High Concentrations Of Glutamate Research Articles

High extracellular concentration of excitatory amino acids glutamate and aspartate in human brain abscess

Brain abscesses often cause symptoms of brain dysfunction, including seizures, suggesting interference with normal neurotransmission. We determined the concentration of extracellular neuroactive amino acids in brain abscesses from 16 human patients. Glutamate was present at 3.6mmol/L (median value, range 0.5–10.8), aspartate at 1.0mmol/L (range 0.09–6.8). For comparison, in cerebroventricular fluid glutamate was ∼0.6μmol/L, and aspartate was not different from zero. The total concentration of amino acids was higher in eight patients with seizures: 66mmol/L (median value, range 19–109) vs. 21mmol/L (range 4–52) in eight patients without seizures (p=0.026). The concentration of aspartate and essential amino acids tryptophan, phenylalanine, tyrosine, leucine, and isoleucine was higher in pus from patients with seizures (p⩽0.040), whereas that of glutamate was not (p=0.095). The median concentration of the non-proteinogenic, inhibitory amino acid taurine was similar in the two groups, 0.7–0.8mmol/L (range 0.1–6.1). GABA could not be detected in pus. The patient groups did not differ with respect to abscess volume, the cerebral lobe affected, age, or time from symptom onset to surgery. Seven patients with extracerebral, intracranial abscesses had significantly lower pus concentration of glutamate (352μmol/L, range 83–1368) and aspartate (71μmol/L, range 22–330) than intracerebral abscesses (p<0.001). We conclude that excitatory amino acids glutamate and aspartate may reach very high concentrations in brain abscesses, probably contributing to symptoms through activation of glutamate receptors in the surrounding brain tissue.

Effect of the Sigma-1 receptor on neurite outgrowth

Neurite outgrowth is one of the essential processes underlying development and plasticity of the nervous system. Enhancing neurite outgrowth, along with neural protection, is one of the most prominent therapeutic strategies against neuronal degeneration or damage. The sigma-1 receptor (Sig-1R) is a brain-enriched a receptor chaperone expressed in the ER membrane. As Sig-1R is involved in the mode of action of several neurotherapeutic drugs, it is important to elucidate the molecular mechanism of Sig-1R. This would increase our understanding of the pathology of neuronal diseases as well as aid in establishing new approaches for their treatment. In this review, we focus on the findings that Sig-1R contributes to neural protection and neurite outgrowth even in pathological conditions, such as in high glutamate concentration. Although a large part of molecular mechanisms of Sig-1R remains unknown, the interaction of Sig-1R and neurotrophin receptors has been recently reported. Activation of Sig-1R leads to an increase in the secretion of neurotrophin. Meanwhile, Sig-1R enhances neurotrophin receptor-mediated neurite outgrowth upon activation. We discuss the neurite outgrowth effect of Sig-1R, especially in relation to neurotrophin-mediated neurite outgrowth.

Glutamate Concentration in the Serum of Patients with Schizophrenia

Glutamate is the major neurotransmitter with multiple functions in the central nervous system. Glutamate-mediated excitotoxicity is involved in the pathophysiological processes in schizophrenia. The purpose of this study was to determine the concentration of glutamate in the serum of patients with paranoid schizophrenia compared with healthy individuals, and depending on the duration of the schizophrenic process and leading clinical symptoms. We investigated the level of glutamate in the serum of 158 patients with paranoid schizophrenia and 94 healthy persons. Higher concentrations of glutamate in schizophrenic patients compared with healthy persons have been found. The maximum concentrations of glutamate were detected in patients with disease duration of more than ten years. Glutamate level in the serum does not depend on the prevailing negative or positive clinical symptoms. The increased concentration of glutamate can hypothetically contribute to dopaminergic and glutamatergic imbalance, leading to the development of psychotic symptoms and cognitive dysfunction.

