Mechanisms of glutamate and aspartate release in the ischemic rat cerebral cortex

Elevated levels of glutamate and aspartate have been implicated in the pathogenesis of neural injury and death induced by ischemia. The mechanism(s) whereby they escape into the extracellular environment have been a subject of controversy. This study evaluated the contribution of phospholipases and protein kinases to ischemia-evoked glutamate and aspartate release from the ischemic/reperfused rat cerebral cortex. Changes in the extracellular levels of these amino acids during four-vessel occlusion elicited global cerebral ischemia were examined using a cortical cup technique. Ischemia-evoked amino acid release was compared in control vs. drug treated animals, in which selective inhibitors of phospholipases and protein kinases were applied topically onto the cerebral cortex. The phospholipase inhibitors tested included 4-bromophenacyl bromide, a non-selective inhibitor; 7,7-dimethyleicosadienoic (DEDA), an inhibitor of secretory type phospholipase A₂ (PLA₂); AACOCF₃, an inhibitor of the Ca²⁺-dependent cytoplasmic form of PLA₂, HELSS, which inhibits a Ca²⁺-independent cytoplasmic PLA₂, and U73122, a selective inhibitor of phospholipase C (PLC). All five phospholipase inhibitors significantly attenuated glutamate and aspartate release into the extracellular milieu, indicating the possibility that several forms of the enzyme are likely to be involved. The protein kinase C (PKC) inhibitor, chelerythrine chloride, also reduced excitatory amino acid efflux, whereas the PKC activator phorbol 12-myristate 13-acetate (PMA) enhanced their release. The non-selective kinase inhibitor, staurosporine, and H-89, which selectively inhibits protein kinase A, did not reduce ischemia-evoked amino acid efflux. These results suggest that ischemia-evoked release of the excitatory transmitters amino acids is a result, in part, of the activation of phospholipases A₂ and C, with PKC involvement in the transduction process. Destabilization and deterioration of the plasma membrane, as a consequence of phospholipid hydrolysis, may allow these transmitter amino acids to diffuse down their concentration gradients into the extracellular fluid.     

Kappa-Opioid and NMDA glutamate receptors are differentially targeted within rat medial prefrontal cortex

Activation of kappa-opioid receptors (KOR) in the medial prefrontal cortex (mPFC) modulates excitatory transmission, which may involve interactions with N-methyl-D-aspartate (NMDA) glutamate receptors. We investigated possible anatomical correlates of this modulation by using dual labeling electron microscopy to examine the cellular distributions of antibodies raised against KOR and the R1 subunit of the NMDA receptor (NR1). KOR immunoreactivity primarily was localized to plasma and vesicular membranes of axons and axon terminals that were morphologically heterogeneous. A small proportion of KOR immunoreactivity was associated with cytosolic compartments of dendrites and membranes of glial processes. NR1 labeling was mainly postsynaptic, associated most often with membranes of cytoplasmic organelles in cell bodies and large dendrites and plasmalemmal surfaces of distal dendrites. The remaining NR1-labeled profiles were axonal profiles and glial processes. Of all cellular associations between labeled profiles, the majority were KOR-labeled axons that contacted NR1-immunoreactive dendrites or cell bodies. Occasionally the two antigens were colocalized in axon terminals that formed either asymmetric synapses or displayed varicose morphology. KOR and NR1 also were colocalized within dendrites, and rarely were observed in the same cell bodies. Occasionally glial processes coursing adjacent to axo-spinous appositions expressed both KOR and NR1 immunoreactivity. These results indicate that ligand activation of KOR or NMDA receptors differentially modulates excitatory transmission in the mPFC through pre- and postsynaptic mechanisms, respectively. The data also suggest more minor roles for colocalized KOR and NMDA receptors in shared regulation of presynaptic transmitter release, postsynaptic responsivity, and glial function.     

