Low Glutamate Concentrations Research Articles

Modulation of Orientational Dynamics of Excitatory Amino Acid Transporter-1 by Cholesterol

Glutamate is the most important excitatory neurotransmitter in the human brain. Its concentration controls synaptic transmission, thus maintaining low concentration of glutamate in the synapse is of critical importance. The synaptic concentration of glutamate in mammalian brain is regulated by excitatory amino acid transporter-1 (EAAT1), which harvests the energy of sodium, proton, and potassium gradients to uptake glutamate from the synapse. The transport mechanism involves outward-facing (OF) to inward-facing (IF) conformational changes of EAAT1, where glutamate is transported from the extracellular to the intracellular region. EAAT1 is reported to partition into cholesterol-rich regions of the membrane, suggesting that cholesterol might play an important role in its function. Each monomer of EAAT1 consists of two distinct domains, the trimerization domain and the transport domain. The interconversion between the two can be described as nearly a rigid body movement of the transport domain in lipid bilayer with respect to the trimerization domain, following an elevator-like motion, involving both translational and orientational components. In this study we have performed extensive all-atom simulations of recently crystalized human EAAT1 in cholesterol-rich and cholesterol-free lipid-bilayers. Our results suggest that compared to the modeled POPC membranes, the cholesterol-rich bilayer modifies the orientational dynamics of EAAT1, which we attribute to the increased lateral pressure of the lipid bilayer. Distribution of lipids around the protein shows cholesterol binding to the interface between the trimerization and transport domains of EAAT1. A combination of equilibrium simulations, string calculations and 1D-BEUS free energy calculations demonstrate that cholesterol has a high affinity towards the interface and alters the OF-IF transitions in EAAT1. Remarkably, the same interface forms the binding pocket for an allosteric inhibitor UCPH, thus we suggest cholesterol might play an important role in governing the dynamics and kinetics of EAAT1.

Voltage Dependent Inhibitor Binding to Plasma-Membrane Glutamate Transporters

Plasma-membrane glutamate transporters of the excitatory amino acid transporter (EAAT) family are important for maintaining a low glutamate concentration in the extracellular space of the mammalian brain. Glutamate is believed to be transported in its negatively-charged form, energetically driven by the co-transport of three sodium ions. At least two of these sodium ions are bound within the dielectric of the membrane, resulting in voltage dependent association and dissociation kinetics. It was speculated that glutamate binding is also electrogenic because the binding site of the transported substrate, glutamate, is located somewhat close to the center of the membrane. Furthermore, it was hypothesized that competitive inhibitor binding is voltage dependent for the same reason. Here, we rapidly applied a low-affinity competitive inhibitor, kainate, to the glutamate transporter subtype EAAT2, resulting in outward transient current caused by movement of net negative charge of the inhibitor into the low dielectric of the protein/membrane. Consistently, inhibitor dissociation kinetics are voltage dependent, accelerating with increasingly negative transmembrane potential. In contrast, binding kinetics of a high-affinity, slow binding inhibitor, TFB-TBOA, showed the opposite voltage dependence compared to dissociation. Consistent with previous studies, our results show that the substrate and inhibitor binding site is located within the membrane environment with low dielectric constant. This work was supported by the National Science Foundation Grant 1515028 awarded to C.G.

Modeling of excitatory amino acid transporters and clearance of synaptic cleft on millisecond time scale

Excitatory Amino Acid Transporters (EAATs) operate over wide time scales in the brain. They maintain low ambient concentrations of the primary excitatory amino acid neurotransmitter glutamate, but they also seem to play a significant role in clearing glutamate from the synaptic cleft in the millisecond time-scale process of chemical communication that occurs between neurons. The detailed kinetic mechanisms underlying glutamate uptake and clearance remain incompletely understood. In this work we used a combination of methods to model EAAT kinetics and gain insight into the impact of transport on glutamate dynamics in a general sense. We derive reliable estimates of the turnover rates of the three major EAAT subtypes expressed in the mammalian cerebral cortex. Previous studies have provided transporter kinetic estimates that vary over an order of magnitude. The values obtained in this study are consistent with estimates that suggest the unitary transporter rates are approximately 20-fold slower than the time course of glutamate in the synapse. A combined diffusion/transport model provides a possible mechanism for the apparent discrepancy.

A Novel Two-Component System, GluR-GluK, Involved in Glutamate Sensing and Uptake in Streptomyces coelicolor.

