lam, D M

    1983-01-01

    We have combined immunocytochemical localization of L-aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1; glutamic-oxaloacetic transaminase) with autoradiographic localization of high-affinity uptake sites for L-glutamate or L-aspartate to identify the neurotransmitters of mammalian photoreceptors. In both human and rat retinas, high aspartate aminotransferase immunoreactivity is found in cones but not in rods; certain putative bipolar and amacrine cells are also heavily stained. In the human retina, and perhaps also in the rat retina, cones possess a high-affinity uptake mechanism for L-glutamate but not L-aspartate, whereas rods and Müller (glial) cells take up both L-glutamate and L-aspartate. Taken together, our results indicate that (i) L-glutamate is much more likely than L-aspartate to be the transmitter for human cones, and possibly for cones of other mammalian species as well, and (ii) major differences exist between mammalian cones and rods in the transport and metabolism or utilization of L-aspartate and L-glutamate. Images PMID:6136039

  12. Cardiovascular afferents cause the release of 5-HT in the nucleus tractus solitarii; this release is regulated by the low- (PMAT) not the high-affinity transporter (SERT).

    PubMed

    Hosford, Patrick S; Millar, Julian; Ramage, Andrew G

    2015-04-01

    The nucleus tractus solitarii (NTS) integrates inputs from cardiovascular afferents and thus is crucial for cardiovascular homeostasis. These afferents primarily release glutamate, although 5-HT has also been shown to play a role in their actions. Using fast-cyclic voltammetry, an increase in 5-HT concentrations (range 12-50 nm) could be detected in the NTS in anaesthetized rats in response to electrical stimulation of the vagus and activation of cardiopulmonary, chemo- and baroreceptor reflexes. This 5-HT signal was not potentiated by the serotonin transporter (SERT) or the noradrenaline transporter (NET) inhibitors citalopram and desipramine (1 mg kg(-1) ). However, decynium-22 (600 μg kg(-1) ), an organic cation 3 transporter (OCT3)/plasma membrane monoamine transporter (PMAT) inhibitor, increased the 5-HT signal by 111 ± 21% from 29 ± 10 nm. The effectiveness of these inhibitors was tested against the removal time of 5-HT and noradrenaline applied by microinjection to the NTS. Citalopram and decynium-22 attenuated the removal of 5-HT but not noradrenaline, whereas desipramine had the reverse action. The OCT3 inhibitor corticosterone (10 mg kg(-1) ) had no effect. Blockade of glutamate receptors with topical kynurenate (10-50 nm) reduced the vagally evoked 5-HT signal by 50%, indicating that this release was from at least two sources. It is concluded that vagally evoked 5-HT release is under the regulation of the high-capacity, low-affinity transporter PMAT, not the low-capacity, high-affinity transporter SERT. This is the first demonstration that PMAT may be playing a physiological role in the regulation of 5-HT transmission and this could indicate that 5-HT is acting, in part, as a volume transmitter within the NTS. PMID:25694117

  13. 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

  14. Local synaptic release of glutamate from neurons in the rat hypothalamic arcuate nucleus.

    PubMed Central

    Belousov, A B; van den Pol, A N

    1997-01-01

    1. The hypothalamic arcuate nucleus (ARC) contains neuroendocrine neurons that regulate endocrine secretions by releasing substances which control anterior pituitary hormonal release into the portal blood stream. Many neuroactive substances have been identified in the ARC, but the existence of excitatory neurons in the ARC and the identity of an excitatory transmitter have not been investigated physiologically. 2. In the present experiments using whole-cell current- and voltage-clamp recording of neurons from cultures and slices of the ARC, we demonstrate for the first time that some of the neurons in the ARC secrete glutamate as their transmitter. 3. Using microdrop stimulation of presynaptic neurons in ARC slices, we found that local axons from these glutamatergic neurons make local synaptic contact with other neurons in the ARC and that all evoked excitatory postsynaptic potentials could be blocked by the selective ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and D,L-2-amino-5-phosphonovalerate (AP5; 100 microM). To determine the identity of ARC neurons postsynaptic to local glutamatergic neurons, we used antidromic stimulation to reveal that many of these cells were neuroendocrine neurons by virtue of their maintaining axon terminals in the median eminence. 4. In ARC cultures, postsynaptic potentials, both excitatory and inhibitory, were virtually eliminated by the glutamate receptor antagonists AP5 and CNQX, underlining the functional importance of glutamate within this part of the neuroendocrine brain. 5. GABA was secreted by a subset of ARC neurons from local axons. The GABAA receptor antagonist bicuculline released glutamatergic neurons from chronic inhibition mediated by synaptically released GABA, resulting in further depolarization and an increase in the amplitude and frequency of glutamate-mediated excitatory postsynaptic potentials. Images Figure 1 PMID:9130170

  15. 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

  16. Different pools of glutamate receptors mediate sensitivity to ambient glutamate in the cochlear nucleus

    PubMed Central

    Yang, Yang

    2015-01-01

    Ambient glutamate plays an important role in pathological conditions, such as stroke, but its role during normal activity is not clear. In addition, it is not clear how ambient glutamate acts on glutamate receptors with varying affinities or subcellular localizations. To address this, we studied “endbulb of Held” synapses, which are formed by auditory nerve fibers onto bushy cells (BCs) in the anteroventral cochlear nucleus. When ambient glutamate was increased by applying the glutamate reuptake inhibitor TFB-TBOA, BCs depolarized as a result of activation of N-methyl-d-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs). Application of antagonists against NMDARs (in 0 Mg2+) or mGluRs caused hyperpolarization, indicating that these receptors were bound by a tonic source of glutamate. AMPA receptors did not show these effects, consistent with their lower glutamate affinity. We also evaluated the subcellular localization of the receptors activated by ambient glutamate. The mGluRs were not activated by synaptic stimulation and thus appear to be exclusively extrasynaptic. By contrast, NMDARs in both synaptic and extrasynaptic compartments were activated by ambient glutamate, as shown using the use-dependent antagonist MK-801. Levels of ambient glutamate appeared to be regulated in a spike-independent manner, and glia likely play a major role. These low levels of ambient glutamate likely have functional consequences, as even low concentrations of TBOA caused significant increases in BC spiking following synaptic stimulation. These results indicate that normal resting potential appears to be poised in the region of maximal sensitivity to small changes in ambient glutamate. PMID:25855696

