Glutamate-induced Increase Research Articles

Increased glutamate in type 2 diabetes in the Korean population is associated with increased plasminogen levels.

Glutamate is a major neurotransmitter, although it causes cytotoxicity and inflammation in nonneuronal organs. This study aimed to investigate the metabolic disorders in which glutamate, associated with type 2 diabetes onset, is induced in the liver. An analysis of Korean community-based Ansan-Ansung cohort study data as well as functional research using in vitro and mouse models were performed. Groups with high plasma glutamate levels (T2, T3) had a significantly increased risk of diabetes incidence after 8 years, compared to the group with relatively low glutamate levels (T1). Analysis of the effect of glutamate on diabetes onset in vitro showed that glutamate induces insulin resistance by increasing glucose-related protein 78 (GRP78) and phosphoenolpyruvate carboxykinase (PEPCK) expression in SK-Hep-1 human liver cells. In addition, three different genes, FRMB4B, PLG, and PARD3, were significantly associated with glutamate and were identified via genome-wide association studies. Among glutamate-related genes, plasminogen (PLG) levels were most significantly increased in several environments in which insulin resistance was induced, and was also upregulated by glutamate. Glutamate-induced increase in PLG in liver cells was caused by metabotropic glutamate receptor 5 activation, and PLG levels were also upregulated after extracellular secretion. Moreover, glutamate increased the expression of plasminogen activator inhibitor-1 (PAI-1). Thus, extracellular secreted PLG cannot be converted to plasmin (fibrinolytic enzyme) by increased PAI-1. Increased glutamate is closely associated with the development of diabetes, and it may cause metabolic disorders by inhibiting the fibrinolytic system, which plays an important role in determining blood clots, a hallmark of diabetes.

Peripheral N-methyl-d-aspartate receptor activation contributes to monosodium glutamate-induced headache but not nausea behaviours in rats

Monosodium glutamate induces behaviors thought to reflect headache and nausea in rats. We explored the effects of the N-methyl-d-aspartate receptor antagonist (2R)-amino-5-phosphonovaleric acid, the inotropic glutamate receptor antagonist kynurenic acid, and the CGRP receptor antagonist olcegepant, on monosodium glutamate-induced increases in nocifensive, headache-like and nausea behaviours. Effects of these antagonists on motor function were examined with a rotarod. The effect of the dopamine receptor antagonist metoclopramide and the serotonin 3 receptor antagonist ondansetron on nausea behaviour was also assessed. (2R)-amino-5-phosphonovaleric acid, and to a lesser extent, kynurenic acid and olcegepant, reduced nocifensive and headache-like behaviours evoked by monosodium glutamate. No alteration in motor function by (2R)-amino-5-phosphonovaleric acid, kynurenic acid or olcegepant was observed. No sex-related differences in the effectiveness of these agents were identified. Nausea behaviour was significantly more pronounced in male than in female rats. Olcegepant, ondansetron and metoclopramide ameliorated this nausea behaviour in male rats. Ondansetron and metoclopramide also reduced headache-like behaviour in male rats. These findings suggest that peripheral N-methyl-d-aspartate receptor activation underlies monosodium glutamate-induced headache-like behaviour but does not mediate the nausea behaviour in rats.

Bacopa monnieri attenuates glutamate-induced nociception and brain mitochondrial toxicity in Zebrafish.

Bacopa monnieri L. (BM; Family: Scrophulariaceae), commonly known as Brahmi, is traditionally used as a nootropic agent. BM also exhibits significant analgesic activity in experimental models of pain. However, the effect of Bacopa monnieri against glutamate-induced nociception in zebrafish is yet to be explored in experimental condition. Therefore, the present study was designed to evaluate the effect of BM against glutamate-induced nociception and brain mitochondrial toxicity in adult zebrafish (Danio rerio). BM at 0.625, 1.25 and 2.5mg/ml was administered to adult zebrafish and after half an hour glutamate was injected through i.m. route of administration. Indomethacin was used as standard drug. After behavioral analysis, the fish were euthanized and the brain was isolated and stored for further biochemical analysis. BM (1.25 and 2.5mg/ml) and indomethacin significantly attenuated the glutamate-induced increase in number of line crossing compared to control group animals. Additionally, BM (1.25 and 2.5mg/ml) and indomethacin significantly reduced the glutamate induced increase in cytosolic calcium level. Further, there was a substantial improvement in mitochondrial function, integrity and bioenergetics in term of respiratory control rate and ADP/O in zebrafish brain. Moreover, BM (1.25 and 2.5mg/ml) and indomethacin significantly reduced the glutamate-induced mitochondria-dependent apoptosis in zebrafish brain. Therefore, BM could be a potential alternative drug candidate in the management of pain.