Two-component systems (TCSs), the predominant signal transduction pathways employed by bacteria, play important roles in physiological metabolism in Streptomyces Here, a novel TCS, GluR-GluK (encoded by SCO5778-SCO5779), which is located divergently from the gluABCD operon encoding a glutamate uptake system, was identified as being involved in glutamate sensing and uptake as well as antibiotic biosynthesis in Streptomyces coelicolor Under the condition of minimal medium (MM) supplemented with different concentrations of glutamate, deletion of the gluR-gluK operon (gluR-K) resulted in enhanced actinorhodin (ACT) but reduced undecylprodigiosin (RED) and yellow type I polyketide (yCPK) production, suggesting that GluR-GluK plays a differential role in antibiotic biosynthesis. Furthermore, we found that the response regulator GluR directly promotes the expression of gluABCD under the culture condition of MM with a high concentration of glutamate (75 mM). Using the biolayer interferometry assay, we demonstrated that glutamate acts as the direct signal of the histidine kinase GluK. It was therefore suggested that upon sensing high concentrations of glutamate, GluR-GluK would be activated and thereby facilitate glutamate uptake by increasing gluABCD expression. Finally, we demonstrated that the role of GluR-GluK in antibiotic biosynthesis is independent of its function in glutamate uptake. Considering the wide distribution of the glutamate-sensing (GluR-GluK) and uptake (GluABCD) module in actinobacteria, it could be concluded that the GluR-GluK signal transduction pathway involved in secondary metabolism and glutamate uptake should be highly conserved in this bacterial phylum.IMPORTANCE In this study, a novel two-component system (TCS), GluR-GluK, was identified to be involved in glutamate sensing and uptake as well as antibiotic biosynthesis in Streptomyces coelicolor A possible GluR-GluK working model was proposed. Upon sensing high glutamate concentrations (such as 75 mM), activated GluR-GluK could regulate both glutamate uptake and antibiotic biosynthesis. However, under a culture condition of MM supplemented with low concentrations of glutamate (such as 10 mM), although GluR-GluK is activated, its activity is sufficient only for the regulation of antibiotic biosynthesis. To the best of our knowledge, this is the first report describing a TCS signal transduction pathway for glutamate sensing and uptake in actinobacteria.

Dual Effects of TARP γ-2 on Glutamate Efficacy Can Account for AMPA Receptor Autoinactivation

SummaryFast excitatory transmission in the CNS is mediated mainly by AMPA-type glutamate receptors (AMPARs) associated with transmembrane AMPAR regulatory proteins (TARPs). At the high glutamate concentrations typically seen during synaptic transmission, TARPs slow receptor desensitization and enhance mean channel conductance. However, their influence on channels gated by low glutamate concentrations, as encountered during delayed transmitter clearance or synaptic spillover, is poorly understood. We report here that TARP γ-2 reduces the ability of low glutamate concentrations to cause AMPAR desensitization and enhances channel gating at low glutamate occupancy. Simulations show that, by shifting the balance between AMPAR activation and desensitization, TARPs can markedly facilitate the transduction of spillover-mediated synaptic signaling. Furthermore, the dual effects of TARPs can account for biphasic steady-state glutamate concentration-response curves—a phenomenon termed “autoinactivation,” previously thought to reflect desensitization-mediated AMPAR/TARP dissociation.

Longitudinal imaging reveals subhippocampal dynamics in glutamate levels associated with histopathologic events in a mouse model of tauopathy and healthy mice.