  17. Different pools of glutamate receptors mediate sensitivity to ambient glutamate in the cochlear nucleus.

    PubMed

    Yang, Yang; Xu-Friedman, Matthew A

    2015-06-01

    Ambient glutamate plays an important role in pathological conditions, such as stroke, but its role during normal activity is not clear. In addition, it is not clear how ambient glutamate acts on glutamate receptors with varying affinities or subcellular localizations. To address this, we studied "endbulb of Held" synapses, which are formed by auditory nerve fibers onto bushy cells (BCs) in the anteroventral cochlear nucleus. When ambient glutamate was increased by applying the glutamate reuptake inhibitor TFB-TBOA, BCs depolarized as a result of activation of N-methyl-D-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs). Application of antagonists against NMDARs (in 0 Mg(2+)) or mGluRs caused hyperpolarization, indicating that these receptors were bound by a tonic source of glutamate. AMPA receptors did not show these effects, consistent with their lower glutamate affinity. We also evaluated the subcellular localization of the receptors activated by ambient glutamate. The mGluRs were not activated by synaptic stimulation and thus appear to be exclusively extrasynaptic. By contrast, NMDARs in both synaptic and extrasynaptic compartments were activated by ambient glutamate, as shown using the use-dependent antagonist MK-801. Levels of ambient glutamate appeared to be regulated in a spike-independent manner, and glia likely play a major role. These low levels of ambient glutamate likely have functional consequences, as even low concentrations of TBOA caused significant increases in BC spiking following synaptic stimulation. These results indicate that normal resting potential appears to be poised in the region of maximal sensitivity to small changes in ambient glutamate. PMID:25855696

  18. Metabotropic glutamate receptors promote disinhibition of olfactory bulb glomeruli that scales with input strength

    PubMed Central

    Zak, Joseph D.; Whitesell, Jennifer D.

    2014-01-01

    Increasing evidence indicates that the neural circuitry within glomeruli of the olfactory bulb plays a major role in affecting information flow between olfactory sensory neurons (OSNs) and output mitral cells (MCs). Glutamatergic external tufted (ET) cells, located at glomeruli, can act as intermediary cells in excitation between OSNs and MCs, whereas activation of MCs by OSNs is, in turn, suppressed by inhibitory synapses onto ET cells. In this study, we used patch-clamp recordings in rat olfactory bulb slices to examine the function of metabotropic glutamate receptors (mGluRs) in altering these glomerular signaling mechanisms. We found that activation of group II mGluRs profoundly reduced inhibition onto ET cells evoked by OSN stimulation. The mGluRs that mediated disinhibition were located on presynaptic GABAergic periglomerular cells and appeared to be activated by glutamate transients derived from dendrites in glomeruli. In terms of glomerular output, the mGluR-mediated reduction in GABA release led to a robust increase in the number of action potentials evoked by OSN stimulation in both ET cells and MCs. Importantly, however, the enhanced excitation was specific to when a glomerulus was strongly activated by OSN inputs. By being selective for strong vs. weak glomerular activation, mGluR-mediated disinhibition provides a mechanism to enhance the contrast in odor signals that activate OSN inputs into a single glomerulus at varying intensities. PMID:25552635

  19. Potentiation of acid-sensing ion channel activity by peripheral group I metabotropic glutamate receptor signaling.

    PubMed

    Gan, Xiong; Wu, Jing; Ren, Cuixia; Qiu, Chun-Yu; Li, Yan-Kun; Hu, Wang-Ping

    2016-05-01

    Glutamate activates peripheral group I metabotropic glutamate receptors (mGluRs) and contributes to inflammatory pain. However, it is still not clear the mechanisms are involved in group I mGluR-mediated peripheral sensitization. Herein, we report that group I mGluRs signaling sensitizes acid-sensing ion channels (ASICs) in dorsal root ganglion (DRG) neurons and contributes to acidosis-evoked pain. DHPG, a selective group I mGluR agonist, can potentiate the functional activity of ASICs, which mediated the proton-induced events. DHPG concentration-dependently increased proton-gated currents in DRG neurons. It shifted the proton concentration-response curve upwards, with a 47.3±7.0% increase of the maximal current response to proton. Group I mGluRs, especially mGluR5, mediated the potentiation of DHPG via an intracellular cascade. DHPG potentiation of proton-gated currents disappeared after inhibition of intracellular Gq/11 proteins, PLCβ, PKC or PICK1 signaling. Moreover, DHPG enhanced proton-evoked membrane excitability of rat DRG neurons and increased the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, peripherally administration of DHPG dose-dependently exacerbated nociceptive responses to intraplantar injection of acetic acid in rats. Potentiation of ASIC activity by group I mGluR signaling in rat DRG neurons revealed a novel peripheral mechanism underlying group I mGluRs involvement in hyperalgesia. PMID:26946972

  20. Pre- and Postnatal Exposure to Moderate Levels of Ethanol Can Have Long-Lasting Effects on Hippocampal Glutamate Uptake in Adolescent Offspring

    PubMed Central

    de Souza, Daniela F.; Lopes, Fernanda M.; Leite, Marina C.; Gonçalves, Carlos-Alberto

    2015-01-01

    The developing brain is vulnerable to the effects of ethanol. Glutamate is the main mediator of excitatory signals in the brain and is probably involved in most aspects of normal brain function during development. The aim of this study was to investigate vulnerability to and the impact of ethanol toxicity on glutamate uptake signaling in adolescent rats after moderate pre and postnatal ethanol exposure. Pregnant female rats were divided into three groups and treated only with water (control), non-alcoholic beer (vehicle) or 10% (v/v) beer solution (moderate prenatal alcohol exposure—MPAE). Thirty days after birth, adolescent male offspring were submitted to hippocampal acute slice procedure. We assayed glutamate uptake and measured glutathione content and also quantified glial glutamate transporters (EAAT 1 and EAAT 2). The glutamate system vulnerability was tested with different acute ethanol doses in naïve rats and compared with the MPAE group. We also performed a (lipopolysaccharide-challenge (LPS-challenge) with all groups to test the glutamate uptake response after an insult. The MPAE group presented a decrease in glutamate uptake corroborating a decrease in glutathione (GSH) content. The reduction in GSH content suggests oxidative damage after acute ethanol exposure. The glial glutamate transporters were also altered after prenatal ethanol treatment, suggesting a disturbance in glutamate signaling. This study indicates that impairment of glutamate uptake can be dose-dependent and the glutamate system has a higher vulnerability to ethanol toxicity after moderate ethanol exposure In utero. The effects of pre- and postnatal ethanol exposure can have long-lasting impacts on the glutamate system in adolescence and potentially into adulthood. PMID:25978644

  1. 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

  2. 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

  3. Metabolic fate and function of dietary glutamate in the gut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutamate is a major constituent of dietary protein and is also consumed in many prepared foods as an additive in the form of monosodium glutamate. Evidence from human and animal studies indicates that glutamate is a major oxidative fuel for the gut and that dietary glutamate is extensively metabol...