Sensory adaptation at ribbon synapses in the zebrafish lateral line.

Adaptation is used by sensory systems to adjust continuously their sensitivity to match changes in environmental stimuli. In the auditory and vestibular systems, the release properties of glutamate-containing vesicles at the hair-cell ribbon synapses play a crucial role in sensory adaptation, thus shaping the neural response to sustained stimulation. How ribbon synapses regulate the release of glutamate and how they modulate afferent responses in vivo is still largely unknown. Here, we have used two-photon imaging and electrophysiology to investigate the synaptic transfer characteristics of the hair cells in the context of sensory adaptation in live zebrafish. Prolonged and repeated water-jet stimulation of the hair-cell stereociliary bundles caused adaptation of the action potential firing rate elicited in the afferent neurons. By monitoring glutamate at ribbon synapses using time-lapse imaging, we identified two kinetically distinct release components: a rapid response that was exhausted within 50-100 ms and a slower and sustained response lasting the entire stimulation. After repeated stimulations, the recovery of the fast component followed a biphasic time course. Depression of glutamate release was largely responsible for the rapid firing rate adaptation recorded in the afferent neurons. However, postsynaptic Ca2+ responses had a slower recovery time course than that of glutamate release, indicating that they are also likely to contribute to the afferent firing adaptation. Hair cells also exhibited a form of adaptation during inhibitory bundle stimulations. We conclude that hair cells have optimised their synaptic machinery to encode prolonged stimuli and to maintain their sensitivity to new incoming stimuli.

Neuroprotective Effect of Gallocatechin Gallate on Glutamate-Induced Oxidative Stress in Hippocampal HT22 Cells.

Oxidative stress leads to protein degeneration or mitochondrial dysfunction, causing neuronal cell death. Glutamate is a neurotransmitter that nerve cells use to send signals. However, the excess accumulation of glutamate can cause excitotoxicity in the central nervous system. In this study, we deciphered the molecular mechanism of catechin-mediated neuroprotective effect on glutamate-induced oxidative stress in mouse hippocampal neuronal HT22 cells. Cellular antioxidant activity was determined using the 1,1-diphenyl-picryl hydrazyl (DPPH) assay and 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) staining. Furthermore, the levels of intracellular calcium (Ca2+) as well as nuclear condensation and protein expression related to neuronal damage were assessed. All five catechins (epigallocatechin gallate, gallocatechin gallate (GCG), gallocatechin, epicatechin gallate, and epicatechin) showed strong antioxidant effects. Among them, GCG exhibited the highest neuroprotective effect against glutamate excitotoxicity and was used for further mechanistic studies. The glutamate-induced increase in intracellular Ca2+ was reduced after GCG treatment. Moreover, GCG reduced nuclear condensation and the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinases (JNK) involved in cell death. The neuroprotective effect of GCG against glutamate-induced oxidative stress in HT22 cells was attributed to the reduction in intracellular free radicals and Ca2+ influx and also the inhibition of phosphorylation of ERK and JNK. Furthermore, the antioxidant effect of GCG was found to be likely due to the inhibition of phosphorylation of ERK and JNK that led to the effective suppression of neurocytotoxicity caused by glutamate in HT22 cells.

