Excessive Extracellular Glutamate Research Articles

Role of glutamate in the development of visual pathways.

Glutamate is an important amino acid, metabolite and excitatory neurotransmitter, which is found in its free form in the extracellular spaces of the central nervous system (CNS). More than half of all synapses in CNS release glutamate. It is the main neurotransmitter driving the light responses in the retina. All types of photoreceptors, bipolar, ganglion and one type of glycinergic amacrine cells express specific subtypes of vesicular glutamate transporters and are the main source of endogenous glutamate in retina, besides Müller glia that are responsible for glutamate homeostasis, release and reuptake. Reduced or excessive extracellular glutamate was detected in the synaptic clefts of several naturally occurring or transgenic eye disease models, in which network rewiring and altered functions were observed. These led to the hypothesis that glutamate is one of the extrinsic signals for visual pathway development. This minireview examines experimental evidences supporting, or refuting, the influence of glutamate on prenatal and postnatal retinal development.

Development of neuroprotection approaches for long-term space missions

The study aimed to develop a strategy and methodology for neuroprotection during long-term space missions, which is based on a comprehensive study of the impact of therapeutic hypothermia combined with the action of neuroactive drugs on the key characteristics of synaptic transmission in brain nerve terminals, which change under the influence of planetary dust and conditions of altered gravity. Development of neurotoxicity under conditions of altered gravity may result from excess extracellular glutamate caused by the reverse functioning of glutamate transporters. Under conditions of moderate and deep hypothermia, a gradual decrease in the transporter-mediated release of L-[14C]glutamate from nerve terminals was demonstrated, which is stimulated by plasma membrane depolarization with KCl and dissipation of the proton gradient of synaptic vesicles by the protonophore FCCP. This fact indicates a neuroprotective effect, which increases when hypothermia changes from moderate to deep. The possible risks of using hypothermia in space medicine have been determined. Hypothermia is not able to reduce the extracellular level of L-[14C]glutamate and [3H]GABA, which increases under the conditions of exposure to carbon-containing planetary dust. Hypothermia can lead to a further decrease in the rate of accumulation of neurotransmitters in the presence of carbon-containing planetary dust and to contribute to the development of neurotoxicity, which is a possible risk of using hypothermia in space medicine. In this context, it is important to choose the optimal individual temperature regime for each astronaut.

EphA4/ephrinA3 reverse signaling mediated downregulation of glutamate transporter GLAST in Müller cells in an experimental glaucoma model.

Deficiency of glutamate transporter GLAST in Müller cells may be culpable for excessive extracellular glutamate, which involves in retinal ganglion cell (RGC) damage in glaucoma. We elucidated how GLAST was regulated in rat chronic ocular hypertension (COH) model. Western blot and whole-cell patch-clamp recordings showed that GLAST proteins and GLAST-mediated current densities in Müller cells were downregulated at the early stages of COH. In normal rats, intravitreal injection of the ephrinA3 activator EphA4-Fc mimicked the changes of GLAST in COH retinas. In purified cultured Müller cells, EphA4-Fc treatment reduced GLAST expression at mRNA and protein levels, which was reversed by the tyrosine kinase inhibitor PP2 or transfection with ephrinA3-siRNA (Si-EFNA3), suggesting that EphA4/ephrinA3 reverse signaling mediated GLAST downregulation. EphA4/ephrinA3 reverse signaling-induced GLAST downregulation was mediated by inhibiting PI3K/Akt/NF-κB pathways since EphA4-Fc treatment of cultured Müller cells reduced the levels of p-Akt/Akt and NF-κB p65, which were reversed by transfecting Si-EFNA3. In Müller cells with ephrinA3 knockdown, the PI3K inhibitor LY294002 still decreased the protein levels of NF-κB p65 in the presence of EphA4-Fc, and the mRNA levels of GLAST were reduced by LY294002 and the NF-κB inhibitor SN50, respectively. Pre-injection of the PI3K/Akt pathway activator 740 Y-P reversed the GLAST downregulation in COH retinas. Western blot and TUNEL staining showed that transfecting of Si-EFNA3 reduced Müller cell gliosis and RGC apoptosis in COH retinas. Our results suggest that activated EphA4/ephrinA3 reverse signaling induces GLAST downregulation in Müller cells via inhibiting PI3K/Akt/NF-κB pathways, thus contributing to RGC damage in glaucoma.