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

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

Neuroprotective biflavonoids of Chamaecyparis obtusa leaves against glutamate-induced oxidative stress in HT22 hippocampal cells

Four biflavonoids (1–4), five flavonoids glycosides (5–9), two catechins (10, 11), two lignans (12–13), neolignan glycoside (14) and phenylpropanoid glycoside (15) were isolated from the leaves of Chamaecyparis obtusa (Cupressaceae). Neuroprotective effects of the isolated compounds were evaluated employing HT22 mouse hippocampal cells, a model system to study glutamate-induced oxidative stress. The glutamate injured HT22 cells were protected significantly by amentoflavone (3), ginkgetin (4) and (−)-epitaxifolin 3-O-β-D-xylopyranoside (9). The reduced activities of antioxidant enzymes, superoxide dismutase (SOD), glutathione reductase (GR) in response to high concentration of glutamate were preserved by pre-treatment of 3, 4 or 9, while the activities of glutathione peroxidase (Gpx) and catalase (CAT) were little affected. The reduced content of GSH induced by glutamate was also recovered by 3, 4 or 9 in accommodation with the decrease in ROS production. In addition, the phosphorylation of ERK1/2 induced by glutamate insult was clearly prevented by 3, while little changed by 4. Taken together, amentoflavone (3), ginkgetin (4) and (−)-epitaxifolin 3-O-β-D-xylopyranoside (9) derived from C. obtusa could protect HT22 neuronal cells against glutamate-induced oxidative damage through preserving antioxidant enzymes activities and/or inhibiting ERK1/2 activation.

Resting-State Glutamate and GABA Concentrations Predict Task-Induced Deactivation in the Default Mode Network

Deactivation of the human brain's default mode network (DMN) is regarded as suppression of endogenous activity to support exogenous task-related processes. This phenomenon has important functional relevance and insufficient DMN deactivation has been implicated in several neuropsychiatric disorders. However, the neurochemical mechanism of the DMN's deactivation remains largely unknown. In the present study, we test the hypothesis that the major excitatory and inhibitory neurotransmitters, glutamate and GABA, respectively, are associated with DMN deactivation. We used magnetic resonance spectroscopy to measure neurotransmitter concentrations in the posterior cingulate cortex/precuneus (PCC/PCu), a key component of the DMN, and functional magnetic resonance imaging to evaluate DMN deactivation induced by an n-back working memory task. Our results demonstrate significant associations of glutamate and GABA with DMN deactivation. Specifically, high regional GABA concentration in the PCC/PCu area is associated with enhanced deactivation induced by the task in the same region, whereas high glutamate concentration is associated with reduced deactivation. Furthermore, the association between GABA and DMN deactivation increases with the cognitive loads. These neurochemical characteristics of DMN deactivation may provide novel insights toward better understanding of the DMN's functions under normal physiological conditions and dysfunctions in neuropsychiatric disorders.

Presenilin-1/γ-Secretase Controls Glutamate Release, Tyrosine Phosphorylation, and Surface Expression of N-Methyl-d-aspartate Receptor (NMDAR) Subunit GluN2B

Abnormally high concentrations of extracellular glutamate in the brain may cause neuronal damage via excitotoxicity. Thus, tight regulation of glutamate release is critical to neuronal function and survival. Excitotoxicity is caused mainly by overactivation of the extrasynaptic NMDA receptor (NMDAR) and results in specific cellular changes, including calcium-induced activation of calpain proteases. Here, we report that presenilin-1 (PS1) null mouse cortical neuronal cultures have increased amounts of calpain-dependent spectrin breakdown products (SBDPs) compared with WT cultures. NMDAR antagonists blocked accumulation of SBDPs, suggesting abnormal activation of this receptor in PS1 null cultures. Importantly, an increase in SBDPs was detected in cultures of at least 7 days in vitro but not in younger cultures. Conditioned medium from PS1 null neuronal cultures at 8 days in vitro contained higher levels of glutamate than medium from WT cultures and stimulated production of SBDPs when added to WT cultures. Use of glutamate reuptake inhibitors indicated that accumulation of this neurotransmitter in the media of PS1 null cultures was due to increased rates of release. PS1 null neurons showed decreased cell surface expression and phosphorylation of the GluN2B subunit of NMDAR, indicating decreased amounts of extrasynaptic NMDAR in the absence of PS1. Inhibition of γ-secretase activity in WT neurons caused changes similar to those observed in PS1 null neurons. Together, these data indicate that the PS1/γ-secretase system regulates release of glutamate, tyrosine phosphorylation, and surface expression of GluN2B-containing NMDARs.