Blockade of glutamate reuptake in the rat nucleus accumbens increases locomotor activity

The effect on locomotor activity of blocking glutamate reuptake in the nucleus accumbens (NAcc) was investigated in the rat. Bilateral intracranial microinjections into the NAcc of the selective glutamate reuptake blocker, l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), were made in the freely moving rat and locomotor activity subsequently measured for 2 h. Different groups of rats injected with one of three doses of PDC (0.5, 5 or 10 nmole/0.5 microl/side) showed significant dose-dependent increases in both horizontal and vertical locomotor activity relative to control rats that received injections of the saline vehicle. These findings indicate that glutamate in the NAcc plays an important role in the production of locomotor behaviors.     

Normobaric hyperoxia induces ischemic tolerance and upregulation of glutamate transporters in the rat brain and serum TNF-alpha level

Recent studies suggest that intermittent and prolonged normobaric hyperoxia (HO) results in ischemic tolerance to reduce ischemic brain injury. In this research, we attempted to see changes in excitatory amino acid transporters (EAATs) and TNF-α levels following prolonged and intermittent hyperoxia preconditioning. Rats were divided into four experimental groups, each of 21 animals. The first two were exposed to 95% inspired HO for 4 h/day for 6 consecutive days (intermittent HO, InHO) or for 24 continuous hours (prolonged HO, PrHO). The second two groups acted as controls, and were exposed to 21% oxygen in the same chamber. Each main group was subdivided to middle cerebral artery occlusion (MCAO-operated), sham-operated (without MCAO), and intact (without any surgery) subgroups. After 24 h from pretreatment, MCAO-operated subgroups were subjected to 60 min of right MCAO. After 24 h reperfusion, neurologic deficit score (NDS) and infarct volume were measured in MCAO-operated subgroups. EAATs expression and serum TNF-α levels were assessed in sham-operated and intact subgroups. Preconditioning with prolonged and intermittent HO decreased NDS and upregulated EAAT1, EAAT2, and EAAT3 and increased serum TNF-α levels significantly. Although further studies are needed to clarify the mechanisms of ischemic tolerance, the intermittent and prolonged HO seems to partly exert their effects via increase serum TNF-α levels and upregulation of EAATs.     

Neuroprotective effects of the glutamate release inhibitor 619C89 in temporary middle cerebral artery occlusion

619C89 is a use-dependent Na⁺ channel antagonist that decreases the release of glutamate during ischemia. The efficacy of this drug in reducing infarction volume 72 h after occlusion of the middle cerebral artery (MCA) for 2 h in rats (n = 93) was determined by analysis of TTC-stained coronal section of the brain. Doses of 10, 20, 30 and 50 mg/kg of study drug given i.v. prior to MCA occlusion significantly (P < 0.05−0.01) reduced infarction volume in cortex compared to vehicle controls. Only the 50 mg/kg dose reduced infarction volume in the striatum (P < 0.05). Administration of 50 mg/kg of 619C89 30 and 60 min after the onset of ischemia reduced cortical infarction volume (P < 0.05), but there was no effect when the drug was given 5 min after reperfusion. No post-treatment regimen reduced striatal infarction volume. These results confirm the neuroprotective effects of 619C89 in temporary focal ischemia.     

Role of astrocytic glutamate transporter in alcohol use disorder

Alcohol use disorder (AUD) is one of the most widespread neuropsychiatric conditions, having a significant health and socioeconomic impact. According to the 2014 World Health Organization global status report on alcohol and health, the harmful use of alcohol is responsible for 5.9% of all deaths worldwide. Additionally, 5.1% of the global burden of disease and injury is ascribed to alcohol (measured in disability adjusted life years, or disability adjusted life years). Although the neurobiological basis of AUD is highly complex, the corticostriatal circuit contributes significantly to the development of addictive behaviors. In-depth investigation into the changes of the neurotransmitters in this circuit, dopamine, gamma-aminobutyricacid, and glutamate, and their corresponding neuronal receptors in AUD and other addictions enable us to understand the molecular basis of AUD. However, these discoveries have also revealed a dearth of knowledge regarding contributions from non-neuronal sources. Astrocytes, though intimately involved in synaptic function, had until recently been noticeably overlooked in their potential role in AUD. One major function of the astrocyte is protecting neurons from excitotoxicity by removing glutamate from the synapse via excitatory amino acid transporter type 2. The importance of this key transporter in addiction, as well as ethanol withdrawal, has recently become evident, though its regulation is still under investigation. Historically, pharmacotherapy for AUD has been focused on altering the activity of neuronal glutamate receptors. However, recent clinical evidence has supported the animal-based findings, showing that regulating glutamate homeostasis contributes to successful management of recovery from AUD.     