Polygalasaponin F protects hippocampal neurons against glutamate-induced cytotoxicity.

Excess extracellular glutamate leads to excitotoxicity, which induces neuronal death through the overactivation of N-methyl-D-aspartate receptors (NMDARs). Excitotoxicity is thought to be closely related to various acute and chronic neurological disorders, such as stroke and Alzheimer's disease. Polygalasaponin F (PGSF) is a triterpenoid saponin monomer that can be isolated from Polygala japonica, and has been reported to protect cells against apoptosis. To investigate the mechanisms underlying the neuroprotective effects of PGSF against glutamate-induced cytotoxicity, PGSF-pretreated hippocampal neurons were exposed to glutamate for 24 hours. The results demonstrated that PGSF inhibited glutamate-induced hippocampal neuron death in a concentration-dependent manner and reduced glutamate-induced Ca2+ overload in the cultured neurons. In addition, PGSF partially blocked the excess activity of NMDARs, inhibited both the downregulation of NMDAR subunit NR2A expression and the upregulation of NMDAR subunit NR2B expression, and upregulated the expression of phosphorylated cyclic adenosine monophosphate-responsive element-binding protein and brain-derived neurotrophic factor. These findings suggest that PGSF protects cultured hippocampal neurons against glutamate-induced cytotoxicity by regulating NMDARs. The study was approved by the Institutional Animal Care Committee of Nanchang University (approval No. 2017-0006) on December 29, 2017.

Amelioration of lipopolysaccharide-induced memory impairment in equilibrative nucleoside transporter-2 knockout mice is accompanied by the changes in glutamatergic pathways

Neuroinflammation has been implicated in cognitive deficits in neurological and neurodegenerative diseases. Lipopolysaccharide (LPS)-induced neuroinflammation and the breakdown of the blood–brain barrier can be attenuated in mice with equilibrative nucleoside transporter-2 (ENT2/Ent2) deletion. The present study was aimed to investigate the role of ENT2 in cognitive and neuronal functions under physiological and inflammatory conditions, in terms of behavioral performance and synaptic plasticity in saline- and LPS-treated Ent2 knockout (KO) mice and their wild-type (WT) littermate controls. Repeated administrations of LPS significantly impaired spatial memory formation in Morris water maze and hippocampal-dependent long-term potentiation (LTP) in WT mice. The LPS-treated WT mice exhibited significant synaptic and neuronal damage in the hippocampus. Notably, the LPS-induced impairment in spatial memory and LTP performance were attenuated in Ent2 KO mice, along with the preservation of neuronal survival. The beneficial effects were accompanied by the normalization of excessive extracellular glutamate and aberrant downstream signaling of glutamate receptor activation, including the upregulation of phosphorylated p38 mitogen-activated protein kinase and the downregulation of phosphorylated cyclic adenosine monophosphate-response element-binding protein. There was no significant difference in behavioral outcome and all tested parameters between these two genotypes under physiological condition. These results suggest that ENT2 plays an important role in regulating inflammation-associated cognitive decline and neuronal damage.

S1P2-Gα12 Signaling Controls Astrocytic Glutamate Uptake and Mitochondrial Oxygen Consumption.