N-Adamantyl-4-methylthiazol-2-amine suppresses glutamate-induced autophagic cell death via PI3K/Akt/mTOR signaling pathways in cortical neurons

We recently reported that N-adamantyl-4-methylthiazol-2-amine (KHG26693) attenuates glutamate-induced oxidative stress and inflammation in the brain. In this study, we investigated KHG 26693 as a therapeutic agent against glutamate-induced autophagic death of cortical neurons. Treatment with KHG26693 alone did not affect the viability of cultured cortical neurons but was protective against glutamate-induced cytotoxicity in a concentration-dependent manner. KHG26693 attenuated the glutamate-induced increase in protein levels of LC3, beclin-1, and p62. Whereas glutamate decreased the phosphorylation of PI3K, Akt, and mTOR, these levels were restored by treatment with KHG26693. These results suggest that KHG26693 inhibits glutamate-induced autophagy by regulating PI3K/Akt/mTOR signaling. Finally, KHG26693 treatment also attenuated glutamate-induced increases in reactive oxygen species, glutathione, glutathione peroxidase, and superoxide dismutase levels in cortical neurons, indicating that KHG26693 also protects cortical neurons against glutamate-induced autophagy by regulating the reactive oxygen species scavenging system.

Changes in the morphofunctional development of the neuronal network in a dissociated cell culture of rat cerebral cortical neurons

Introduction. Study of the morphofunctional neuronal development in a dissociated cerebrocortical cell culture, using modern cell technologies, is a priority in experimental neurology, which is required for successful in vitro modelling of acute and chronic forms of cerebral pathology. Aim. A morphofunctional study of the in vitro changes in neuronal differentiation of rat cerebral cortical neurons, using a range of analysis methods, including immunohistochemistry, fluorescence, and electrophysiology. Materials and methods. We investigated the degree of culture differentiation on day 3–4 and day 10–11 of in vitro cultivation, measured by the intensity of the PSA-NCAM protein expression and the level of neuronal glutamate-induced calcium overload. That was then compared with the functional activity of the neuronal network cultivated on a microelectrode array, and with changes of the neuronal network’s activity in response to glutamate receptor overstimulation. Results. A significant glutamate-induced increase of the intracellular calcium concentration was typical for mature neurons (day 10–11 of cultivation), along with a lack of PSA-NCAM paranuclear accumulation, which was only found in immature cells (day 3–4 of cultivation). There was a glutamate suppression of the neuronal network burst activity, formed in vitro by day 10–11, with had no effect on the generation of single action potentials. At the same time, kainate, the exogenous selective agonist of the one of the glutamate subtypes, completely blocked spontaneous activity of the mature neurons. Conclusion. Neocortical rat neurons reach the differentiation level necessary for the modelling of the cerebral pathologies by day 10–11 of in vitro cultivation. At this point, the process of disruption of the microelectrode array cultivated neuronal network by the glutamate receptor overactivation, has become multilayered: excitotoxic glutamate-induced damage produces selective disruption of neuronal burst activity, and with the greater cytotoxicity caused by kainate, spontaneous bioelectrical activity is completely blocked.

Achyranthes bidentata polypeptides prevent apoptosis by inhibiting the glutamate current in cultured hippocampal neurons.