Effects of recombinant human erythropoietin on the expression of glutamine synthetase of cultured retinal Miiller cells under high glucose condition

Objective To investigate the effect of recombinant human erythropoietin （rhEPO） on the expression of glutamine synthetase （GS） of cultured rat retinal Mtiller cells in high glucose environment in vitro. Methods Mtiller cells were isolated from retinas of 10 Sprague-Dawley rats at postnatal day three to five by trypsin digestion, and were randomly divided into six groups, including normal control group, high glucose group, high glucose ＋U/ml rhEPO group, high glucose＋10 U/ml rhEPO group, high glucose＋20 U/ml rhEPO group, high glucose＋ 40 U/ml rhEPO groups. After 48 hours, the apoptosis of retinal MUller cells were assayed by terminal transferase-mediated DNA end labelling assay, and the expression levels of GS protein were detected with semi-quantitative immunoeytochemistry. Results Compared with the normal control group, the cell viability and GS protein were reduced while the cell death increased in Mtiller cells cultured in high glucose, the difference was statistically significant （t= 27.4, P~0.01）. Compared with the high glucose group, rhEPO treatment reduced the apoptotic Mtiller cells （t= 857.2, 2 374.6, 2 473.2, 2 537.7; P〈0.01）, induced the expression of GS proteins （t=3.2, 18.0, 22.5, 26.4; P〈0.05）. Conclusions rhEPO can protect Muller cells from apoptosis under high glucose condition. The mechanism may be related to its function to up-regulate the GS protein expression, promote glutamic acid cycle, and reduce the excitotoxicity effects of high concentration of glutamate. Key words: Erythropoietin/pharmacology; Microglia; Glutamate-ammonia ligase

Neuroprotective effects of liquiritigenin isolated from licorice roots on glutamate-induced apoptosis in hippocampal neuronal cells

The progressive death of neurons following exposure to high concentrations of glutamate leads to loss of learning and memory and pathogenesis of neurodegenerative disorders. Therefore, identification of drugs that protect against glutamate-mediated neuronal cell death is a good strategy for prevention and treatment of neurodegenerative diseases. In this study, we isolated liquiritigenin, an active compound found in licorice roots, by column chromatography and examined its protective effects against glutamate-mediated apoptotic stimuli in a mouse hippocampus-derived neuronal cell line (HT22 cells). Cell viability was significantly recovered following treatment with 50μM liquiritigenin up to 77.50±1.93% over the control (100.00±5.62%), whereas cell viability following 5mM glutamate treatment was decreased to 52.52±4.82%. Liquiritigenin effectively reduced glutamate-induced early apoptosis through inhibition of Ca2+ influx, intracellular reactive oxygen species (ROS) production, and lipid peroxidation. In addition, the levels of Bcl-2 and full-length Bid were protected, and that of mitochondrial Bax was reduced by liquiritigenin. Liquiritigenin suppressed not only the release of apoptosis-inducing factor (AIF), but also activation of mitogen-activated protein kinases (MAPKs) such as p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Therefore, the active component in licorice roots, liquiritigenin, might facilitate development of drug leads for neurodegenerative disorders.

Amelioration of ischemic brain damage by peritoneal dialysis

Ischemic stroke is a devastating condition, for which there is still no effective therapy. Acute ischemic stroke is associated with high concentrations of glutamate in the blood and interstitial brain fluid. The inability of the tissue to retain glutamate within the cells of the brain ultimately provokes neuronal death. Increased concentrations of interstitial glutamate exert further excitotoxic effects on healthy tissue surrounding the infarct zone. We developed a strategy based on peritoneal dialysis to reduce blood glutamate levels, thereby accelerating brain-to-blood glutamate clearance. In a rat model of stroke, this simple procedure reduced the transient increase in glutamate, consequently decreasing the size of the infarct area. Functional magnetic resonance imaging demonstrated that the rescued brain tissue remained functional. Moreover, in patients with kidney failure, peritoneal dialysis significantly decreased glutamate concentrations. Our results suggest that peritoneal dialysis may represent a simple and effective intervention for human stroke patients.