Glutamate is the principal excitatory neurotransmitter in the human brain. Following neurotransmission, astrocytes remove excess extracellular glutamate to prevent neurotoxicity. Glutamate neurotoxicity has been reported in multiple neurologic diseases including multiple sclerosis (MS), representing a shared neurodegenerative mechanism. A potential modulator of glutamate neurotoxicity is the bioactive lysophospholipid sphingosine 1-phosphate (S1P) that signals through five cognate G-protein-coupled receptors, S1P1-S1P5; however, a clear link between glutamate homeostasis and S1P signaling has not been established. Here, S1P receptor knock-out mice, primary astrocyte cultures, and receptor-selective chemical tools were used to examine the effects of S1P on glutamate uptake. S1P inhibited astrocytic glutamate uptake in a dose-dependent manner and increased mitochondrial oxygen consumption, primarily through S1P2 Primary cultures of wild-type mouse astrocytes expressed S1P1,2,3 transcripts, and selective deletion of S1P1 and/or S1P3 in cerebral cortical astrocytes, did not alter S1P-mediated, dose-dependent inhibition of glutamate uptake. Pharmacological antagonists, S1P2-null astrocytes, and Gα12 hemizygous-null astrocytes indicated that S1P2-Gα12-Rho/ROCK signaling was primarily responsible for the S1P-dependent inhibition of glutamate uptake. In addition, S1P exposure increased mitochondrial oxygen consumption rates (OCRs) in wild-type astrocytes and reduced OCRs in S1P2-null astrocytes, implicating receptor selective metabolic consequences of S1P-mediated glutamate uptake inhibition. Astrocytic S1P-S1P2 signaling increased extracellular glutamate, which could contribute to neurotoxicity. This effect was not observed with the FDA-approved S1P receptor modulators, siponimod and fingolimod. Development and use of S1P2-selective antagonists may provide a new approach to reduce glutamate neurotoxicity in neurologic diseases.

Disturbance of the Glutamate-Glutamine Cycle, Secondary to Hepatic Damage, Compromises Memory Function.

Glutamate fulfils many vital functions both at a peripheral level and in the central nervous system (CNS). However, hyperammonemia and hepatic failure induce alterations in glutamatergic neurotransmission, which may be the main cause of hepatic encephalopathy (HE), an imbalance which may explain damage to both learning and memory. Cognitive and motor alterations in hyperammonemia may be caused by a deregulation of the glutamate-glutamine cycle, particularly in astrocytes, due to the blocking of the glutamate excitatory amino-acid transporters 1 and 2 (EAAT1, EAAT2). Excess extracellular glutamate triggers mechanisms involving astrocyte-mediated inflammation, including the release of Ca2+-dependent glutamate from astrocytes, the appearance of excitotoxicity, the formation of reactive oxygen species (ROS), and cell damage. Glutamate re-uptake not only prevents excitotoxicity, but also acts as a vital component in synaptic plasticity and function. The present review outlines the evidence of the relationship between hepatic damage, such as that occurring in HE and hyperammonemia, and changes in glutamine synthetase function, which increase glutamate concentrations in the CNS. These conditions produce dysfunction in neuronal communication. The present review also includes data indicating that hyperammonemia is related to the release of a high level of pro-inflammatory factors, such as interleukin-6, by astrocytes. This neuroinflammatory condition alters the function of the membrane receptors, such as N-methyl-D-aspartate (NMDA), (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) AMPA, and γ-aminobutyric acid (GABA), thus affecting learning and spatial memory. Data indicates that learning and spatial memory, as well as discriminatory or other information acquisition processes in the CNS, are damaged by the appearance of hyperammonemia and, moreover, are associated with a reduction in the production of cyclic guanosine monophosphate (cGMP). Therefore, increased levels of pharmacologically controlled cGMP may be used as a therapeutic tool for improving learning and memory in patients with HE, hyperammonemia, cerebral oedema, or reduced intellectual capacity.

Transient coating of γ-Fe2O3 nanoparticles with glutamate for its delivery to and removal from brain nerve terminals.