Tauopathies are neurodegenerative disorders characterized by abnormal intracellular aggregates of tau protein, and include Alzheimer's disease, corticobasal degeneration, frontotemporal dementia, and traumatic brain injury. Glutamate metabolism is altered in neurodegenerative disorders manifesting in higher or lower concentrations of glutamate, its transporters or receptors. Previously, glutamate chemical exchange saturation transfer (GluCEST) magnetic resonance imaging (MRI) demonstrated that glutamate levels are reduced in regions of synapse loss in the hippocampus of a mouse model of late-stage tauopathy. We performed a longitudinal GluCEST imaging experiment paired with a cross-sectional study of histologic markers of tauopathy to determine whether (1) early GluCEST changes are associated with synapse loss before volume loss occurs in the hippocampus, and whether (2) subhippocampal dynamics in GluCEST are associated with histopathologic events related to glutamate alterations in tauopathy. Live imaging of the hippocampus in three serial slices was performed without exogenous contrast agents, and subregions were segmented based on a k-means cluster model. Subregions of the hippocampus were analyzed (cornu ammonis CA1, CA3, dentate gyrus DG, and ventricle) in order to associate local MRI-observable changes in glutamate with histological measures of glial cell proliferation (GFAP), synapse density (synaptophysin, VGlut1) and glutamate receptor (NMDA-NR1) levels. Early differences in GluCEST between healthy and tauopathy mice were measured in the CA1 and DG subregions (30% reduction, P≤0.001). Synapse density was also significantly reduced in every subregion of the hippocampus in tauopathy mice by 6months. Volume was not significantly reduced in any subregion until 13months. Further, a gradient in glutamate levels was observed in vivo along hippocampal axes that became polarized as tauopathy progressed. Dynamics in hippocampal glutamate levels throughout lifetime were most closely correlated with combined changes in synaptophysin and GFAP, indicating that GluCEST imaging may be a surrogate marker of glutamate concentration in glial cells and at the synaptic level. © 2016 Wiley Periodicals, Inc.

Brain glutamate in anorexia nervosa: a magnetic resonance spectroscopy case control study at 7 Tesla

RationaleAnorexia nervosa (AN) is a serious psychiatric disorder with high morbidity and mortality. There are no established pharmacological treatments and the neurobiology of the condition is poorly understood. Previous studies using magnetic resonance spectroscopy (MRS) have shown that AN may be associated with reductions in indices of brain glutamate; however, at conventional field strengths (≤3 T), it is difficult to separate glutamate from its precursor and metabolite, glutamine.ObjectivesThe objective of the present study was to use high field (7 T) MRS to measure concentrations of glutamate, in three separate brain voxels, in women with AN.MethodsWe studied 13 female participants with AN and 12 healthy female controls who underwent MRS scanning at 7 T with voxels placed in anterior cingulate cortex, occipital cortex and putamen. Neurometabolites were calculated using the unsuppressed water signal as a reference and corrected for individual cerebrospinal fluid concentration in the voxel.ResultsWe found that participants with AN had significantly lower concentrations of glutamate in all three voxels (mean reduction 8%, p = 0.002) but glutamine levels were not altered. Concentrations of N-acetylaspartate, creatine, GABA and glutathione were also unchanged. However, inositol was lower in AN participants in anterior cingulate (p = 0.022) and occipital cortex (p = 0.002).ConclusionsWomen with AN apparently have widespread reductions in brain glutamate. Further work will be needed to assess if this change has pathophysiological relevance or whether it is a consequence of the many physical changes produced in AN by food restriction.

Glutamate concentrations vary with antiepileptic drug use and mental slowing.

Although antiepileptic drugs (AEDs) are effective in suppressing epileptic seizures, they also induce (cognitive) side effects, with mental slowing as a general effect. This study aimed to assess whether concentrations of MR detectable neurotransmitters, glutamate and GABA, are associated with mental slowing in patients with epilepsy taking AEDs. Cross-sectional data were collected from patients with localization-related epilepsy using a variety of AEDs from three risk categories, i.e., AEDs with low, intermediate, and high risks of developing cognitive problems. Patients underwent 3T MR spectroscopy, including a PRESS (n=55) and MEGA-PRESS (n=43) sequence, to estimate occipital glutamate and GABA concentrations, respectively. The association was calculated between neurotransmitter concentrations and central information processing speed, which was measured using the Computerized Visual Searching Task (CVST) and compared between the different risk categories. Combining all groups, patients with lower processing speeds had lower glutamate concentrations. Patients in the high-risk category had a lower glutamate concentration and lower processing speed compared with patients taking low-risk AEDs. Patients taking intermediate-risk AEDs also had a lower glutamate concentration compared with patients taking low-risk AEDs, but processing speed did not differ significantly between those groups. No associations were found between the GABA concentration and risk category or processing speed. For the first time, a relation is shown between glutamate concentration and both mental slowing and AED use. It is suggested that the reduced excitatory action, reflected by lowered glutamate concentrations, may have contributed to the slowing of information processing in patients using AEDs with higher risks of cognitive side effects.