Glutamate-induced excitotoxicity plays a critical role in the neurological impairment caused by middle cerebral artery occlusion. Achyranthes bidentata polypeptides have been shown to protect against neurological functional damage caused by middle cerebral artery occlusion, but the underlying neuroprotective mechanisms and the relationship to glutamate-induced excitotoxicity remain unclear. Therefore, in the current study, we investigated the protective effects of Achyranthes bidentata polypeptides against glutamate-induced excitotoxicity in cultured hippocampal neurons. Hippocampal neurons were treated with Mg2+-free extracellular solution containing glutamate (300 µM) for 3 hours as a model of glutamate-mediated excitotoxicity (glutamate group). In the normal group, hippocampal neurons were incubated in Mg2+-free extracellular solution. In the Achyranthes bidentata polypeptide group, hippocampal neurons were incubated in Mg2+-free extracellular solution containing glutamate (300 µM) and Achyranthes bidentata polypeptide at different concentrations. At 24 hours after exposure to the agents, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Hoechst 33258 staining were used to assess neuronal viability and nuclear morphology, respectively. Caspase-3 expression and activity were evaluated using western blot assay and colorimetric enzymatic assay, respectively. At various time points after glutamate treatment, reactive oxygen species in cells were detected by H2DCF-DA, and mitochondrial membrane potential was detected by rhodamine 123 staining. To examine the effect of Achyranthes bidentata polypeptides on glutamate receptors, electrophysiological recording was used to measure the glutamate-induced inward current in cultured hippocampal neurons. Achyranthes bidentata polypeptide decreased the percentage of apoptotic cells and reduced the changes in caspase-3 expression and activity induced by glutamate. In addition, Achyranthes bidentata polypeptide attenuated the amplitude of the glutamate-induced current. Furthermore, the glutamate-induced increase in intracellular reactive oxygen species and reduction in mitochondrial membrane potential were attenuated by Achyranthes bidentata polypeptide treatment. These findings collectively suggest that Achyranthes bidentata polypeptides exert a neuroprotective effect in cultured hippocampal neurons by suppressing the overactivation of glutamate receptors and inhibiting the caspase-3-dependent mitochondrial apoptotic pathway. All animal studies were approved by the Animal Care and Use Committee, Nantong University, China (approval No. 20120216-001) on February 16, 2012.

Antioxidant activity of phenylethanoid glycosides on glutamate-induced neurotoxicity.

Exposure of PC12 cells to 10 mM glutamate caused significant viability loss, cell apoptosis, decreased activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as well as increased levels of malondialdehyde (MDA). In parallel, glutamate significantly increased the intracellular levels of ROS and intracellular calcium. However, pretreatment of the cells with acteoside and isoacteoside significantly suppressed glutamate-induced cellular events. Moreover, acteoside and isoacteoside reduced the glutamate-induced increase of caspase-3 activity and also ameliorated the glutamate-induced Bcl-2/Bax ratio reduction in PC12 cells. Furthermore, acteoside and isoacteoside significantly inhibited glutamate-induced DNA damage. In the mouse model, acteoside significantly attenuated cognitive deficits in the Y maze test and attenuated neuronal damage of the hippocampal CA1 regions induced by glutamate. These data indicated that acteoside and isoacteoside play neuroprotective effects through anti-oxidative stress, anti-apoptosis, and maintenance of steady intracellular calcium.

Interleukin-16 inhibits sodium channel function and GluA1 phosphorylation via CD4- and CD9-independent mechanisms to reduce hippocampal neuronal excitability and synaptic activity

Interleukin 16 (IL-16) is a cytokine that is primarily associated with CD4+ T cell function, but also exists as a multi-domain PDZ protein expressed within cerebellar and hippocampal neurons. We have previously shown that lymphocyte-derived IL-16 is neuroprotective against excitotoxicity, but evidence of how it affects neuronal function is limited. Here, we have investigated whether IL-16 modulates neuronal excitability and synaptic activity in mouse primary hippocampal cultures. Application of recombinant IL-16 impairs both glutamate-induced increases in intracellular Ca2+ and sEPSC frequency and amplitude in a CD4- and CD9-independent manner. We examined the mechanisms underlying these effects, with rIL-16 reducing GluA1 S831 phosphorylation and inhibiting Na+ channel function. Taken together, these data suggest that IL-16 reduces neuronal excitability and synaptic activity via multiple mechanisms and adds further evidence that alternative receptors may exist for IL-16.

Extracellular ATP and glutamate drive pyruvate production and energy demand to regulate mitochondrial respiration in astrocytes.