Glutamate is the main excitatory neurotransmitter in the central nervous system and excessive extracellular glutamate concentration is a characteristic feature of stroke, brain trauma, and epilepsy. Also, glutamate is a potential tumor growth factor. Using radiolabeled ʟ-[14C]glutamate and magnetic fields, we developed an approach for monitoring the biomolecular coating (biocoating) with glutamate of the surface of maghemite (γ-Fe2O3) nanoparticles. The nanoparticles decreased the initial rate of ʟ-[14C]glutamate uptake, and increased the ambient level of ʟ-[14C]glutamate in isolated cortex nerve terminals (synaptosomes). The nanoparticles exhibit a high capability to adsorb glutamate/ʟ-[14C]glutamate in water. Some components of the incubation medium of nerve terminals, that is, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and NaH2PO4, decreased the ability of γ-Fe2O3 nanoparticles to form a glutamate biocoating by about 50% and 90%, respectively. Only 15% of the amount of glutamate biocoating obtained in water was obtained in blood plasma. Albumin did not prevent the formation of a glutamate biocoating. It was shown that the glutamate biocoating is a temporal dynamic structure at the surface of γ-Fe2O3 nanoparticles. Also, components of the nerve terminal incubation medium and physiological fluids responsible for the desorption of glutamate were identified. Glutamate-coated γ-Fe2O3 nanoparticles can be used for glutamate delivery to the nervous system or for glutamate adsorption (but with lower effectiveness) in stroke, brain trauma, epilepsy, and cancer treatment following by its subsequent removal using a magnetic field. γ-Fe2O3 nanoparticles with transient glutamate biocoating can be useful for multifunctional theranostics.

Abstract 3561: HIF-1α stabilisation in triple receptor negative breast cancer is sensitive to glutaminase inhibition

Abstract Increased expression of Hypoxia-Inducible Factor 1α (HIF-1α) in breast tumours is associated with poor survival outcome. HIF-1α promotes phenotypic adaptations including metabolic reorganisation, angiogenesis and increased cell invasiveness. Notably, HIF-1α activity is increased in Triple receptor Negative Breast Cancer (TNBC) compared with other breast cancer subtypes. One reason for this elevated HIF-1α stabilisation in TNBC has been described; intracellular cysteine depletion which leads to impaired prolyl hydroxylase-domain (PHD) enzyme activity. Normally, cystine is acquired in exchange for intracellular glutamate using the xCT antiporter. However, in TNBC the excessive secretion and extracellular accumulation of glutamate impairs cystine exchange. Since the majority of glutamate produced in TNBC is derived from glutaminase-catalysed hydrolysis of glutamine, we hypothesised that glutaminase inhibition using a potent and selective inhibitor, CB-839, would improve glutamate/cystine exchange and reduce HIF-1α levels. We detected normoxic HIF-1α expression in BT549, Hs578T and SUM159PT TNBC cell cultures. In these cases HIF-1α protein was destabilised upon treatment with CB-839 and the expression of some HIF-1α-target genes was significantly decreased (e.g. CA9, BNIP3, PDK1, LDHA) but not others (e.g. ADM and VEGFA). HIF-1α destabilisation corresponded with increased PHD-dependent hydroxylation, supporting the hypothesis that glutaminase inhibition can improve PHD function. Consistently, WT but not a proline hydroxylation-null mutant HIF-1α protein was depleted by CB-839. We confirmed that CB-839 decreased glutamate secretion but, unexpectedly, HIF-1α levels were not restabilised by addition of excess extracellular glutamate. Thus glutamine metabolism drives normoxic HIF-1α stabilisation via an additional mechanism distinct from perturbation of the glutamate/cystine exchange system. To elucidate the underlying process we focused on the glutamine-derived metabolite (S)-2-Hydroxyglutarate [(S)-2HG], a competitive inhibitor of 2-Oxoglutarate (2OG)-dependent enzymes, including the PHD enzymes. Glutaminase inhibition decreased the levels of (S)-2HG. Furthermore, growing CB-839-treated cells in culture medium containing cell permeable (S)-2HG, but not (R)-2HG, could restabilise HIF-1α. In addition, increasing 2OG levels in these cells further stabilised HIF-1α, an effect that was blunted by inhibition of lactate dehydrogenase A (LDHA), a promiscuous source of (S)-2HG generated via reduction of 2OG. Conclusion: CB-839 has demonstrated encouraging clinical responses in patients with heavily pretreated and therapy-resistant metastatic TNBC. Our work demonstrates that high levels of glutamine metabolism provoke HIF-1α stabilisation through the LDHA-catalysed production of (S)-2HG. Assessment of HIF-1α activity may be a useful approach to identify glutamine-addicted tumours and predict responsiveness to glutaminase inhibitor therapy. Citation Format: Laura Cussonneau, Anne-Lise Dechaume, Pamela M. Murray, Henegama Liyanage, Tet-Woo Lee, Frederik Pruijn, Euphemia Y. Leung, Dean C. Singleton. HIF-1α stabilisation in triple receptor negative breast cancer is sensitive to glutaminase inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3561.