NMDA receptor activition by residual glutamate in glutamine preparations: a cautionary note regarding weak NMDA receptor agonists

During an investigation of excitatory amino acids on cultured embryonic Xenopus neurons, we observed that commercial preparations of glutamine had weal agonist activity on NMDA-type glutamate receptors. Threshold responses were observed at 100 μM glutamine, and the dose-response relation did not show inflection or saturation at concentrations of up to 10 mM. However, NMDA receptor activation induced by glutamine probably represented activity of residual glutamate because: (a) recrystallization of glutamine reduced residual glutamate levels (measured by HPLC analysis) and NMDA receptor activation by comparable amounts; and (b) glutamate at concentrations close to those predicted to be present in glutamine preparations elicited currents of similar amplitudes. Our data indicate that residual glutamate at levels of less than 0.05% are sufficient to confound studies of weak NMDA receptor agonists.     

应激对大鼠海马谷氨酸、天冬氨酸和γ-氨基丁酸含量的影响

目的　探讨应激对大鼠海马谷氨酸、天冬氨酸和γ 氨基丁酸 (GABA)含量的动态影响。方法　将 72只健康雄性大鼠随机分为 5个应激暴露不同时间组和对照组 ,每组 12只。利用高效液相色谱仪 紫外检测法 ,分别于应激第 1,3,7,14和 2 8天观察应激对大鼠海马谷氨酸、天冬氨酸及GABA含量的影响。结果　应激第 1天组大鼠海马谷氨酸和天冬氨酸含量与对照组相比 ,差异无显著性 ;但GABA含量 [(2 74 7± 0 339) μmol/g]低于对照组 [(3 719± 0 5 2 8) μmol/g;P <0 0 5 ]。应激第 3,7,14和 2 8天组谷氨酸含量 [分别为 (7 818± 0 799) μmol/g ,(9 0 0 7± 0 5 2 0 ) μmol/g,(8 0 4 9± 0 733) μmol/g和 (8 12 9± 1 5 5 6 ) μmol/g]高于对照组 [(6 4 11± 0 6 38) μmol/g];天冬氨酸含量 [分别为 (2 717± 0 2 5 8)μmol/g,(2 6 96± 0 317) μmol/g,(2 82 8± 0 4 6 8) μmol/g和 (4 6 4 9± 0 6 37) μmol/g]也高于对照组 [(2 0 0 3± 0 2 71) μmol/g];均P <0 0 1。应激第 14天组和 2 8天组GABA含量 [分别为 (4 4 6 2± 0 883) μmol/g和(4 4 97± 0 85 7) μmol/g]高于对照组 (P <0 0 5～0 .0 0 1) ,应激第 3天组和 7天组的GABA含量与对照组间的差异无显著性。结论　应激第 3天开始     