  4. 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...

  5. 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…

  6. 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

  7. Glutamate Receptor Dynamics in Dendritic Microdomains

    PubMed Central

    Newpher, Thomas M.; Ehlers, Michael D.

    2008-01-01

    Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity. PMID:18498731

  8. 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

  9. 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.

  10. 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

  11. 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

  12. [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

  13. 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

  14. 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

  15. Therapeutic Potential of Metabotropic Glutamate Receptor Modulators

    PubMed Central

    Hovelsø, N; Sotty, F; Montezinho, L.P; Pinheiro, P.S; Herrik, K.F; Mørk, A

    2012-01-01

    Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS) and is a major player in complex brain functions. Glutamatergic transmission is primarily mediated by ionotropic glutamate receptors, which include NMDA, AMPA and kainate receptors. However, glutamate exerts modulatory actions through a family of metabotropic G-protein-coupled glutamate receptors (mGluRs). Dysfunctions of glutamatergic neurotransmission have been implicated in the etiology of several diseases. Therefore, pharmacological modulation of ionotropic glutamate receptors has been widely investigated as a potential therapeutic strategy for the treatment of several disorders associated with glutamatergic dysfunction. However, blockade of ionotropic glutamate receptors might be accompanied by severe side effects due to their vital role in many important physiological functions. A different strategy aimed at pharmacologically interfering with mGluR function has recently gained interest. Many subtype selective agonists and antagonists have been identified and widely used in preclinical studies as an attempt to elucidate the role of specific mGluRs subtypes in glutamatergic transmission. These studies have allowed linkage between specific subtypes and various physiological functions and more importantly to pathological states. This article reviews the currently available knowledge regarding the therapeutic potential of targeting mGluRs in the treatment of several CNS disorders, including schizophrenia, addiction, major depressive disorder and anxiety, Fragile X Syndrome, Parkinson’s disease, Alzheimer’s disease and pain. PMID:22942876

  16. SYNAPTIC VESICLE PROTEIN TRAFFICKING AT THE GLUTAMATE SYNAPSE

    PubMed Central

    Santos, Magda S.; Li, Haiyan; Voglmaier, Susan M.

    2009-01-01

    Expression of the integral and associated proteins of synaptic vesicles is subject to regulation over time, by region, and in response to activity. The process by which changes in protein levels and isoforms result in different properties of neurotransmitter release involves protein trafficking to the synaptic vesicle. How newly synthesized proteins are incorporated into synaptic vesicles at the presynaptic bouton is poorly understood. During synaptogenesis, synaptic vesicle proteins sort through the secretory pathway and are transported down the axon in precursor vesicles that undergo maturation to form synaptic vesicles. Changes in protein content of synaptic vesicles could involve the formation of new vesicles that either mix with the previous complement of vesicles or replace them, presumably by their degradation or inactivation. Alternatively, new proteins could individually incorporate into existing synaptic vesicles, changing their functional properties. Glutamatergic vesicles likely express many of the same integral membrane proteins and share certain common mechanisms of biogenesis, recycling, and degradation with other synaptic vesicles. However, glutamatergic vesicles are defined by their ability to package glutamate for release, a property conferred by the expression of a vesicular glutamate transporter (VGLUT). VGLUTs are subject to regional, developmental, and activity-dependent changes in expression. In addition, VGLUT isoforms differ in their trafficking, which may target them to different pathways during biogenesis or after recycling, which may in turn sort them to different vesicle pools. Emerging data indicate that differences in the association of VGLUTs and other synaptic vesicle proteins with endocytic adaptors may influence their trafficking. These observations indicate that independent regulation of synaptic vesicle protein trafficking has the potential to influence synaptic vesicle protein composition, the maintenance of synaptic vesicle

  17. AGC1/2, the mitochondrial aspartate-glutamate carriers.

    PubMed

    Amoedo, N D; Punzi, G; Obre, E; Lacombe, D; De Grassi, A; Pierri, C L; Rossignol, R

    2016-10-01

    In this review we discuss the structure and functions of the aspartate/glutamate carriers (AGC1-aralar and AGC2-citrin). Those proteins supply the aspartate synthesized within mitochondrial matrix to the cytosol in exchange for glutamate and a proton. A structure of an AGC carrier is not available yet but comparative 3D models were proposed. Moreover, transport assays performed by using the recombinant AGC1 and AGC2, reconstituted into liposome vesicles, allowed to explore the kinetics of those carriers and to reveal their specific transport properties. AGCs participate to a wide range of cellular functions, as the control of mitochondrial respiration, calcium signaling and antioxydant defenses. AGC1 might also play peculiar tissue-specific functions, as it was found to participate to cell-to-cell metabolic symbiosis in the retina. On the other hand, AGC1 is involved in the glutamate-mediated excitotoxicity in neurons and AGC gene or protein alterations were discovered in rare human diseases. Accordingly, a mice model of AGC1 gene knock-out presented with growth delay and generalized tremor, with myelinisation defects. More recently, AGC was proposed to play a crucial role in tumor metabolism as observed from metabolomic studies showing that the asparate exported from the mitochondrion by AGC1 is employed in the regeneration of cytosolic glutathione. Therefore, given the central role of AGCs in cell metabolism and human pathology, drug screening are now being developed to identify pharmacological modulators of those carriers. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou. PMID:27132995

  18. MDMA increases glutamate release and reduces parvalbumin-positive GABAergic cells in the dorsal hippocampus of the rat: role of cyclooxygenase

    PubMed Central

    Anneken, John H.; Cunningham, Jacobi I.; Collins, Stuart A.; Yamamoto, Bryan K.; Gudelsky, Gary A.

    2012-01-01

    3,4-Methylenedioxymethamphetamine (MDMA; Ecstasy) is a popular drug of abuse with well-documented acute effects on serotonergic, dopaminergic, and cholinergic transmitter systems, as well as evidence of long-term disruption of serotoninergic systems in the rat brain. Recently, it was demonstrated that MDMA evokes a delayed and sustained increase in glutamate release in the hippocampus. The purpose of the present study was to determine the role of inflammatory mediators in the MDMA-induced increase in glutamate release, as well as the contribution of inflammatory pathways in the persistent neurochemical toxicity associated with repeated MDMA treatment. Treatment with the non-selective cyclooxygenase (COX) inhibitor ketoprofen and the COX-2 selective inhibitor nimesulide attenuated the increase in extracellular glutamate in the hippocampus evoked by repeated MDMA exposure (10 mg/kg, i.p., every 2 h); no attenuation was observed in rats treated with the COX-1 selective inhibitor piroxicam. Reverse dialysis of a major product of COX activity, prostaglandin E2, also resulted in a significant increase in extracellular glutamate in the hippocampus. Repeated exposure to MDMA diminished the number of parvalbumin-positive GABA interneurons in the dentate gyrus of the hippocampus, an effect that was attenuated by ketoprofen treatment. However, COX inhibition with ketoprofen did not prevent the long-term depletion of 5-HT in the hippocampus evoked by MDMA treatment. These data are supportive of the view that cyclooxygenase activity contributes to the mechanism underlying both the increased release of glutamate and decreased number of GABA interneurons in the rat hippocampus produced by repeated MDMA exposure. PMID:23179355

  19. Auditory and visual evoked potentials during hyperoxia

    NASA Technical Reports Server (NTRS)

    Smith, D. B. D.; Strawbridge, P. J.