Glutamate induced neonatal excitotoxicity modifies the expression level of EAAT1 (GLAST) and EAAT2 (GLT-1) proteins in various brain regions of the adult rat

Glutamate-mediated excitatory synaptic signalling is primarily controlled by excitatory amino acid transporters (EAATs), such as EAAT1 and EAAT2, which are located mostly on astrocytes and, together, uptake more than 95 % of extracellular glutamate. Alterations in the functional expression levels of EAATs can lead to excessive extracellular glutamate accumulation, potentially triggering excitotoxicity and seizures, among other neurological disorders. Excitotoxicity induced in early developmental stages can lead to lasting changes in several neurotransmission systems, including the glutamatergic system, which could make the brain more susceptible to a second insult. In this study, the expression levels of EAAT1 (GLAST) and EAAT2 (GLT-1) proteins were assessed in the cerebral motor cortex (CMC), striatum, hippocampus and entorhinal cortex (EC) of male adult rats following the neonatal excitotoxic process triggered by monosodium glutamate (MSG)-treatment (4 g/kg of body weight at postnatal days 1,3,5 and 7, subcutaneously). Western blot analysis showed that neonatal MSG-treatment decreased EAAT1 expression levels in the CMC, striatum and hippocampus, while EAAT2 levels were increased in the striatum and EC and decreased in the CMC. Immunofluorescence staining confirmed the changes in EAAT1 and EAAT2 expression induced by neonatal MSG-treatment, which were accompanied by an increase in the glial fibrillary acidic protein (GFAP) immunofluorescence signalthat was particularly significant in the hippocampus. Our results show that a neonatal excitotoxic processes can induce lasting changes in the expression levels of EAAT1 and EAAT2 proteins and suggest that although astrogliosis occurs, glutamate uptake could be deficient, particularly in the CMC and hippocampus.

Comprehensive Insight of Neurodegenerative Diseases and The Role of Neurotoxin Agents — Glutamate

The increased concentration of extracellular glutamate has been reported to play a key role in most of the neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease, even though its importance as an amino acid neurotransmitter in mammalian. Glutamate toxicity, which can be caused by excessive intake of monosodium glutamate (MSG), is the major contributor to pathological neuronal cell death. It causes neuronal dysfunction and degeneration in the central nervous system (CNS). Glutamate neurotoxicity can be categorized into two forms, which are receptor-mediated glutamate excitotoxicity and non-receptor mediated glutamate oxidative toxicity. The receptor-mediated glutamate excitotoxicity involved excessive stimulation of glutamate receptors (GluRs) which lead to excessive ion calcium (Ca2+) influx and activates a cell death cascade involving the accumulation of mitochondrially generated reactive oxygen species (ROS). Studies showed excessive extracellular glutamate leads to nerve cell death via the activation of N-methyl-Daspartate (NMDA) receptors in the cases of trauma or stroke. Whereas non-receptor mediated oxidative toxicity involved the breakdown of the cystine/glutamate antiporter (xc - ) mechanism, which leads to the depletion of glutathione (GSH) and causes oxidative stress and cell death. The cystine/glutamate antiporter couples the import of cystine to the export of glutamate. The increased concentration of extracellular glutamate could inhibit the uptake of cystine, which is required for the synthesis of the intracellular antioxidant GSH. GSH plays an important role in the disposal of peroxides by brain cells and in the protection against ROS. Depletion of GSH renders the cell to oxidative stress and ultimately leading to cell death. This article aims to provide a comprehensive review of neurodegenerative diseases and the role of neurotoxin agents, glutamate in these diseases.