Astrocytes respond to energetic demands by upregulating glycolysis, lactate production, and respiration. This study addresses the role of respiration and calcium regulation of respiration as part of the astrocyte response to the workloads caused by extracellular ATP and glutamate. Extracellular ATP (100 μM to 1 mM) causes a Ca2+ -dependent workload and fall of the cytosolic ATP/ADP ratio which acutely increases astrocytes respiration. Part of this increase is related to a Ca2+ -dependent upregulation of cytosolic pyruvate production. Conversely, glutamate (200 μM) causes a Na+ , but not Ca2+ , dependent workload even though glutamate-induced Ca2+ signals readily reach mitochondria. The glutamate workload triggers a rapid fall in the cytosolic ATP/ADP ratio and stimulation of respiration. These effects are mimicked by D-aspartate a nonmetabolized agonist of the glutamate transporter, but not by a metabotropic glutamate receptor agonist, indicating a major role of Na+ -dependent workload in stimulated respiration. Glutamate-induced increase in respiration is linked to a rapid increase in glycolytic pyruvate production, suggesting that both glutamate and extracellular ATP cause an increase in astrocyte respiration fueled by workload-induced increase in pyruvate production. However, glutamate-induced pyruvate production is partly resistant to glycolysis blockers (iodoacetate), indicating that oxidative consumption of glutamate also contributes to stimulated respiration. As stimulation of respiration by ATP and glutamate are similar and pyruvate production smaller in the first case, the results suggest that the response to extracellular ATP is a Ca2+ -dependent upregulation of respiration added to glycolysis upregulation. The global contribution of astrocyte respiratory responses to brain oxygen consumption is an open question.

Sanguiin H-11 from Sanguisorbae radix protects HT22 murine hippocampal cells against glutamate-induced death

Excessive glutamate level induces neuronal death in acute brain injuries and chronic neurodegenerative diseases. Natural compounds from medicinal and food plants have been attracting interest as a treatment for neurological disorders. Sanguiin H-11 (SH-11), a hydrolysable ellagitannin, inhibits neutrophil movement and nitric oxide -production. However, its neuroprotective effect has not been studied. Therefore, the present study examined the protective effect of SH-11 from Sanguisorbae radix and its mechanism against glutamate-induced death in HT22 cells. Our results showed that SH-11 possessed a strong antioxidant activity and prevented glutamate-induced death in HT22 cells. As a strong antioxidant, SH-11 significantly reduced glutamate-induced increases in intracellular reactive oxygen species accumulation and calcium ion influx. Western blotting analysis showed that glutamate-induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-related kinases 1/2, c-Jun N-terminal kinase, and p38, was significantly decreased by SH-11. Furthermore, SH-11 significantly decreased the number of annexin V-positive HT22 cells, which is indicating apoptotic cell death. In conclusion, our results suggested that SH-11 exerted a potent neuroprotective activity against glutamate-mediated apoptotic cell death by inhibiting oxidative stress-mediated MAPK activation.

Neuroprotective effect of zolpidem against glutamate-induced toxicity is mediated via the PI3K/Akt pathway and inhibited by PK11195

Excitotoxicity is a pathological process in which neuronal dysfunction and death are induced by excessive glutamate stimulation, the major fast excitatory neurotransmitter in the mammalian brain. Excitotoxicity-induced neurodegeneration is a contributing factor in ischemia-induced brain damage, traumatic brain injury, and various neurodegenerative diseases. It is triggered by calcium overload due to prolonged over-activation of ionotropic N-methyl-d-aspartate (NMDA) receptors. Enhanced Ca2+ release results in neuronal vulnerability through several intertwined mechanisms, including activation of proteolytic enzymes, increased production of reactive oxygen species (ROS), mitochondrial dysfunction and modulation of intracellular signalling pathways. We investigated the neuroprotective effect of hypnotic zolpidem, a drug that exerts its central effects at the GABAA receptor complex, against glutamate-induced toxicity in P19 neurons. Zolpidem prevented death of P19 neurons exposed to glutamate, and abolished the glutamate-induced increase in ROS production, p53 and Bax expression, and caspase-3/7 activity. Zolpidem effects were mediated by marked over-activation of Akt kinase. The pro-survival effect, as well as the pAkt induction, were prevented in the presence of wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K) that functions upstream of Akt. The beneficial effect of zolpidem on neuronal survival was not prevented by flumazenil, a GABAA receptor antagonist. PK11195, a drug that modulates the mitochondrial translocator protein 18 kDa (TSPO) and F0F1-ATPase, prevented the beneficial effect of zolpidem, indicating that the mechanism of zolpidem action involves preservation of mitochondrial function and integrity. Zolpidem effects were further mediated by prevention of glutamate-induced increase in the expression of the NR2B subunit of NMDA receptor. The obtained results suggest the promising therapeutic potential of zolpidem against excitotoxic insults and highlight the importance of mitochondria and the Akt pathway as valuable targets for therapeutic interventions in glutamate-mediated neuropathological conditions.