    1974-01-01

    Experimental study of the auditory and visual averaged evoked potentials (AEPs) recorded during hyperoxia, and investigation of the effect of hyperoxia on the so-called contingent negative variation (CNV). No effect of hyperoxia was found on the auditory AEP, the visual AEP, or the CNV. Comparisons with previous studies are discussed.

  20. SENSORY EVOKED POTENTIALS: MEASURES OF NEUROTOXICITY

    EPA Science Inventory

    There is a need for tests of sensory function to be incorporated in laboratory and toxicity testing. t is clear that sensory dysfunction may frequently occur, but go undetected, in standard animal toxicological testing protocols. ensory evoked potential technology can be employed...

  1. 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

  2. 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 ß-galactosidase and wheat germ agglutinin from a vesicular glutamate transporter-1 promoter

    PubMed Central

    Zhang, Guo-rong; Cao, Haiyan; Li, Xu; Zhao, Hua; Geller, Alfred I.

    2010-01-01

    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 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 ß-galactosidase (ß-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 ß-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. PMID:20849834

  3. The Degradation of 14C-Glutamic Acid by L-Glutamic Acid Decarboxylase.

    ERIC Educational Resources Information Center

    Dougherty, Charles M; Dayan, Jean

    1982-01-01

    Describes procedures and semi-micro reaction apparatus (carbon dioxide trap) to demonstrate how a particular enzyme (L-Glutamic acid decarboxylase) may be used to determine the site or sites of labeling in its substrate (carbon-14 labeled glutamic acid). Includes calculations, solutions, and reagents used. (Author/SK)

  4. Photoreduction of α-Ketoglutarate to Glutamate by Vicia faba Chloroplasts 1

    PubMed Central

    Givan, Curtis V.; Givan, Alice L.; Leech, Rachel M.

    1970-01-01

    Intact chloroplasts isolated from leaves of Vicia faba L. var. the Sutton show a decline in the endogenous level of α-ketoglutarate upon illumination. α-Ketoglutarate supplied to the chloroplasts is similarly utilized in this light-dependent reaction, and its consumption is paralleled by a concomitant increase in the level of glutamate. There is no photostimulation of glutamate synthesis in chloroplasts broken by osmotic shock, but it can be somewhat restored by addition of ferredoxin and NADP. These results suggest that in the isolated chloroplast the synthesis of glutamate from α-ketoglutarate is regulated by the availability of reduced pyridine nucleotide generated by photosynthetic electron transport. This conclusion is supported by the finding of an apparent competition between the photoreduction of phosphoglycerate to triose phosphate and the photoutilization of α-ketoglutarate. PMID:16657357

  5. An essential role of acetylcholine-glutamate synergy at habenular synapses in nicotine dependence

    PubMed Central

    Frahm, Silke; Antolin-Fontes, Beatriz; Görlich, Andreas; Zander, Johannes-Friedrich; Ahnert-Hilger, Gudrun; Ibañez-Tallon, Ines

    2015-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.11396.001 PMID:26623516

  6. Glutamine synthetase activity and glutamate uptake in hippocampus and frontal cortex in portal hypertensive rats

    PubMed Central

    Acosta, Gabriela Beatriz; Fernández, María Alejandra; Roselló, Diego Martín; Tomaro, María Luján; Balestrasse, Karina; Lemberg, Abraham

    2009-01-01

    AIM: To study glutamine synthetase (GS) activity and glutamate uptake in the hippocampus and frontal cortex (FC) from rats with prehepatic portal vein hypertension. METHODS: Male Wistar rats were divided into sham-operated group and a portal hypertension (PH) group with a regulated stricture of the portal vein. Animals were sacrificed by decapitation 14 d after portal vein stricture. GS activity was determined in the hippocampus and FC. Specific uptake of radiolabeled L-glutamate was studied using synaptosome-enriched fractions that were freshly prepared from both brain areas. RESULTS: We observed that the activity of GS increased in the hippocampus of PH rats, as compared to control animals, and decreased in the FC. A significant decrease in glutamate uptake was found in both brain areas, and was more marked in the hippocampus. The decrease in glutamate uptake might have been caused by a deficient transport function, significantly and persistent increase in this excitatory neurotransmitter activity. CONCLUSION: The presence of moderate ammonia blood levels may add to the toxicity of excitotoxic glutamate in the brain, which causes alterations in brain function. Portal vein stricture that causes portal hypertension modifies the normal function in some brain regions. PMID:19533812

  7. A patch clamp study of the effects of ciprofloxacin and biphenyl acetic acid on rat hippocampal neurone GABAA and ionotropic glutamate receptors.

    PubMed

    Halliwell, R F; Davey, P G; Lambert, J J

    1995-12-01

    The neurotoxic effects of 4-quinolones alone and in combination with certain non-steroidal anti-inflammatory drugs (NSAIDs) may be related to an interaction at GABAA and/or ionotropic glutamate receptors. In the present study, the effects of the fluoroquinolone, ciprofloxacin, alone and in combination with the NSAID, biphenyl acetic acid (BPAA), were examined on GABAA-, NMDA-, AMPA-, and kainate-evoked current responses recorded from cultured rat hippocampal neurones, using the whole cell patch clamp technique. GABA-evoked currents were reversibly inhibited by bicuculline (3 microM) and ciprofloxacin (100 microM) to 11 +/- 5 and 38 +/- 7% of control, respectively. BPAA (100 microM) had little affect on the GABA current (the response was 82 +/- 4% of control) but enhanced the inhibitory potency of ciprofloxacin by approx. 3000-fold. The antagonist effects of ciprofloxacin (30 microM) and ciprofloxacin (0.03 microM) together with BPAA (100 microM) on the GABA-evoked current were not voltage-dependent. Whole cell currents evoked by NMDA, AMPA or kainate were little influenced by ciprofloxacin (100 microM), BPAA (100 microM), or ciprofloxacin plus BPAA (both at 100 microM); the responses being > or = 90% of control in all cases. These data suggest that the proconvulsant effects of quinolones when combined with BPAA may be related to antagonism of central GABAA receptors but not to an interaction at ionotropic glutamate receptors. PMID:8788959