Effect of treatment with simvastatin and cyclosporine on neurotransmitter concentrations in cerebrospinal fluid after subarachnoid hemorrhage in dogs

To measure concentrations of glutamate, aspartate, γ-aminobutyric acid (GABA), and glycine in CSF of dogs with experimentally induced subarachnoid hemorrhage (SAH) and to assess effects of cyclosporine and simvastatin on these concentrations. CSF samples from 13 dogs. In a previous study, SAH was induced in dogs via 2 injections of autologous blood into the cerebellomedullary cistern 24 hours apart. Dogs were untreated (control; n = 5) or received simvastatin alone (4) or simvastatin in combination with cyclosporine (4). Samples of CSF were collected before the first blood injection (baseline; time 0), before the second blood injection, and on days 3, 7, and 10. For the study reported here, neurotransmitter concentrations in CSF were analyzed via high-performance liquid chromatography. Data were analyzed with a repeated-measures model with adjustments for multiple comparisons by use of the Tukey method. In control dogs, the glutamate concentration peaked on day 3 and there was a significant increase in GABA and glutamate concentrations. Glutamate concentrations were significantly lower and glycine concentrations significantly higher on day 3 after administration of simvastatin alone or simvastatin in combination with cyclosporine, compared with concentrations for the control group. No significant differences in GABA and aspartate concentrations were detected among treatment groups at any time point. Glutamate concentrations were increased in the CSF of dogs with SAH. Simvastatin administration attenuated high glutamate concentrations. A combination of immunosuppression and upregulation of nitric oxide synthase may be useful in lowering high glutamate concentrations in ischemic CNS conditions.

Comparison of concentrations of γ-aminobutyric acid and glutamate in cerebrospinal fluid of dogs with idiopathic epilepsy with and without seizure-related magnetic resonance imaging hyperintense areas in the limbic system

To investigate differences in CSF concentrations of excitatory and inhibitory neurotransmitters in dogs with and without T2-weighted (T2W) MRI hyperintense areas in the limbic system. Archived CSF samples and stored brain MRI images of 5 healthy research dogs (group 1), 8 dogs with idiopathic epilepsy (IE) with no abnormal MRI findings (group 2), and 4 dogs with IE with hyperintense areas in the limbic system detected by means of T2W MRI (group 3). Archived CSF samples and stored MRI images obtained from all dogs were evaluated. Dogs in groups 2 and 3 were matched on the basis of age and breed. High-performance liquid chromatography was used to evaluate glutamate and γ-aminobutyric acid (GABA) concentrations in CSF samples. Glutamate concentrations were higher in CSF of both groups of dogs with IE than in healthy dogs. However, glutamate concentrations in CSF were not significantly higher in dogs with IE and with hyperintense areas than in dogs with IE but no abnormal MRI findings. Concentrations of GABA in CSF were higher in group 3 than in group 2 and in group 2 than in group 1. No significant difference was evident between glutamate concentrations in CSF of dogs with IE and with and without hyperintense areas detected by means of T2W MRI. However, glutamate concentrations typically were higher in CSF of dogs with IE and MRI hyperintense areas. Future studies with larger sample sizes should be conducted to confirm this finding and to determine the clinical importance of high glutamate concentrations in CSF of dogs with IE.

Autotypical septal contacts of cerebellar glial cells as protective reaction under toxic action of glutamate and nitric oxide

Structural transformations of the frog cerebellar glial cells (astrocytes) in the presence of high concentrations of glutamate and nitric oxide (NO) were studied by electron microscopy method. It was found that under these conditions mimicking a natural stroke, glial processes storing sufficient amounts of glycogen grains could protect the neurons of the molecular layer. Under these conditions glial processes transformed (stretched out) into strands, virtually deprived of cytoplasm. The distance between two membranes of such strands dropped to 25–30 nm, and cross-bridges appeared inside. These structures are called “autotypic septal contact” (ASC). A modified glial process with ASC could wrap injured synapse or synaptic elements (buttons, dendritic spines) and form a tight capsule. Glial processes with ASC can also intrude between two neurons of the cerebellum granular layer, preventing their pathological fusion. The results suggest that ASC can have a protecting role in the toxic action of glutamate and NO-generating agents.