Phycoerythrin-Derived Tryptic Peptide of a Red Alga Pyropia yezoensis Attenuates Glutamate-Induced ER Stress and Neuronal Senescence in Primary Rat Hippocampal Neurons.

Glutamate excitotoxicity has been observed in association with neurodegenerative disorders. This study aimed to investigate whether a phycoerythrin-derived tryptic peptide of Pyropia yezoensis (PYP) reduces glutamate-induced excitotoxicity and neuronal senescence in primary rat hippocampal neurons. Glutamate exposure (100μm) decreased cell viability and increased expression of endoplasmic reticulum (ER) stress response protein glucose-regulated protein 78 (GRP78) starting at 60min following glutamate exposure, which was prevented by pretreating the neurons with PYP (1μg mL-1 ). The glutamate-induced increase in GRP78 expression was downregulated by blocking N-methyl-d-aspartate (NMDA) receptor with MK801 (10μm) and inhibiting c-Jun N-terminal kinase (JNK) phosphorylation with SP600125 (10μm). Moreover, phosphorylation of JNK was decreased by blockade of NMDA receptor. The PYP pretreatment downregulated glutamate-induced increase in GRP78 expression and JNK phosphorylation, and this effect was abolished by inhibiting tropomyosin-related kinase B (TrkB) receptor, phosphatidylinositiol 3-kinase, and extracellular signal-regulated kinase (ERK)1/2 using cyclotraxin B (200nm), LY294002 (20μm), and SL327 (10μm), respectively. In addition, PYP downregulated increase in GRP78 expression, senescence-associated β-galactosidase activity, and neurite degeneration in aging hippocampal neurons. These findings indicate that activation of TrkB receptor-mediated ERK1/2 by PYP attenuates glutamate-induced ER stress, which may improve the survival of hippocampal neurons with age.

Bupivacaine Indirectly Potentiates Glutamate-induced Intracellular Calcium Signaling in Rat Hippocampal Neurons by Impairing Mitochondrial Function in Cocultured Astrocytes.

Bupivacaine induces central neurotoxicity at lower blood concentrations than cardiovascular toxicity. However, central sensitivity to bupivacaine is poorly understood. The toxicity mechanism might be related to glutamate-induced excitotoxicity in hippocampal cells. The intracellular free Ca concentration ([Ca]i), mitochondrial membrane potential, and reactive oxygen species generation were measured by fluorescence and two-photon laser scanning microscopy in fetal rat hippocampal neurons and astrocytes. In astrocyte/neuron cocultures, 300 μM bupivacaine inhibited glutamate-induced increases in [Ca]i in astrocytes by 40% (P < 0.0001; n = 20) but significantly potentiated glutamate-induced increases in [Ca]i in neurons by 102% (P = 0.0007; n = 10). Ropivacaine produced concentration-dependent effects similar to bupivacaine (0.3 to 300 μM). Tetrodotoxin did not mimic bupivacaine's effects. In pure cell cultures, bupivacaine did not affect glutamate-induced increases in [Ca]i in neurons but did inhibit increased [Ca]i in astrocytes. Moreover, bupivacaine produced a 61% decrease in the mitochondrial membrane potential (n = 20) and a 130% increase in reactive oxygen species generation (n = 15) in astrocytes. Cyclosporin A treatment suppressed bupivacaine's effects on [Ca]i, mitochondrial membrane potential, and reactive oxygen species generation. When astrocyte/neuron cocultures were incubated with 500 μM dihydrokainic acid (a specific glutamate transporter-1 inhibitor), bupivacaine did not potentiate glutamate-induced increases in [Ca]i in neurons but still inhibited glutamate-induced increases in [Ca]i in astrocytes. In primary rat hippocampal astrocyte and neuron cocultures, clinically relevant concentrations of bupivacaine selectively impair astrocytic mitochondrial function, thereby suppressing glutamate uptake, which indirectly potentiates glutamate-induced increases in [Ca]i in neurons.