  8. The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells

    PubMed Central

    Menga, Alessio; Iacobazzi, Vito; Infantino, Vittoria; Avantaggiati, Maria Laura; Palmieri, Ferdinando

    2015-01-01

    The aspartate/glutamate carrier isoform 1 is an essential mitochondrial transporter that exchanges intramitochondrial aspartate and cytosolic glutamate across the inner mitochondrial membrane. It is expressed in brain, heart and muscle and is involved in important biological processes, including myelination. However, the signals that regulate the expression of this transporter are still largely unknown. In this study we first identify a CREB binding site within the aspartate/glutamate carrier gene promoter that acts as a strong enhancer element in neuronal SH-SY5Y cells. This element is regulated by active, phosphorylated CREB protein and by signal pathways that modify the activity of CREB itself and, most noticeably, by intracellular Ca2+ levels. Specifically, aspartate/glutamate carrier gene expression is induced via CREB by forskolin while it is inhibited by the PKA inhibitor, H89. Furthermore, the CREB-induced activation of gene expression is increased by thapsigargin, which enhances cytosolic Ca2+, while it is inhibited by BAPTA-AM that reduces cytosolic Ca2+ or by STO-609, which inhibits CaMK-IV phosphorylation. We further show that CREB-dependent regulation of aspartate/glutamate carrier gene expression occurs in neuronal cells in response to pathological (inflammation) and physiological (differentiation) conditions. Since this carrier is necessary for neuronal functions and is involved in myelinogenesis, our results highlight that targeting of CREB activity and Ca2+ might be therapeutically exploited to increase aspartate/glutamate carrier gene expression in neurodegenerative diseases. PMID:25597433

  9. Gating characteristics control glutamate receptor distribution and trafficking in vivo.

    PubMed

    Petzoldt, Astrid G; Lee, Yü-Hien; Khorramshahi, Omid; Reynolds, Eric; Plested, Andrew J R; Herzel, Hanspeter; Sigrist, Stephan J

    2014-09-01

    Glutamate-releasing synapses dominate excitatory release in the brain. Mechanisms governing their assembly are of major importance for circuit development and long-term plasticity underlying learning and memory. AMPA/Kainate-type glutamate receptors (GluRs) are tetrameric ligand-gated ion channels that open their ion-conducting pores in response to binding of the neurotransmitter. Changes in subunit composition of postsynaptic GluRs are highly relevant for plasticity and development of glutamatergic synapses [1-4]. To date, posttranslational modifications, mostly operating via the intracellular C-terminal domains (CTDs) of GluRs, are presumed to be the major regulator of trafficking [5]. In recent years, structural and electrophysiological analyses have improved our understanding of GluR gating mechanism [6-11]. However, whether conformational changes subsequent to glutamate binding may per se be able to influence GluR trafficking has remained an unaddressed question. Using a Drosophila system allowing for extended visualization of GluR trafficking in vivo, we here provide evidence that mutations changing the gating behavior alter GluR distribution and trafficking. GluR mutants associated with reduced charge transfer segregated from coexpressed wild-type GluRs on the level of individual postsynaptic densities. Segregation was lost upon blocking of evoked glutamate release. Photobleaching experiments suggested increased mobility of mutants with reduced charge transfer, which accumulated prematurely during early steps of synapse assembly, but failed to further increase their level in accordance with assembly of the presynaptic scaffold. In summary, gating characteristics seem to be a new variable for the understanding of GluR trafficking relevant to both development and plasticity. PMID:25131677

  10. Blockade of spinal glutamate recycling produces paradoxical antinociception in rats with orofacial inflammatory pain.

    PubMed

    Yang, Kui Y; Mun, Jun H; Park, Ki D; Kim, Min J; Ju, Jin S; Kim, Seong T; Bae, Yong C; Ahn, Dong K

    2015-03-01

    In our current study, we investigated the role of spinal glutamate recycling in the development of orofacial inflammatory pain. DL-threo-β-benzyloxyaspartate (TBOA) or methionine sulfoximine (MSO) was administered intracisternally to block spinal glutamate transporter and glutamine synthetase activity in astroglia. Intracisternal administration of high dose TBOA (10 μg) produced thermal hyperalgesia in naïve rats but significantly attenuated the thermal hyperalgesia in rats that had been pretreated with interleukin (IL)-1β or Complete Freund's Adjuvant (CFA). In contrast, intracisternal injection of MSO produced anti-hyperalgesic effects against thermal stimuli in CFA-treated rats only. To confirm the paradoxical antinociceptive effects of TBOA and MSO, we examined changes in c-Fos expression in the medullary dorsal horn produced by thermal stimulation in naïve, IL-1β-, or CFA-treated rats, after intracisternal injections of TBOA and MSO. Intracisternal administration of TBOA significantly increased c-Fos immunoreactivity in naïve rats. In contrast, intracisternal administration of TBOA significantly decreased the up-regulation of c-Fos immunoreactivity in the medullary dorsal horn of IL-1β- and CFA-treated rats. However, intracisternal injection of MSO blocked the up-regulation of c-Fos immunoreactivity in CFA-treated rats only. We also investigated the effects of botulinum toxin type A (BoNT-A) on TBOA-induced paradoxical antinociception in CFA-treated rats, as BoNT-A inhibits the release of neurotransmitters, including glutamate. BoNT-A treatment reversed behavioral responses produced by intracisternal administration of TBOA in CFA-treated rats. These results suggest that the paradoxical responses produced by blocking glutamate transporters under inflammatory pain conditions are mediated by the modulation of glutamate release from presynaptic terminals. Moreover, blockade of glutamate reuptake could represent a new therapeutic target for the treatment of

  11. The inhibitory neurotransmitter GABA evokes long-lasting Ca(2+) oscillations in cortical astrocytes.

    PubMed

    Mariotti, Letizia; Losi, Gabriele; Sessolo, Michele; Marcon, Iacopo; Carmignoto, Giorgio