Multifactorial Effects on Different Types of Brain Cells Contribute to Ammonia Toxicity

Effects of ammonia on astrocytes play a major role in hepatic encephalopathy, acute liver failure and other diseases caused by increased arterial ammonia concentrations (e.g., inborn errors of metabolism, drug or mushroom poisoning). There is a direct correlation between arterial ammonia concentration, brain ammonia level and disease severity. However, the pathophysiology of hyperammonemic diseases is disputed. One long recognized factor is that increased brain ammonia triggers its own detoxification by glutamine formation from glutamate. This is an astrocytic process due to the selective expression of the glutamine synthetase in astrocytes. A possible deleterious effect of the resulting increase in glutamine concentration has repeatedly been discussed and is supported by improvement of some pathologic effects by GS inhibition. However, this procedure also inhibits a large part of astrocytic energy metabolism and may prevent astrocytes from responding to pathogenic factors. A decrease of the already low glutamate concentration in astrocytes due to increased synthesis of glutamine inhibits the malate-aspartate shuttle and energy metabolism. A more recently described pathogenic factor is the resemblance between NH4+ and K+ in their effects on the Na+,K+-ATPase and the Na+,K+, 2 Cl- and water transporter NKCC1. Stimulation of the Na+,K+-ATPase driven NKCC1 in both astrocytes and endothelial cells is essential for the development of brain edema. Na+,K+-ATPase stimulation also activates production of endogenous ouabains. This leads to oxidative and nitrosative damage and sensitizes NKCC1. Administration of ouabain antagonists may accordingly have therapeutic potential in hyperammonemic diseases.

Brain metabolite abnormalities in ventromedial prefrontal cortex are related to duration of hypercortisolism and anxiety in patients with Cushing's syndrome.

Chronic exposure to excessive glucocorticoid (GC) concentration in Cushing's syndrome (CS) can affect the brain structurally and functionally; ventromedial prefrontal cortex (vmPFC) is rich in GC receptors and therefore particularly vulnerable to excessive GC concentration. Proton magnetic resonance spectroscopy ((1)H-MRS) is a sensitive, non-invasive imaging technique that provides information on brain metabolites in vivo. Our aim was to investigate metabolite concentrations in vmPFC of CS patients and their relationship with clinical outcome. Twenty-two right-handed CS patients (7 active/15 in remission, 19 females, 41.6±12.3years) and 22 right-handed healthy controls (14 females, 41.7±11years) underwent brain MRI and (1)H-MRS exams at 3 Tesla. Concentrations of glutamate (Glu), glutamate+glutamine (Glx), creatine (Cr), N-Acetyl-aspartate (NAA), N-Acetyl-aspartate+N-acetylaspartylglutamate (total NAA), choline-containing compounds (Cho) and myoinositol (MI) were determined. Moreover, anxiety and depressive symptoms were evaluated with the State-Trait Anxiety Inventory (STAI) and the Beck Depression Inventory-II (BDI-II) test, respectively. CS patients had lower concentrations of glutamate and total NAA in the vmPFC than healthy controls (8.6±1.2 vs. 9.3±0.7mmol/L, and 6.4±0.8 vs. 6.8±0.4mmol/L, respectively; p<0.05). Duration of hypercortisolism was negatively correlated with total NAA (r=-0.488, p<0.05). Moreover, the concentration of total NAA was negatively correlated with anxiety state (r=-0.359, p<0.05). Brain metabolites are abnormal in the vmPFC of patients with CS. Decreased total NAA and glutamate concentrations indicate neuronal dysfunction that appear to be related with duration of hypercortisolism and anxiety.

Glutamate Limitation, BvgAS Activation, and (p)ppGpp Regulate the Expression of the Bordetella pertussis Type 3 Secretion System.