A Novel Tetramethylpyrazine Derivative Prophylactically Protects against Glutamate-Induced Excitotoxicity in Primary Neurons through the Blockage of N-Methyl-D-aspartate Receptor.

The over-activation of NMDA receptor via the excessive glutamate is believed to one of the most causal factors associated with Alzheimer’s disease (AD), a progressive neurodegenerative brain disorder. Molecules that could protect against glutamate-induced neurotoxicity may hold therapeutic values for treating AD. Herein, the neuroprotective mechanisms of dimeric DT-010, a novel derivative of naturally occurring danshensu and tetramethylpyrazine, were investigated using primary rat cerebellar granule neurons (CGNs) and hippocampal neurons. It was found that DT-010 (3–30 μM) markedly prevented excitotoxicity of CGNs caused by glutamate, as evidenced by the promotion of neuronal viability as well as the reversal of abnormal morphological changes. While its parent molecules did not show any protective effects even when their concentration reached 50 μM. Additionally, DT-010 almost fully blocked intracellular accumulation of reactive oxygen species caused by glutamate and exogenous oxidative stimulus. Moreover, Western blot results demonstrated that DT-010 remarkably attenuated the inhibition of pro-survival PI3K/Akt/GSK3β pathway caused by glutamate. Ca2+ imaging with Fluo-4 fluorescence analysis further revealed that DT-010 greatly declined glutamate-induced increase in intracellular Ca2+. Most importantly, with the use of whole-cell patch clamp electrophysiology, DT-010 directly inhibited NMDA-activated whole-cell currents in primary hippocampal neurons. Molecular docking simulation analysis further revealed a possible binding mode that inhibited NMDA receptor at the ion channel, showing that DT-010 favorably binds to Asn602 of NMDA receptor via arene hydrogen bond. These results suggest that DT-010 could be served as a novel NMDA receptor antagonist and protect against glutamate-induced excitotoxicity from blocking the upstream NMDA receptors to the subsequent Ca2+ influx and to the downstream GSK3β cascade.

A Microfluidic Platform for the Characterisation of CNS Active Compounds

New in vitro technologies that assess neuronal excitability and the derived synaptic activity within a controlled microenvironment would be beneficial for the characterisation of compounds proposed to affect central nervous system (CNS) function. Here, a microfluidic system with computer controlled compound perfusion is presented that offers a novel methodology for the pharmacological profiling of CNS acting compounds based on calcium imaging readouts. Using this system, multiple applications of the excitatory amino acid glutamate (10 nM–1 mM) elicited reproducible and reversible transient increases in intracellular calcium, allowing the generation of a concentration response curve. In addition, the system allows pharmacological investigations to be performed as evidenced by application of glutamatergic receptor antagonists, reversibly inhibiting glutamate-induced increases in intracellular calcium. Importantly, repeated glutamate applications elicited significant increases in the synaptically driven activation of the adjacent, environmentally isolated neuronal network. Therefore, the proposed new methodology will enable neuropharmacological analysis of CNS active compounds whilst simultaneously determining their effect on synaptic connectivity.

Glutamate-induced rapid induction of Arc/Arg3.1 requires NMDA receptor-mediated phosphorylation of ERK and CREB

Arc/Arg3.1 is a unique immediate early gene whose expression is highly dynamic and correlated with various forms of synaptic plasticity. Many previous reports highlight the complexity of mechanisms that regulate Arc/Arg3.1 expression in neurons. In the present study, the expression and regulation of Arc/Arg3.1 after glutamate treatment in primary cultured cortical neurons were investigated. We found that both Arc/Arg3.1 mRNA and Arc/Arg3.1 protein dynamically increased within 24h after glutamate treatment. The results of immunostaining showed that abundant amounts of Arc/Arg3.1 protein are presented in both soma and dendrites. The glutamate-induced increase in Arc/Arg3.1 protein levels was partially prevented by the NMDAR inhibitor DL-AP5, but not the AMPAR inhibitor NBQX. The results of calcium imaging showed that glutamate induced significant increases in intracellular calcium levels in a NMDAR-dependent manner. However, the intracellular calcium chelator BAPTA-AM had no effect on glutamate-induced upregulation of Arc/Arg3.1 protein, and alteration of cytosolic calcium ion homeostasis with A23187 and TG did not change Arc/Arg3.1 protein levels. In addition, the phosphorylation of ERK and CREB, two downstream factors of NMDAR signaling, markedly increased after glutamate exposure. Blocking ERK and CREB activation via selective inhibitors partially prevented the glutamate-induced elevation of Arc/Arg3.1 protein levels. Combined observations support a NMDAR-mediated and calcium-independent mechanism by which glutamate increases Arc/Arg3.1 expression in cortical neurons.