    2016-03-01

    Studies over the last decade provided evidence that in a dynamic interaction with neurons glial cell astrocytes contribut to fundamental phenomena in the brain. Most of the knowledge on this derives, however, from studies monitoring the astrocyte Ca(2+) response to glutamate. Whether astrocytes can similarly respond to other neurotransmitters, including the inhibitory neurotransmitter GABA, is relatively unexplored. By using confocal and two photon laser-scanning microscopy the astrocyte response to GABA in the mouse somatosensory and temporal cortex was studied. In slices from developing (P15-20) and adult (P30-60) mice, it was found that in a subpopulation of astrocytes GABA evoked somatic Ca(2+) oscillations. This response was mediated by GABAB receptors and involved both Gi/o protein and inositol 1,4,5-trisphosphate (IP3 ) signalling pathways. In vivo experiments from young adult mice, revealed that also cortical astrocytes in the living brain exibit GABAB receptor-mediated Ca(2+) elevations. At all astrocytic processes tested, local GABA or Baclofen brief applications induced long-lasting Ca(2+) oscillations, suggesting that all astrocytes have the potential to respond to GABA. Finally, in patch-clamp recordings it was found that Ca(2+) oscillations induced by Baclofen evoked astrocytic glutamate release and slow inward currents (SICs) in pyramidal cells from wild type but not IP3 R2(-/-) mice, in which astrocytic GABAB receptor-mediated Ca(2+) elevations are impaired. These data suggest that cortical astrocytes in the mouse brain can sense the activity of GABAergic interneurons and through their specific recruitment contribut to the distinct role played on the cortical network by the different subsets of GABAergic interneurons. PMID:26496414

  12. Long-term potentiation and evoked spike responses in the cingulate cortex of freely mobile rats.

    PubMed

    Gorkin, A G; Reymann, K G; Aleksandrov, Yu I

    2003-10-01

    Long-term potentiation of synaptic efficiency is regarded as a major candidate for the role of the physiological mechanism of long-term memory. However, the limited development of concepts of the cellular and subcellular characteristics of the induction of long-term potentiation in animals in conditions of free behavior does not correspond to the importance of this question. The present study was undertaken to determine whether the characteristics of potentiation in the cingulate cortex in response to stimulation of fibers of the subiculo-cingulate tract are truly long-term, i.e., develop through all known phases and last at least 24 h, in freely moving animals. In addition, the study aims included identification of the effects of application of blockers of different types of glutamate receptors on the development of long-term potentiation and identification of the characteristics of spike responses of single cingulate cortex neurons to stimulation of the subiculo-cingulate tract. Long-term potentiation, lasting more than 24 h, was obtained in freely moving adult rats not treated with GABA blockers. Injection of glutamate NMDA synapse blockers led to significant decreases in evoked cingulate cortex potentials in response to test stimulation. Activatory short-latency spike responses were characterized by a low probability of spike generation, and this increased with increases in the stimulation current. These data demonstrated that it is methodologically possible to compare, in freely moving rats, the involvement of individual neurons in the mechanisms involved in learning one or another type of adaptive behavior and the dynamics of their evoked spike activity during the formation of long-term potentiation. PMID:14635990

  13. Mitochondrial glutamate carriers from Drosophila melanogaster: biochemical, evolutionary and modeling studies.

    PubMed

    Lunetti, Paola; Cappello, Anna Rita; Marsano, René Massimiliano; Pierri, Ciro Leonardo; Carrisi, Chiara; Martello, Emanuela; Caggese, Corrado; Dolce, Vincenza; Capobianco, Loredana

    2013-10-01

    The mitochondrial carriers are members of a family of transport proteins that mediate solute transport across the inner mitochondrial membrane. Two isoforms of the glutamate carriers, GC1 and GC2 (encoded by the SLC25A22 and SLC25A18 genes, respectively), have been identified in humans. Two independent mutations in SLC25A22 are associated with severe epileptic encephalopathy. In the present study we show that two genes (CG18347 and CG12201) phylogenetically related to the human GC encoding genes are present in the D. melanogaster genome. We have functionally characterized the proteins encoded by CG18347 and CG12201, designated as DmGC1p and DmGC2p respectively, by overexpression in Escherichia coli and reconstitution into liposomes. Their transport properties demonstrate that DmGC1p and DmGC2p both catalyze the transport of glutamate across the inner mitochondrial membrane. Computational approaches have been used in order to highlight residues of DmGC1p and DmGC2p involved in substrate binding. Furthermore, gene expression analysis during development and in various adult tissues reveals that CG18347 is ubiquitously expressed in all examined D. melanogaster tissues, while the expression of CG12201 is strongly testis-biased. Finally, we identified mitochondrial glutamate carrier orthologs in 49 eukaryotic species in order to attempt the reconstruction of the evolutionary history of the glutamate carrier function. Comparison of the exon/intron structure and other key features of the analyzed orthologs suggests that eukaryotic glutamate carrier genes descend from an intron-rich ancestral gene already present in the common ancestor of lineages that diverged as early as bilateria and radiata. PMID:23850633

  14. Nitric oxide induces rapid, calcium-dependent release of vesicular glutamate and ATP from cultured rat astrocytes.

    PubMed

    Bal-Price, Anna; Moneer, Zahid; Brown, Guy C

    2002-12-01

    Nitric oxide (NO; 1 microM) or an NO donor (500 microM diethylenetriamine-nitric oxide, DETA-NONOate) caused rapid glutamate and ATP release from cultured rat cortical astrocytes. NO-induced glutamate release was prevented by calcium chelators (EGTA or BAPTA-AM) and an inhibitor of vesicular exocytosis (botulinum neurotoxin C, BoTx-C), but not by a glutamate transport inhibitor, L-trans-pyrrolidine-2,4-dicarboxylate (t-PDC), a cyclooxygenase inhibitor (indomethacin), or an inhibitor of soluble guanylate cyclase 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), and was not induced by mitochondrial respiratory inhibitors (myxothiazol or azide). Similarly to glutamate, NO-induced ATP release was also completely blocked by BAPTA-AM and BoTx-C, suggesting again a vesicular, calcium-dependent mechanism of release. Addition of DETA-NONOate (500 microM) to fura-2-loaded astrocytes induced a rapid, transient increase in intracellular calcium levels followed by a lower, sustained level of calcium entry. The latter was blocked by gadolinium (1 microM), an inhibitor of capacitative Ca(2+) entry. Thus, NO appears to cause rapid exocytosis of vesicular glutamate and ATP from astrocytes by raising intracellular calcium levels. Astrocytes activated by lipopolysaccharide/endotoxin and interferon-gamma to express inducible NO synthase (iNOS) maintained substantially higher extracellular glutamate levels than nonactivated cells or activated cells treated with an iNOS inhibitor (1400W), but the rate of glutamate uptake by these cells was similar. This suggests that NO from inflammatory-activated astrocytes causes release of astrocytic glutamate. NO-induced release of astrocytic glutamate and ATP may be important in physiological or pathological communication between astrocytes and neurons. PMID:12420311