Actions of Riluzole on Glast and GLT<sub>1</sub> Transporters in Rat Migraine Model

Riluzole exerts a neuroprotective effect through different mechanisms including action on glutamatergic transmission. We investigated whether this drug affect glutamate transporter mediated uptake expressing the rat glutamate transporters GLAST, GLT. We found that riluzole significantly increased glutamate uptake in a dose-dependent manner. This may facilitate the buffering of excessive extra cellular glutamate under pathological conditions suggesting that riluzole’s neuroprotective action might be mediated by its activating effect on glutamate uptake.

Glutamate in cancers: from metabolism to signaling.

Glutamine and glutamate are major bioenergy substrates for normal and cancer cell growth. Cancer cells need more biofuel than normal tissues for energy supply, anti-oxidation activity and biomass production. Genes related to metabolic chains in many cancers are somehow mutated, which makes cancer cells more glutamate dependent. Meanwhile, glutamate is an excitatory neurotransmitter for conducting signals through binding with different types of receptors in central neuron system. Interestingly, increasing evidences have shown involvement of glutamate signaling, guided through their receptors, in human malignancy. Dysregulation of glutamate transporters, such as excitatory amino acid transporter and cystine/glutamate antiporter system, also generates excessive extracellular glutamate, which in turn, activates glutamate receptors on cancer cells and results in malignant growth. These features make glutamate an attractive target for anti-cancer drug development with some glutamate targeted but blood brain barrier impermeable anti-psychosis drugs under consideration. We discussed the relevant progressions and drawbacks in this field herein.

Potassium and glutamate transport is impaired in scar-forming tumor-associated astrocytes

Unprovoked recurrent seizures are a serious comorbidity affecting most patients who suffer from glioma, a primary brain tumor composed of malignant glial cells. Cellular mechanisms contributing to the development of recurrent spontaneous seizures include the release of the excitatory neurotransmitter glutamate from glioma into extracellular space. Under physiological conditions, astrocytes express two high affinity glutamate transporters, Glt-1 and Glast, which are responsible for the removal of excess extracellular glutamate. In the context of neurological disease or brain injury, astrocytes become reactive which can negatively affect neuronal function, causing hyperexcitability and/or death. Using electrophysiology, immunohistochemistry, fluorescent in situ hybridization, and Western blot analysis in different orthotopic xenograft and allograft models of human and mouse gliomas, we find that peritumoral astrocytes exhibit astrocyte scar formation characterized by proliferation, cellular hypertrophy, process elongation, and increased GFAP and pSTAT3. Overall, peritumoral reactive astrocytes show a significant reduction in glutamate and potassium uptake, as well as decreased glutamine synthetase activity. A subset of peritumoral astrocytes displayed a depolarized resting membrane potential, further contributing to reduced potassium and glutamate homeostasis. These changes may contribute to the propagation of peritumoral neuronal hyperexcitability and excitotoxic death.

Epileptic Seizures: Glia-Neuron Interactions For Better or For Worse.

Investigations of the mechanisms generating epileptic seizures have primarily focused on neurons. However, more systemic research of brain circuits has highlighted an important role of non-neuronal cells such as glia in the genesis and spreading of generalized seizures in the brain.