Bordetella pertussis is a bacterium that is considered to be highly adapted to humans, and it has not been isolated from the environment. As this bacterium does not utilize sugars, the abundant supply of glutamate in Stainer Scholte (SS) medium enables B. pertussis to grow efficiently in liquid culture in vitro, and as such, SS medium is a popular choice for laboratory experiments. However, the concentration of glutamate in the in vivo niche of B. pertussis is quite low. We investigated the bacterial response to low concentrations of glutamate to elucidate bacterial physiology via the expression of the type 3 secretion system (T3SS), and we discuss its relationship to the Bvg mode in which the two-component regulator of pathogenesis (BvgAS) is activated. Glutamate limitation induced the expression of both the T3SS apparatus and effector genes at the transcriptional level. (p)ppGpp, a modulator of the stringent response, was necessary for maximum expression of the T3SS genes. These observations indicate that the expression of the T3SS is managed by nutrient starvation. In addition, the autoaggregation ability was high in the absence of glutamate and no autoaggregation was observed in glutamate-replete medium. Taken together, glutamate-limited conditions in Bvg(+) mode elicit the high expression of T3SS genes in B. pertussis and promotes its sessile form. Bordetella pertussis is a highly contagious pathogen that causes respiratory infectious disease. In spite of the increasing use of vaccination, the number of patients with pertussis is increasing. The proteins produced in vivo often are different from the protein profile under laboratory conditions; therefore, the development of conditions reflecting the host environment is important to understand native bacterial behavior. In the present study, we examined the effect of glutamate limitation, as its concentration in vivo is much lower than that in the culture medium currently used for B. pertussis experiments. As predicted, the T3SS was induced by glutamate limitation. These results are suggestive of the importance of regulation by nutrient conditions and in the pathogenicity of B. pertussis.

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

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 Q 10 = 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 the presence of chloride are discussed.

Low Dose Administration of Glutamate Triggers a Non-Apoptotic, Autophagic Response in PC12 Cells

Background/Aims: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis. Methods: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level. Results: Administration of glutamate in PC12 cells in doses as low as 10 μM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3. Conclusion: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion.

A preliminary examination of cortical neurotransmitter levels associated with heavy drinking in posttraumatic stress disorder

Posttraumatic stress disorder (PTSD) patients have low cortical concentrations of γ-aminobutyric acid (GABA) and elevated glutamate (Glu) as measured by proton magnetic resonance spectroscopy (1H MRS). Alcohol use disorder (AUD) is highly comorbid with PTSD, but the neurobiological underpinnings are largely unknown. We wanted to determine if PTSD patients with AUD have normalized cortical GABA and Glu levels in addition to metabolite alterations common to AUD. We compared brain metabolite concentrations in 10 PTSD patients with comorbid AUD (PAUD) with concentrtations in 28 PTSD patients without AUD and in 20 trauma-exposed controls (CON) without PTSD symptoms. We measured concentrations of GABA, Glu, N-acetylaspartate (NAA), creatine- (Cr) and choline-containing metabolites (Cho), and myo-Inositol (mI) in three cortical brain regions using 1H MRS and correlated them with measures of neurocognition, insomnia, PTSD symptoms, and drinking severity. In contrast to PTSD, PAUD exhibited normal GABA and Glu concentrations in the parieto-occipital and temporal cortices, respectively, but lower Glu and trends toward higher GABA levels in the anterior cingulate cortex (ACC). Temporal NAA and Cho as well as mI in the ACC were lower in PAUD than in both PTSD and CON. Within PAUD, more cortical GABA and Glu correlated with better neurocognition. Heavy drinking in PTSD is associated with partially neutralized neurotransmitter imbalance, but also with neuronal injury commonly observed in AUD.

Magnetic resonance spectroscopy and tissue protein concentrations together suggest lower glutamate signaling in dentate gyrus in schizophrenia.

Hippocampal dysfunction in schizophrenia is widely acknowledged, yet the mechanism of such dysfunction remains debated. In this study we investigate the excitatory and inhibitory hippocampal neurotransmission using two complementary methodologies, proton magnetic resonance spectroscopy (MRS) and tissue biochemistry, sampling individuals with schizophrenia in vivo and postmortem hippocampal tissue in vitro. The results show significantly lower glutamate concentrations in hippocampus in schizophrenia, an in vivo finding mirrored by lower GluN1 protein levels selectively in the dentate gyrus (DG) in vitro. In a mouse model with a DG knockout of the GRIN1 gene, we further confirmed that a selective decrease in DG GluN1 is sufficient to decrease the glutamate concentrations in the whole hippocampus. Gamma-aminobutyric acid (GABA) concentrations and GAD67 protein were not significantly different in hippocampus in schizophrenia. Similarly, GABA concentrations in the hippocampi of mice with a DG knockout of the GRIN1 gene were not significantly different from wild type. These findings provide strong evidence implicating the excitatory system within hippocampus in the pathophysiology of schizophrenia, particularly indicating the DG as a site of pathology.