Focal reduction of neuronal glutamate transporters in human neocortical epilepsy

PURPOSE: To study the differential expression of excitatory amino acid transporters (EAATs) at localized epileptic foci compared to nonepileptic regions in human neocortical epilepsy. Decreased expression of EAATs, the predominant mechanism to remove synaptic-released glutamate, may explain mechanisms of heightened excitability at these epileptic foci. METHODS: The differential expression of EAAT1-4 at the mRNA and protein levels was measured in electrically mapped human neocortical tissues using quantitative real-time PCR and immunoblotting. This required a novel way to prevent aggregation of EAAT proteins through cold solubilization. Layer-specific neuronal densities were measured to control for potential differences in neuronal density. RESULTS: While focal epileptic brain regions show marked increases in immediate early genes, they have significant reductions in the neuronal glutamate transporter mRNAs (EAAT3 and EAAT4). These changes were not associated with changes in relative neuronal density, suggesting a reduction in EAAT mRNA per neuron. Immunohistochemical staining of epileptic human neocortex confirmed the presence of EAAT1 and EAAT2 proteins in astroglial cells and EAAT3 and EAAT4 proteins in human cortical neurons. At the protein level, western blots of the same epileptic and nonepileptic regions for a subset of these patients showed a similar decrease of EAAT3 and EAAT4. Despite no change in EAAT2 mRNA, EAAT2 protein expression was significantly reduced at epileptic foci. CONCLUSION: Regional reductions in EAAT expression at human neocortical epileptic foci could produce increased local glutamate levels that in turn may contribute to both hyperexcitability and the spontaneous generation of epileptic discharges that characterize human epileptic foci.     

Dynamic expression of cerebral cortex and hippocampal glutamate transporters in a rat model of chest compression-induced global cerebral ischemia

The present study established a rat model of global cerebral ischemia induced by chest compression for six minutes to dynamically observe expressional changes of three glutamate transporters in the cerebral cortex and hippocampus. After 24 hours of ischemia, expression of glutamate transporter-1 significantly decreased in the cerebral cortex and hippocampus, which was accompanied by neuronal necrosis. At 7 days post-ischemia, expression of excitatory amino acid carrier 1 decreased in the hippocampal CA1 region and cortex, and was accompanied by apoptosis. Expression of glutamate-aspartate transporter remained unchanged at 6 hours-7 days after ischemia. These results suggested that glutamate transporter levels were altered at different periods of cerebral ischemia.     

Molecular pharmacology of glutamate transporters, EAATs and VGLUTs

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

Immediate and delayed effects of in vitro ischemia on glutamate efflux from guinea-pig cerebral cortex slices

Immediate and delayed effects of glucose deprivation, oxygen deprivation (hypoxia) and both oxygen and glucose deprivation (in vitro ischemia) on glutamate efflux from guinea pig cerebral cortex slices were studied. Immediate effects were evaluated by measuring changes of glutamate efflux during the metabolic insults. Delayed effects were evaluated by measuring the response of the tissue to a 50 mM KCl pulse applied 60 minafter the metabolic insults. Deprivation of glucose in the medium did not induce either immediate or delayed effects, while hypoxic condition produced an immediate slight stimulation of glutamate efflux without any delayed effect. Conversely, in vitro ischemia produced both immediate and delayed effects on glutamate efflux. During in vitro ischemia glutamate efflux dramatically increased in a calcium-independent and tetrodotoxin-sensitive manner; this effect was potentiated by a low sodium containing medium. The blockade of the sodium/potassium ATP_ase exchanger by ouabain caused a glutamate outflow similar to that induced by in vitro ischemia. On the whole, these data demonstrate the central role played by the sodium electrochemical gradient and by the membrane glutamate uptake system in the glutamate overflow induced by in vitro ischemia. Moreover, in slices previously exposed to both oxygen and glucose deprivation the effect of KCl on glutamate efflux was potentiated. This in vitro ischemia-induced delayed potentiation of neurotransmitter efflux, until now unreported in the literature, was found to be selectively restricted to glutamatergic structures and to be mainly due to an enhancement of the exocytotic component of glutamate release. © 1997 Elsevier Science B.V. All rights reserved.     