  15. Glutamate Metabolism in Major Depressive Disorder

    PubMed Central

    Abdallah, Chadi G.; Jiang, Lihong; De Feyter, Henk M.; Fasula, Madonna; Krystal, John H.; Rothman, Douglas L.; Mason, Graeme F.; Sanacora, Gerard

    2015-01-01

    Objective Emerging evidence suggests abnormalities in amino acid neurotransmitter function and impaired energy metabolism contribute to the underlying pathophysiology of Major Depressive Disorder (MDD). To test whether impairments in energetics and glutamate neurotransmitter cycling are present in MDD we used in vivo 13C magnetic resonance spectroscopy (13C MRS) to measure these fluxes in individuals diagnosed with MDD relative to non-depressed subjects. Method 1H MRS and 13C MRS data were collected on 23 medication-free MDD and 17 healthy subjects. 1H MRS provided total glutamate and GABA concentrations, and 13C MRS, coupled with intravenous infusion of [1-13C]-glucose, provided measures of the neuronal tricarboxylic acid cycle (VTCAN) for mitochondrial energy production, GABA synthesis, and glutamate/glutamine cycling, from voxels placed in the occipital cortex. Results Our main finding was that mitochondrial energy production of glutamatergic neurons was reduced by 26% in MDD subjects (t = 2.57, p = 0.01). Paradoxically we found no difference in the rate of glutamate/glutamine cycle (Vcycle). We also found a significant correlation between glutamate concentrations and Vcycle considering the total sample. Conclusions We interpret the reduction in mitochondrial energy production as being due to either mitochondrial dysfunction or a reduction in proper neuronal input or synaptic strength. Future MRS studies could help distinguish these possibilities. PMID:25073688

  16. Flavor Preferences Conditioned by Dietary Glutamate.

    PubMed

    Ackroff, Karen; Sclafani, Anthony

    2016-07-01

    Our understanding of the molecular basis of umami taste and its appetitive qualities has been greatly aided by studies in laboratory rodents. This review describes methods for testing responses to the prototypical umami substance monosodium glutamate (MSG) in rodents. Two techniques, forced exposure to MSG and 2-bottle choice tests with ascending concentrations, were used to evaluate the responses to the taste of umami itself, and 2 other methods used oral or postoral MSG to modify the responses to other flavors. Intake and preference for MSG are enhanced in mice by experience with MSG and with other nutrients with positive postoral effects. In addition, flavor preferences are enhanced in mice and rats by gastric or intestinal MSG infusions via an associative learning process. Even mice with an impaired or absent ability to taste MSG can learn to prefer a flavor added to an MSG solution, supporting the notion that glutamate acts postorally. The more complex flavor of dashi seasoning, which includes umami substances (inosinate, glutamate), is attractive to rodents, but dashi does not condition flavor preferences. Details of the postoral glutamate detection process and the nature of the signal involved in learned preferences are still uncertain but probably involve gastric or intestinal sensors or both and vagal transmission. Some findings suggest that postoral glutamate effects may enhance food preferences in humans, but this requires further study. PMID:27422522

  17. Loss of Local Astrocyte Support Disrupts Action Potential Propagation and Glutamate Release Synchrony from Unmyelinated Hippocampal Axon Terminals In Vitro

    PubMed Central

    Sobieski, Courtney; Jiang, Xiaoping; Crawford, Devon C.

    2015-01-01

    Neuron–astrocyte interactions are critical for proper CNS development and function. Astrocytes secrete factors that are pivotal for synaptic development and function, neuronal metabolism, and neuronal survival. Our understanding of this relationship, however, remains incomplete due to technical hurdles that have prevented the removal of astrocytes from neuronal circuits without changing other important conditions. Here we overcame this obstacle by growing solitary rat hippocampal neurons on microcultures that were comprised of either an astrocyte bed (+astrocyte) or a collagen bed (−astrocyte) within the same culture dish. −Astrocyte autaptic evoked EPSCs, but not IPSCs, displayed an altered temporal profile, which included increased synaptic delay, increased time to peak, and severe glutamate release asynchrony, distinct from previously described quantal asynchrony. Although we observed minimal alteration of the somatically recorded action potential waveform, action potential propagation was altered. We observed a longer latency between somatic initiation and arrival at distal locations, which likely explains asynchronous EPSC peaks, and we observed broadening of the axonal spike, which likely underlies changes to evoked EPSC onset. No apparent changes in axon structure were observed, suggesting altered axonal excitability. In conclusion, we propose that local astrocyte support has an unappreciated role in maintaining glutamate release synchrony by disturbing axonal signal propagation. SIGNIFICANCE STATEMENT Certain glial cell types (oligodendrocytes, Schwann cells) facilitate the propagation of neuronal electrical signals, but a role for astrocytes has not been identified despite many other functions of astrocytes in supporting and modulating neuronal signaling. Under identical global conditions, we cultured neurons with or without local astrocyte support. Without local astrocytes, glutamate transmission was desynchronized by an alteration of the waveform

  18. Single channel kinetics of a glutamate receptor.

    PubMed Central

    Kerry, C J; Kits, K S; Ramsey, R L; Sansom, M S; Usherwood, P N

    1987-01-01

    The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating. PMID:2436676

  19. Single Channel Kinetics of a Glutamate Receptor

    PubMed Central

    Kerry, Cathryn J.; Kits, Karel S.; Ramsey, Robert L.; Sansom, Mark S. P.; Usherwood, Peter N. R.

    1986-01-01

    The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the presence of 10-4 M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating. PMID:19431683

  20. [Long-term potentiation and unit evoked responses in the cingulate cortex of freely moving rats].

    PubMed

    Gorkin, A G; Reymann, K G; Aleksandrov, Iu I

    2002-01-01

    Long-term potentiation (LTP) of synaptic efficacy is considered to be the most probable physiological mechanism of long-term memory. However, lack of understanding of cellular and subcellular mechanisms of LTP induction in freely behaving animals does not correspond to the importance of the problem. It was tested whether the characteristics of potentiation in the cingulate cortex after tetanization of the subiculocingulate tract (SCT) meet the criteria of true LTP (that passes all known stages in its development and lasts for more than a day in freely-behaving animals). Additionally, characteristics of spike responses to SCT stimulation and the effects of application of different glutamate receptor blockers were studied. Without application of GABA receptor blockers, the LTP lasted for more than 24 hours. Application of NMDA glutamate receptor blockers significantly inhibited field potentials evoke by testing stimulation. Short-latency spike responses to SCT stimulation were recorded with low probability that increased with stimulation intensity. The obtained data reveal the possibility to compare the involvement of cingulate neurons in acquisition of adaptive behavior and changes in their spike responses during the LTP development in freely-moving rats. PMID:12528373