Glutamatergic Transmission Aberration: A Major Cause of Behavioral Deficits in a Murine Model of Down's Syndrome

Trisomy 21, or Down's syndrome (DS), is the most common genetic cause of intellectual disability. Altered neurotransmission in the brains of DS patients leads to hippocampus-dependent learning and memory deficiency. Although genetic mouse models have provided important insights into the genes and mechanisms responsible for DS-specific changes, the molecular mechanisms leading to memory deficits are not clear. We investigated whether the segmental trisomy model of DS, Ts[Rb(12.1716)]2Cje (Ts2), exhibits hippocampal glutamatergic transmission abnormalities and whether these alterations cause behavioral deficits. Behavioral assays demonstrated that Ts2 mice display a deficit in nest building behavior, a measure of hippocampus-dependent nonlearned behavior, as well as dysfunctional hippocampus-dependent spatial memory tested in the object-placement and the Y-maze spontaneous alternation tasks. Magnetic resonance spectra measured in the hippocampi revealed a significantly lower glutamate concentration in Ts2 as compared with normal disomic (2N) littermates. The glutamate deficit accompanied hippocampal NMDA receptor1 (NMDA-R1) mRNA and protein expression level downregulation in Ts2 compared with 2N mice. In concert with these alterations, paired-pulse analyses suggested enhanced synaptic inhibition and/or lack of facilitation in the dentate gyrus of Ts2 compared with 2N mice. Ts2 mice also exhibited disrupted synaptic plasticity in slice recordings of the hippocampal CA1 region. Collectively, these findings imply that deficits in glutamate and NMDA-R1 may be responsible for impairments in synaptic plasticity in the hippocampus associated with behavioral dysfunctions in Ts2 mice. Thus, these findings suggest that glutamatergic deficits have a significant role in causing intellectual disabilities in DS.

Abnormal partitioning of hexokinase 1 suggests disruption of a glutamate transport protein complex in schizophrenia

Excitatory amino acid transporter 2 (EAAT2) belongs to a family of Na+ dependent glutamate transporters that maintain a low synaptic concentration of glutamate by removing glutamate from the synaptic cleft into astroglia and neurons. EAAT2 activity depends on Na+ and K+ gradients generated by Na+/K+ ATPase and ATP. Hexokinase 1 (HK1), an initial enzyme of glycolysis, binds to mitochondrial outer membrane where it couples cytosolic glycolysis to mitochondrial oxidative phosphorylation, producing ATP utilized by the EAAT2/Na+/K+ ATPase protein complex to facilitate glutamate reuptake. In this study, we hypothesized that the protein complex formed by EAAT2, Na+/K+ ATPase and mitochondrial proteins in human postmortem prefrontal cortex may be disrupted, leading to abnormal glutamate transmission in schizophrenia. We first determined that EAAT2, Na+/K+ ATPase, HK1 and aconitase were found in both EAAT2 and Na+/K+ ATPase interactomes by immunoisolation and mass spectrometry in human postmortem prefrontal cortex. Next, we measured levels of glutamate transport complex proteins in subcellular fractions in the dorsolateral prefrontal cortex and found increases in the EAAT2B isoform of EAAT2 in a fraction containing extrasynaptic membranes and increased aconitase 1 in a mitochondrial fraction. Finally, an increased ratio of HK1 protein in the extrasynaptic membrane/mitochondrial fraction was found in subjects with schizophrenia, suggesting that HK1 protein is abnormally partitioned in this illness. Our findings indicate that the integrity of the glutamate transport protein complex may be disrupted, leading to decreased perisynaptic buffering and reuptake of glutamate, as well as impaired energy metabolism in schizophrenia.

In vivo Proton NMR Spectroscopy of Genetic Mouse Models BALB/cJ and C57BL/6By: Variation in Hippocampal Glutamate Level and the Metabotropic Glutamate Receptor, Subtype 7 (Grm7) Gene