Free-Energy Simulations Resolve the Low-Affinity Na+-High-Affinity Asp Binding Paradox in GltPh

Glutamate transporters clear up excess extracellular glutamate by cotransporting three Na+ and one H+ with the countertransport of one K+. The archaeal homologs are selective to aspartate and only cotransport three Na+. The crystal structures of GltPh from archaea have been used in computational studies to understand the transport mechanism. Although some progress has been made with regard to the ligand-binding sites, a consistent picture of transport still eludes us. A major concern is the discrepancy between the computed binding free energies, which predict high-affinity Na+-low-affinity aspartate binding, and the experimental results in which the opposite is observed. Here, we show that the binding of the first two Na+ ions involves an intermediate state near the Na1 site, where two Na+ ions coexist and couple to aspartate with similar strengths, boosting its affinity. Binding free energies for Na+ and aspartate obtained using this intermediate state are in good agreement with the experimental values. Thus, the paradox in binding affinities arises from the assumption that the ligands bind to the sites observed in the crystal structure following the order dictated by their binding free energies with no intermediate states. In fact, the presence of an intermediate state eliminates such a correlation between the binding free energies and the binding order. The intermediate state also facilitates transition of the first Na+ ion to its final binding site via a knock-on mechanism, which induces substantial conformational changes in the protein consistent with experimental observations.

A Novel and Translational Rat Model of Concussion Combining Force and Rotation with In Vivo Cerebral Microdialysis.

Persistent cognitive and motor symptoms are known consequences of concussions/mild traumatic brain injury (mTBIs) that can be partly attributable to altered neurotransmission. Indeed, cerebral microdialysis studies in rodents have demonstrated an excessive extracellular glutamate release in the hippocampus within the first 10 min following trauma. Microdialysis offers the clear advantage of in vivo neurotransmitter continuous sampling while not having to sacrifice the animal. In addition to the aforementioned technique, a closed head injury model that exerts rapid acceleration and deceleration of the head and torso is needed, as such a factor is not available in many other animal models. The Wayne State weight-drop model mimics this essential component of human craniocerebral trauma, allowing the induction of an impact on the head of an unrestrained rodent with a falling weight. Our novel and translational rat model combines cerebral microdialysis with the Wayne State weight-drop model to study, in lightly anesthetized and unrestrained adult rats, the acute changes in extracellular neurotransmitter levels following concussion. In this protocol, the microdialysis probe was inserted inside the hippocampus as region of interest, and was left inserted in the brain at impact. There is a high density of terminals and receptors in the hippocampus, making it a relevant region to document altered neurotransmission following concussion. When applied to adult Sprague-Dawley rats, our combined model induced increases in hippocampal extracellular glutamate concentrations within the first 10 min, consistent with the previously reported post-concussion symptomology. This combined weight-drop model provides a reliable tool for researchers to study early therapeutic responses to concussions in addition to repetitive brain injury, since this protocol induces a closed-head mild trauma.

Erratum: Neuroprotective Effects of Dammarane-Type Saponins from Panax notoginseng on Glutamate-Induced Cell Damage in PC12 Cells.

Dammarane-type saponins, the main active ingredients of Panax notoginseng, have substantial neuroprotective effects in different animal models of neurodegenerative diseases. However, because these compounds have different structures, the level of protection provided by individual compounds varies, and highly active compounds can be selected based on structure-activity relationships. Glutamate is a major excitatory neurotransmitter that plays an important role in synaptic response development. However, excessive extracellular glutamate levels lead to neuronal dysfunctions in the central nervous system. Herein, we investigated the neuroprotective effects of nine saponins (compounds 1 – 9) on glutamate-treated PC12 cells in the concentration range of 0.1 – 10 µM. The MTT assay revealed that these compounds increased cell viability to 65.6, 69.8, 76.9, 91.7, 74.4, 63.3, 59.9, 64.7, and 59.9%, respectively, compared with the glutamate-treated cells (44.6%). Protopanaxatriol (compound 4) was the most neuroprotective compound, and subsequent experiments revealed that pretreatment with compound 4 significantly reverses mitochondrial membrane potential collapse, increases superoxide dismutase activity, and decreases lactate dehydrogenase leakage, malondiadehyde levels, reactive oxygen species generation, and cell apoptosis. Compound 4 also decreased the Bax/Bcl-2 ratio, cleaved caspase-3, N-methyl-D-aspartic receptor 1, and Ca2+-/calmodulin-dependent protein kinase II expression, and inhibited glutamate-induced cytochrome C release and phosphorylation of apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase, and p38. Overall, the results indicate that protopanaxatriol has significant neuroprotective effects, and might be a promising neuroprotective agent for preventing and treating neurodegenerative diseases.