Plasticity of calcium signaling cascades in human embryonic stem cell-derived neural precursors.

Human embryonic stem cell-derived neural precursors (hESC NPs) are considered to be a promising tool for cell-based therapy in central nervous system injuries and neurodegenerative diseases. The Ca(2+) ion is an important intracellular messenger essential for the regulation of various cellular functions. We investigated the role and physiology of Ca(2+) signaling to characterize the functional properties of CCTL14 hESC NPs during long-term maintenance in culture (in vitro). We analyzed changes in cytoplasmic Ca(2+) concentration ([Ca(2+)]i) evoked by high K(+), adenosine-5'-triphosphate (ATP), glutamate, γ-aminobutyric acid (GABA), and caffeine in correlation with the expression of various neuronal markers in different passages (P6 through P10) during the course of hESC differentiation. We found that only differentiated NPs from P7 exhibited significant and specific [Ca(2+)]i responses to various stimuli. About 31% of neuronal-like P7 NPs exhibited spontaneous [Ca(2+)]i oscillations. Pharmacological and immunocytochemical assays revealed that P7 NPs express L- and P/Q-type Ca(2+) channels, P2X2, P2X3, P2X7, and P2Y purinoreceptors, glutamate receptors, and ryanodine (RyR1 and RyR3) receptors. The ATP- and glutamate-induced [Ca(2+)]i responses were concentration-dependent. Higher glutamate concentrations (over 100 μM) caused cell death. Responses to ATP were observed in the presence or in the absence of extracellular Ca(2+). These results emphasize the notion that with time in culture, these cells attain a transient period of operative Ca(2+) signaling that is predictive of their ability to act as stem elements.

Increase of extracellular glutamate concentration increases its oxidation and diminishes glucose oxidation in isolated mouse hippocampus: Reversible by TFB‐TBOA

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.

The utility of ionotropic glutamate receptor antagonists in the treatment of nociception induced by epidural glutamate infusion in rats

Background:The authors have previously demonstrated that human herniated disc material contains high concentrations of free glutamate. In an experimental model, elevated epidural glutamate concentrations in the lumbar spine can cause a focal hyperesthetic state.Methods:Rats underwent epidural glutamate infusion in the lumbar spine by a miniosmotic pump over a 72-hour period. Some rats underwent coinfusion with glutamate and ionotropic glutamate antagonists. Nociception was assessed by von Frey fibers and by assessment of glutamate receptor expression in the corresponding dorsal horn of the spinal cord.Results:The kainic acid antagonist, UBP 301, decreased epidural glutamate-based hyperesthesia in a dose dependent manner. Concordant with these findings, there was significant decrease in kainate receptor expression in the dorsal horn. The N-Methyl-4-isoxazoleproionic acid (NMDA) antagonist Norketamine also significantly diminished hyperesthesia and decreased receptor expression in the dorsal horn.Conclusions:Both UBP 301, the kainic acid receptor antagonist and Norketamine, an NMDA receptor antagonist, dampened epidural glutamate-based nociception. Focal epidural injections of Kainate or NMDA receptor antagonists could be effective treatments for disc herniation-based lumbar radiculopathy.

Identification of two‐component system AfsQ1/Q2 regulon and its cross‐regulation with GlnR in Streptomyces coelicolor

The two-component system AfsQ1/Q2 of Streptomyces coelicolor was identified in our previous work as a pleiotropic regulator for antibiotic biosynthesis and morphological differentiation under the condition of a minimal medium supplemented with 75 mM glutamate. In this work, we report the dissection of the mechanism underlying the function of AfsQ1/Q2 on antibiotic production and also the identification of the AfsQ1/Q2 regulon. The results showed that AfsQ1/Q2 stimulated antibiotic ACT, RED and CDA production directly through the pathway-specific activator genes actII-ORF4, redZ and cdaR respectively. In addition, expression of sigQ that encodes a sigma factor and is divergently transcribed from afsQ1 was also subject to direct regulation by AfsQ1/Q2. The precise AfsQ1 binding sites in the upstream regions of these target genes were determined by DNase I footprinting assays coupled with site-directed DNA mutagenesis. By computational prediction and functional analysis, at least 17 new AfsQ1 targets were identified, including pstS gene encoding a high-affinity phosphate-binding protein and two developmental genes whiD, bldM. For the AfsQ1/Q2 regulon, an AfsQ1 binding motif comprising the sequence GTnAC-n(6) -GTnAC has been defined. Interestingly, we found from electrophoretic mobility shift assays and transcriptional analysis that AfsQ1/Q2 can also function as a repressor for nitrogen assimilation, and AfsQ1 can compete with GlnR for the promoter regions of glnA and nirB, suggesting the cross-regulation between AfsQ1/Q2 and GlnR in nitrogen metabolism. These findings suggested that AfsQ1/Q2 is important not only for antibiotic biosynthesis but also in maintaining the metabolic homeostasis of nutrient utilization under the stress of high concentration of glutamate in S. coelicolor.