Glutamatergic neurotransmission in the brain is modulated by metabotropic glutamate receptors (mGluR). In recent studies, we identified a cis-regulated variant of a gene (Grm7) which codes for mGluR subtype 7 (mGluR7), a presynaptic inhibitory receptor. The genetic variant derived from the BALB/cJ mouse strain (Grm7 (BALB/cJ)) codes for higher abundance of mGluR7 mRNA in the hippocampus than the C57BL/6By strain-derived variant (Grm7 (C57BL/6By)). Here, we used localized in vivo (1)H NMR spectroscopy to test the hypothesis that Grm7 (BALB/cJ) is also associated with lower glutamate concentration in the same brain region. All data were obtained on a 7.0 T Agilent (Santa Clara, CA, USA) 40-cm bore system using experimentally naive adult male inbred C57BL/6By, BALB/cJ, and congenic mice (B6By.C.6.132.54) constructed in our laboratory carrying Grm7 (BALB/cJ) on C57BL/6By genetic background. The voxel of interest size was 6 μL (1 × 2 × 3 mm(3)) placed in the hippocampal CA1 region. The results showed that the hippocampal level of glutamate in the congenic mouse strain was significantly lower than that in the background C57BL/6By strain which carried the Grm7 (C57BL/6By) allele. Because the two inbred strains are genetically highly similar except at the region of the Grm7 gene, the results raise the possibility that allelic variation at the Grm7 locus contributes to the strain differences in both hippocampal mRNA abundance and glutamate level which may modulate complex behavioral traits, such as learning and memory, addiction, epilepsy, and mood disorders.

Modulation of homomeric and heteromeric kainate receptors by the auxiliary subunit Neto1

The ionotropic glutamate receptors are primary mediators of fast excitatory neurotransmission, and their properties are determined both by their subunit composition and their association with auxiliary subunits. The neuropilin and tolloid-like 1 and 2 proteins (Neto1 and Neto2) have been recently identified as auxiliary subunits for kainate-type glutamate receptors. Heteromeric kainate receptors (KARs) can be assembled from varying combinations of low-affinity (GluK1-GluK3) and high-affinity (GluK4-GluK5) subunits. To better understand the functional impact of auxiliary subunits on KARs, we examined the effect of Neto1 on the responses of recombinant homomeric and heteromeric KARs to varying concentrations of glutamate. We found that co-expression of Neto1 with homomeric GluK2 receptors had a small effect on sensitivity of the receptors to glutamate, but decreased the onset of desensitization while speeding recovery from desensitization. In the absence of Neto1, addition of GluK5 subunits to form GluK2/GluK5 heteromeric receptors slowed the onset of desensitization at low glutamate concentrations, compared with GluK2 homomers. Co-expression of Neto1 with GluK2/GluK5 receptors further enhanced these effects, essentially eliminating desensitization at μm glutamate concentrations without altering the EC50 for activation by glutamate. In addition, a prominent rebound current was observed upon removal of the agonist. The rate of recovery from desensitization was increased to the same degree by Neto1 for both homomeric GluK2 and heteromeric GluK2/GluK5 receptors. Expression of Neto1 with GluK1/GluK5, GluK3/GluK5 or GluK2/GluK4 receptors produced qualitatively similar effects on whole-cell currents, suggesting that the impact of Neto1 on the desensitization properties of heteromeric receptors was not subunit dependent. These results provide greater insight into the functional effects of the auxiliary subunit Neto1 on both homomeric and heteromeric KARs. Alteration of the characteristics of desensitization at both sub-maximal and saturating glutamate concentrations could influence the responsiveness of these receptors to repeated stimuli. As a result, assembly of KARs with the Neto auxiliary subunits could change the kinetic properties of the neuronal response to glutamatergic input.

Glutamate induces H2O2 synthesis in nonsynaptic brain mitochondria

Mitochondrial reactive oxygen species regulate many important biological processes. We studied H2O2 formation by nonsynaptic brain mitochondria in response to the addition of low concentrations of glutamate, an excitatory neurotransmitter. We demonstrated that glutamate at concentrations from 10 to 50μM stimulated the H2O2 generation in mitochondria up to 4-fold, in a dose-dependent manner. The effect of glutamate was observed only in the presence of Ca2+ (20μM) in the incubation medium, and the rate of calcium uptake by the brain mitochondria was increased by up to 50% by glutamate. Glutamate-dependent effects were sensitive to the NMDA receptor inhibitors MK-801 (10μM) and D-AP5 (20μM) and the inhibitory neurotransmitter glycine (5mM). We have shown that the H2O2 formation caused by glutamate is associated with complex II and is dependent on the mitochondrial potential. We have found that nonsynaptic brain mitochondria are a target of direct glutamate signaling, which can specifically activate H2O2 formation through mitochondrial respiratory chain complex II. The H2O2 formation induced by glutamate can be blocked by glycine, an inhibitory neurotransmitter that prevents the deleterious effects of glutamate in brain mitochondria.