Effect of upregulation of AMPA glutamate receptors on cerebral O2 consumption and blood flow in rat

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptors, in cerebral cortex, underwent upregulation (35% increase) following chronic blockade with a non-competitive AMPA receptor antagonist, GYKI 52466 (1-(aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2,3-benzodiazepine). Such upregulation did not alter basal cerebrocortical blood flow or O(2) consumption. There was a much higher increase in blood flow and O(2) consumption in the upregulated, agonist (AMPA) stimulated cortices of anesthetized rats.     

Release of cholecystokinin from rat cerebral cortex in vivo: role of GABA and glutamate receptor systems

Using cortical cups in chloralose-urethanized rats, the in vivo release of cholecystokinin-like immunoreactivity (CCK-LI) from cerebral cortex was examined. Resting levels of cholecystokinin-like immunoreactivity ranged from 20 to 30 pg/20 min sample. The addition of potassium (40 mM) in excess, resulted in a highly significant elevation in the levels of CCK-LI in the cortical superfusate. Deletion of calcium and the substitution of cobalt (10 mM), resulted in a significant reduction in both resting release and the release otherwise evoked by the addition of potassium. Focal electrical stimulation of the cortex (20 Hz), resulted in a significant (1.9 +/- 0.2-fold, n = 8) increase in the levels of CCK-LI. The addition of glutamate (10(-6)-10(-4) M) of kainic acid (10(-8)-10(-6) M), also resulted in significant elevations in the levels of CCK-LI. The co-administration of a putative glutamate receptor antagonist, kynurenic acid (10(-4) M) resulted in a significant reduction in the levels of release otherwise evoked by the addition of glutamate, but not by electrical stimulation. The addition of GABA (10(-5)-10(-3) M) resulted in a dose-dependent decrease in the resting release of CCK-LI, and the release evoked by glutamate. Picrotoxin (10(-6)-10(-4) M), resulted in a highly significant increase in the levels of CCK-LI in the cortical effluent. These results are consistent with a tonic GABAergic inhibition of CCK-releasing neurons. The treatment of the animal with diazepam (30 mg/kg, i.p.) also resulted in a significant reduction in resting release and the release otherwise evoked by focal cortical stimulation.     

Indomethacin modulates ischemia-evoked release of glutamate and adenosine from the rat cerebral cortex

The effects of indomethacin (10 mg/kg) on the release of the transmitter amino acids, glutamate, aspartate, GABA, and of the purines, adenosine and inosine, from the cerebral cortex was studied in a four-vessel occlusion rat model of cerebral ischemia/reperfusion. In comparison with the control group, indomethacin significantly attenuated the ischemia-evoked release of glutamate and aspartate, but not of GABA. Adenosine levels in the cortical superfusates were significantly elevated following indomethacin administration. As indomethacin is a potent inhibitor of adenosine uptake, these results suggest that, by blocking adenosine uptake, indomethacin could elevate extracellular adenosine levels and depress glutamate and asparte efflux as a consequence of the activation of adenosine A₁ receptors.     

Local anesthetics inhibit glutamate release from rat cerebral cortex synaptosomes

Local anesthetics have been widely used for regional anesthesia and the treatment of cardiac arrhythmias. Recent studies have also demonstrated that low‐dose systemic local anesthetic infusion has neuroprotective properties. Considering the fact that excessive glutamate release can cause neuronal excitotoxicity, we investigated whether local anesthetics might influence glutamate release from rat cerebral cortex nerve terminals (synaptosomes). Results showed that two commonly used local anesthetics, lidocaine and bupivacaine, exhibited a dose‐dependent inhibition of 4‐AP‐evoked release of glutamate. The effects of lidocaine or bupivacaine on the evoked glutamate release were prevented by the chelation of extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl ‐threo‐beta‐benzyl‐oxyaspartate did not have any effect on the action of lidocaine or bupivacaine. Both lidocaine and bupivacaine reduced the depolarization‐induced increase in [Ca²⁺]_C but did not alter 4‐AP‐mediated depolarization. Furthermore, the inhibitory effect of lidocaine or bupivacaine on evoked glutamate release was prevented by blocking the Ca_v2.2 (N‐type) and Ca_v2.1 (P/Q‐type) channels, but it was not affected by blocking of the ryanodine receptors or the mitochondrial Na⁺/Ca²⁺ exchange. Inhibition of protein kinase C (PKC) and protein kinase A (PKA) also prevented the action of lidocaine or bupivacaine. These results show that local anesthetics inhibit glutamate release from rat cortical nerve terminals. This effect is linked to a decrease in [Ca²⁺]_C caused by Ca²⁺ entry through presynaptic voltage‐dependent Ca²⁺ channels and the suppression of the PKA and PKC signaling cascades. Synapse 67:568–579, 2013 . © 2013 Wiley Periodicals, Inc.     