Chemical Gradients within Brain Extracellular Space Measured using Low Flow Push–Pull Perfusion Sampling in Vivo

Although populations of neurons are known to vary on the micrometer scale, little is known about whether basal concentrations of neurotransmitters also vary on this scale. We used low-flow push-pull perfusion to test if such chemical gradients exist between several small brain nuclei. A miniaturized polyimide-encased push-pull probe was developed and used to measure basal neurotransmitter spatial gradients within brain of live animals with 0.004 mm(3) resolution. We simultaneously measured dopamine (DA), norepinephrine, serotonin (5-HT), glutamate, γ-aminobutyric acid (GABA), aspartate (Asp), glycine (Gly), acetylcholine (ACh), and several neurotransmitter metabolites. Significant differences in basal concentrations between midbrain regions as little as 200 μm apart were observed. For example, dopamine in the ventral tegmental area (VTA) was 4.8 ± 1.5 nM but in the red nucleus was 0.5 ± 0.2 nM. Regions of high glutamate concentration and variability were found within the VTA of some individuals, suggesting hot spots of glutamatergic activity. Measurements were also made within the nucleus accumbens core and shell. Differences were not observed in dopamine and 5-HT in the core and shell; but their metabolites homovanillic acid (460 ± 60 nM and 130 ± 60 nM respectively) and 5-hydroxyindoleacetic acid (720 ± 200 nM and 220 ± 50 nM respectively) did differ significantly, suggesting differences in dopamine and 5-HT activity in these brain regions. Maintenance of these gradients depends upon a variety of mechanisms. Such gradients likely underlie highly localized effects of drugs and control of behavior that have been found using other techniques.

NMR metabolomic of frontal cortex extracts: First study comparing two neurodegenerative diseases, Alzheimer disease and amyotrophic lateral sclerosis

ObjectiveThis study was designed to assess the brain metabolites’ variability between two neurodegenerative diseases in frontal cortex samples obtained post-mortem. NMR metabolomics was used for the first time in this context. Materials and methods1H NMR metabolomic was applied to tissue extracts from patients with Alzheimer disease (ALZ) and patients with amyotrophic lateral sclerosis (ALS) to investigate qualitative and quantitative variations of brain metabolites. ResultsThe Alzheimer disease metabolic signature was characterized by a high concentration of alanine, acetate, glutamate and glutamine, and low concentrations of lactate and creatine, while the ALS metabolic signature appears to be marked by high concentrations of lactate, N-acetyl aspartate, creatine, choline and myo-inositol. Moreover, in vitro1H NMR could detect metabolites such as 3-hydroxybutyrate, alanine, succinate and aspartate that cannot be detected with in vivo NMR. DiscussionThe neurodegenerative diseases exhibit diverging metabolic pathways. Some of the metabolites responsible for the discrimination between the two diseases were detected before in vivo. However, this in vitro metabolomic investigation demonstrates the involvement of metabolites not detected with in vivo studies. ConclusionUpon these findings, in vitro metabolomics appears to be an efficient tool to investigate the fundamentals of the metabolic pathway modulations in these neurodegenerative diseases to help the interpretation of clinical data obtained with in vivo NMR spectroscopy.

Autoinactivation of the Stargazin–AMPA Receptor Complex: Subunit-Dependency and Independence from Physical Dissociation

Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This “autoinactivation” has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1–A4 AMPA receptors (all flip isoform) expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.