Astaxanthin attenuates glutamate-induced apoptosis via inhibition of calcium influx and endoplasmic reticulum stress

Astaxanthin (AST) is a carotenoid that has been shown to have neuroprotective effects. In this study, it was found that AST significantly inhibited glutamate-induced loss of cell viability and apoptosis. AST pretreatment attenuated glutamate-induced activation of caspase-3, reduction of anti-apoptotic protein Bcl-2, and increase of pro-apoptotic protein Bak. In addition, AST pretreatment suppressed the production of intracellular reactive oxygen species. AST treatment also prevented glutamate-induced increase of the level of activated p38 mitogen-activated protein kinase (MAPK), which has been shown to promote apoptotic events. Furthermore, AST treatment greatly reduced the elevation of intracellular calcium level induced by glutamate and inhibited the activity of calpain, a calcium-dependent protease that plays an important role in mediating apoptosis stimulated by calcium overload in cytoplasm. Both oxidative stress and calcium overload can lead to endoplasmic reticulum (ER) stress. C/EBP-homologous protein (CHOP) is a bZIP transcription factor that can be activated by ER stress and promotes apoptosis. Here we found that AST attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein (GRP78). Overall, these results suggested that AST might protect cells against glutamate-induced apoptosis through maintaining redox balance and inhibiting glutamate-induced calcium influx and ER stress.

Glutamate affects the production of epoxyeicosanoids within the brain: The up-regulation of brain CYP2J through the MAPK-CREB signaling pathway.

Glutamate is the major excitatory neurotransmitter in the brain, and chronic glutamate excitotoxicity has been thought to be involved in numerous neurodegenerative diseases. We investigated the effects of glutamate at concentrations lower than the usual extrasynaptic concentrations on the production of epoxyeicosanoids mediated by brain CYP2J. Glutamate increased CYP2J2 mRNA levels in astrocytes in a dose-dependent manner, while an antagonist of the metabotropic glutamate receptor subtype 5 (mGlu5 receptor) attenuated the glutamate-induced increases in CYP2J2 levels by glutamate. Glutamate increased the binding of cAMP response element-binding protein (CREB) with the CYP2J2 promoter, and the inhibition of the MAPK signaling pathway (ERK1/2, p38, and JNK) decreased the binding of CREB with the CYP2J2 promoter following the glutamate treatment. CREB activated the CYP2J2 promoter located at -1522 to -1317bp, and CREB overexpression significantly increased CYP2J2 mRNA levels. The CYP2J2 and mGlu5 mRNA levels were higher in the frontal cortex, hippocampus, cerebellum, and brainstem in adult rats that received a subcutaneous injection of monosodium l-glutamate at 1, 3, 5, and 7days of age. The data from the partial least-squares-discriminant analysis showed the epoxyeicosanoid profile of the hippocampus from the cerebellum, brain stem, and frontal cortex. The sum of the epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs) was increased by 1.16-fold, 1.18-fold, and 1.19-fold in the frontal cortex, cerebellum, and brain stem, respectively, in rats treated with monosodium l-glutamate compared with the control group. The results suggest that brain CYP2J levels and CYP2J-mediated epoxyeicosanoid production can be regulated by extrasynaptic glutamate. The glutamate receptors expressed in astrocytes may mediate the regulation of drug-metabolizing enzymes and the metabolome of endogenous substances by glutamate.