  1. Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

    PubMed Central

    Rouach, Nathalie

    2012-01-01

    Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents. PMID:23222635

  2. The safety evaluation of monosodium glutamate.

    PubMed

    Walker, R; Lupien, J R

    2000-04-01

    L-Glutamic acid and its ammonium, calcium, monosodium and potassium salts were evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1988. The Committee noted that intestinal and hepatic metabolism results in elevation of levels in systemic circulation only after extremely high doses given by gavage (>30mg/kg body weight). Ingestion of monosodium glutamate (MSG) was not associated with elevated levels in maternal milk, and glutamate did not readily pass the placental barrier. Human infants metabolized glutamate similarly to adults. Conventional toxicity studies using dietary administration of MSG in several species did not reveal any specific toxic or carcinogenic effects nor were there any adverse outcomes in reproduction and teratology studies. Attention was paid to central nervous system lesions produced in several species after parenteral administration of MSG or as a consequence of very high doses by gavage. Comparative studies indicated that the neonatal mouse was most sensitive to neuronal injury; older animals and other species (including primates) were less so. Blood levels of glutamate associated with lesions of the hypothalamus in the neonatal mouse were not approached in humans even after bolus doses of 10 g MSG in drinking water. Because human studies failed to confirm an involvement of MSG in "Chinese Restaurant Syndrome" or other idiosyncratic intolerance, the JECFA allocated an "acceptable daily intake (ADI) not specified" to glutamic acid and its salts. No additional risk to infants was indicated. The Scientific Committee for Food (SCF) of the European Commission reached a similar evaluation in 1991. The conclusions of a subsequent review by the Federation of American Societies for Experimental Biology (FASEB) and the Federal Drug Administration (FDA) did not discount the existence of a sensitive subpopulation but otherwise concurred with the safety evaluation of JECFA and the SCF. PMID:10736380

  3. Mood disorders: regulation by metabotropic glutamate receptors.

    PubMed

    Pilc, Andrzej; Chaki, Shigeyuki; Nowak, Gabriel; Witkin, Jeffrey M

    2008-03-01

    Medicinal therapies for mood disorders neither fully serve the efficacy needs of patients nor are they free of side-effect issues. Although monoamine-based therapies are the primary current treatment approaches, both preclinical and clinical findings have implicated the excitatory neurotransmitter glutamate in the pathogenesis of major depressive disorders. The present commentary focuses on the metabotropic glutamate receptors and their relationship to mood disorders. Metabotropic glutamate (mGlu) receptors regulate glutamate transmission by altering the release of neurotransmitter and/or modulating the post-synaptic responses to glutamate. Convergent biochemical, pharmacological, behavioral, and clinical data will be reviewed that establish glutamatergic neurotransmission via mGlu receptors as a biologically relevant process in the regulation of mood and that these receptors may serve as novel targets for the discovery of small molecule modulators with unique antidepressant properties. Specifically, compounds that antagonize mGlu2, mGlu3, and/or mGlu5 receptors (e.g. LY341495, MGS0039, MPEP, MTEP) exhibit biochemical effects indicative of antidepressant effects as well as in vivo activity in animal models predictive of antidepressant efficacy. Both preclinical and clinical data have previously been presented to define NMDA and AMPA receptors as important targets for the modulation of major depression. In the present review, we present a model suggesting how the interplay of glutamate at the mGlu and at the ionotropic AMPA and NMDA receptors might account for the antidepressant-like effects of glutamatergic- and monoaminergic-based drugs affecting mood in patients. The current data lead to the hypothesis that mGlu-based compounds and conventional antidepressants impact a network of interactive effects that converge upon a down regulation of NMDA receptor function and an enhancement in AMPA receptor signaling. PMID:18164691

  4. Spinal evoked potentials following transcranial magnetic stimulation.

    PubMed

    Nemoto, J; Sasaki, T; Kikuchi, Y; Konno, Y; Sakuma, J; Kodama, N

    2001-06-01

    Motor evoked potentials by magnetic stimulation is less invasive and causes no pain as opposed to high current electric stimulation. However, the distribution of the magnetic field generated by the round coil has not been fully studied. In this report, we mapped the extent of the magnetic induction flux density, and then the evoked potentials from the spinal cord were investigated by transcranial magnetic stimulation. We also examined the origin of the evoked potentials obtained by the magnetic stimulation. The following results were obtained. The magnetic induction flux density was at its maximum at the edge of the coil. The potentials consisted of a first negative wave and subsequent multiphasic waves. The first negative wave was similar to a response of the subcorticospinal tract in the lower brain stem, while the subsequent multiphasic waves were similar to those of the pyramidal tract. Although magnetic stimulation has certain advantages over electric stimulation, several problems remain to be solved for the monitoring of motor functions in the clinical settings. PMID:11764415

  5. Circuit Mapping by UV Uncaging of Glutamate

    PubMed Central

    Shepherd, Gordon M. G.

    2014-01-01

    In laser photostimulation, small clusters of neurons in brain slices are induced to fire action potentials by focal glutamate uncaging, and synaptic connectivity between photoexcited presynaptic neurons and individual postsynaptic neurons is assessed by intracellular recording of synaptic events. With a scanner, this process can be repeated sequentially across a patterned array of stimulus locations, generating maps of neurons’ local sources of synaptic inputs. Laser scanning photostimulation (LSPS) based on patterned glutamate uncaging offers an efficient, quantitative, optical-electrophysiological way to map synaptic circuits in brain slices. PMID:22949715

  6. P2X and NMDA receptor involvement in temporomandibular joint-evoked reflex activity in rat jaw muscles.

    PubMed

    Watanabe, T; Tsuboi, Y; Sessle, B J; Iwata, K; Hu, J W

    2010-07-30

    We have previously shown that injection of the excitatory amino glutamate into the rat temporomandibular joint (TMJ) evokes reflex activity in both anterior digastric (DIG) and masseter (MASS) muscles that can be attenuated by prior TMJ injection of an N-methyl-d-aspartate (NMDA) receptor antagonist. The aim of the present study was to test if jaw muscle activity could also be evoked by P2X receptor agonist injection into the rat TMJ region and if the reflex activity could be modulated by TMJ injection of P2X receptor antagonist or NMDA receptor antagonist. The selective P2X subtype agonist alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-me ATP) and vehicle (phosphate-buffered saline) or the selective P2X antagonist, 2'-(or-3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) or the selective NMDA antagonist (+/-)-d-2-amino-5-phosphonovalerate(APV) were injected into the rat TMJ region. Electromyographic (EMG) reflex activity was recorded in both DIG and MASS muscles. Compared with the baseline EMG activity, alpha,be