Effect of repeated methamphetamine administrations on dopamine and glutamate efflux in rat prefrontal cortex

Pretreatment with psychostimulants such as methamphetamine (METH) results in augmented mesostriatal dopamine transmission upon a challenge administration of the drug. This effect can be blocked by dopamine antagonists and excitatory amino acid antagonists. However, no direct comparisons have been made with respect to the effects of a low-dose pretreatment regimen of METH on impulse and transporter-mediated dopamine release or to what extent glutamate release is altered by a pretreatment regimen with METH. The purpose of this study was to examine dopamine and glutamate efflux in the prefrontal cortex and striatum in rats pretreated with METH following either high potassium (80 mM) infusion or after a systemic injection of a low dose of METH. Extracellular dopamine and glutamate concentrations in the prefrontal cortex and striatum were measured in vivo by microdialysis. Potassium infusion increased extracellular dopamine and glutamate concentrations to a greater extent in the prefrontal cortex than in the striatum of METH-pretreated rats compared to saline-pretreated controls. A low dose METH challenge significantly increased extracellular dopamine but not glutamate concentrations in both prefrontal cortex and striatum of all animals. Moreover, the acute METH-induced increase in cortical dopamine efflux was significantly greater in rats pretreated with METH. Overall, these data are the first evidence that repeated METH administrations can enhance cortical glutamate efflux and indicate that a low dose pretreatment regimen of METH enhances dopamine transmission in the prefrontal cortex through both transporter and depolarization-induced mechanisms.     

Ceftriaxone-mediated upregulation of the glutamate transporter GLT-1 contrasts neurotoxicity evoked by kainate in rat organotypic spinal cord cultures

Excitotoxicity is a major pathological trigger of neurodegenerative diseases like amyotrophic lateral sclerosis. This process is caused by excessive release of the transmitter glutamate that overwhelms the capacity of astroglia transporters to maintain a low extracellular level of this aminoacid and strongly stimulates neurons. Using an in vitro model of rat organotypic spinal slice culture, we explored if the excitotoxicity caused by the potent glutamate analogue kainate, widely employed as a paradigm to evoke neurotoxicity in the central nervous system, was prevented by the antibiotic ceftriaxone known to enhance glutamate transporter expression. We also tested if excitotoxicity was made worse by inhibiting glutamate uptake with dl-threo-β-benzyloxyaspartate (TBOA). These experiments were aimed at clarifying the relative contribution to neurotoxicity by kainate-activation of glutamate receptors or kainate-mediated release of glutamate. Neither ceftriaxone nor TBOA alone had adverse effects. Ceftriaxone (10 μM; 3 days) significantly decreased delayed cell death induced by kainate (100 μM; 1 h) and limited neuronal damage especially to motoneurons. This effect was associated to stronger astrocytic immunostaining of the glutamate transporter GLT-1. Conversely, pharmacological inhibition of glutamate uptake with TBOA was per se unable to induce neurotoxicity, yet it intensified cell death evoked by kainate. These data indicate that kainate-mediated glutamate release was critical to damage neurons, an effect prevented by up regulating glutamate uptake. These data suggest that modulating glutamate uptake is an important strategy to preserve neuronal networks.