MiR-124 upregulates astrocytic glutamate transporter-1 via the Akt and mTOR signaling pathway post ischemic stroke

High-concentration glutamic acid (Glu) induced by ischemic stroke can be inhibited by glutamate transporter-1 (GLT-1), which is the main mechanism for preventing excessive extracellular glutamate accumulation in the central nervous system. Upregulation of miR-124 could reduce the infarct area and promote the recovery of neurological function after ischemic stroke. A previous study investigated whether miR-124 could regulate GLT-1 expression in normal culture conditions. However, the role of miR-124 in the regulation of GLT-1 expression and further mechanisms after ischemic stroke remain unclear. In this study, the effects of miR-124 on GLT-1 expression in astrocytes after ischemic stroke were explored using an in vitro model of ischemic stroke (oxygen-glucose deprivation/reperfusion, OGD/reperfusion). The expression of GLT-1 was significantly decreased with lower expression of miR-124 in astrocytes injured by OGD/reperfusion. When miR-124 expression was improved, the expression of GLT-1 was notably increased in astrocytes injured by OGD/reperfusion. The results revealed that GLT-1 expression in astrocytes had a relationship with miR-124 after OGD/reperfusion. However, a direct interaction could not be confirmed with a luciferase reporter assay. Further results demonstrated that an inhibitor of Akt could decrease the increased protein expression of GLT-1 induced by miR-124 mimics, and an inhibitor of mTOR could increase the reduced protein expression of GLT-1 caused by a miR-124 inhibitor in astrocytes injured by different OGD/reperfusion conditions. These results indicated that miR-124 could regulate GLT-1 expression in astrocytes after OGD/reperfusion through the Akt and mTOR pathway.

Neuroprotective Effects of Dammarane-Type Saponins from Panax notoginseng on Glutamate-Induced Cell Damage in PC12 Cells.

Dammarane-type saponins, the main active ingredients of Panax notoginseng, have substantial neuroprotective effects in different animal models of neurodegenerative diseases. However, because these compounds have different structures, the level of protection provided by individual compounds varies, and highly active compounds can be selected based on structure-activity relationships. Glutamate is a major excitatory neurotransmitter that plays an important role in synaptic response development. However, excessive extracellular glutamate levels lead to neuronal dysfunctions in the central nervous system. Herein, we investigated the neuroprotective effects of nine saponins (compounds 1: - 9: ) on glutamate-treated PC12 cells in the concentration range of 0.1 - 10 µM. The MTT assay revealed that these compounds increased cell viability to 65.6, 69.8, 76.9, 91.7, 74.4, 63.3, 59.9, 64.7, and 59.9%, respectively, compared with the glutamate-treated cells (44.6%). Protopanaxatriol (compound 4: ) was the most neuroprotective compound, and subsequent experiments revealed that pretreatment with compound 4: significantly reverses mitochondrial membrane potential collapse, increases superoxide dismutase activity, and decreases lactate dehydrogenase leakage, malondiadehyde levels, reactive oxygen species generation, and cell apoptosis. Compound 4: also decreased the Bax/Bcl-2 ratio, cleaved caspase-3, N-methyl-D-aspartic receptor 1, and Ca2+-/calmodulin-dependent protein kinase II expression, and inhibited glutamate-induced cytochrome C release and phosphorylation of apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase, and p38. Overall, the results indicate that protopanaxatriol has significant neuroprotective effects, and might be a promising neuroprotective agent for preventing and treating neurodegenerative diseases.