Increase In Extracellular Glutamate Concentration Research Articles

Increase of glutamate in satellite glial cells of the trigeminal ganglion in a rat model of craniofacial neuropathic pain.

Satellite glial cells (SGCs) that envelop the cell bodies of neurons in sensory ganglia have been shown to both release glutamate, and be activated by glutamate in the context of nociceptive signaling. However, little is known about the subpopulations of SGCs that are activated following nerve injury and whether glutamate mechanisms in the SGCs are involved in the pathologic pain. To address this issue, we used light and electron microscopic immunohistochemistry to examine the change in the glutamate levels in the SGCs and the structural relationship between neighboring neurons in the trigeminal ganglion (TG) in a rat model of craniofacial neuropathic pain, CCI-ION. Administration of ionomycin, ATP and Bz-ATP induced an increase of extracellular glutamate concentration in cultured trigeminal SGCs, indicating a release of glutamate from SGCs. The level of glutamate immunostaining in the SGCs that envelop neurons of all sizes in the TG was significantly higher in rats with CCI-ION than in control rats, suggesting that SGCs enveloping nociceptive as well as non-nociceptive mechanosensitive neurons are activated following nerve injury, and that the glutamate release from SGCs increases in pathologic pain state. Close appositions between substance-P (SP)-immunopositive (+) or calcitonin gene-related peptide (CGRP)+, likely nociceptive neurons, between Piezo1+, likely non-nociceptive, mechanosensitive neurons and SP+ or CGRP+ neurons, and between SGCs of neighboring neurons were frequently observed. These findings suggest that glutamate in the trigeminal SGCs that envelop all types of neurons may play a role in the mechanisms of neuropathic pain, possibly via paracrine signaling.

The Computer Simulation of Therapy with the NMDA Antagonist in Excitotoxic Neurodegeneration in an Alzheimer's Disease-like Pathology.

(1) Background: The use of uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists results in neuroprotective benefits in patients with moderate to severe Alzheimer’s disease. In this study, we demonstrated mathematical and computer modelling of the excitotoxicity phenomenon and performed virtual memantine therapy. (2) Methods: A computer simulation environment of the N-methyl-D-aspartate receptor combining biological mechanisms of channel activation by means of excessive extracellular glutamic acid concentration in three models of excitotoxicity severity. The simulation model is based on sliding register tables, where each table is associated with corresponding synaptic inputs. Modelling of the increase in extracellular glutamate concentration, through over-stimulation of NMDA receptors and exacerbation of excitotoxicity, is performed by gradually increasing the parameters of phenomenological events by the power function. Pathological models were virtually treated with 3–30 µM doses of memantine compared to controls. (3) Results: The virtual therapy results of memantine at doses of 3–30 µM in the pathological models of excitotoxicity severity show statistically significant neuroprotective benefits in AD patients with moderate severity, 1.25 (95% CI, 1.18–1.32) vs. 1.76 (95% CI, 1.71–1.80) vs. 1.53 (95% CI, 1.48–1.59), (p < 0.001), to severe, 1.32 (95% CI, 1.12–1.53) vs. 1.77 (95% CI, 1.72–1.82) vs. 1.73 (95% CI, 1.68–1.79), (p < 0.001), in the area of effects on memory. A statistically significant benefit of memantine was demonstrated for all neuronal parameters in pathological models. In the mild severity model, a statistically significant increase in frequency was obtained relative to virtual memantine treatment with a dose of 3 µM, which was 23.5 Hz (95% CI, 15.5–28.4) vs. 38.8 Hz (95% CI, 34.0–43.6), (p < 0.0001). In the intermediate excitotoxicity severity model, a statistically significant increase in frequency was obtained relative to virtual memantine therapy with a 3 µM dose of 26.0 Hz (95% CI, 15.7–36.2) vs. 39.0 Hz (95% CI, 34.2–43.8) and a 10 µM dose of 26.0 Hz (95% CI, 15.7–36.2) vs. 30.9 Hz (95% CI, 26.4–35.4), (p < 0.0001). A statistically significant increase in frequency was obtained in the advanced excitotoxicity severity model as in the medium. (4) Conclusions: The NMDA antagonist memantine causes neuroprotective benefits in patients with moderate to severe AD. One of the most important benefits of memantine is the improvement of cognitive function and beneficial effects on memory. On the other hand, memantine provides only symptomatic and temporary support for AD patients. Memantine is prescribed in the US and Europe if a patient has moderate to severe AD. Memantine has also been approved for mild to moderate AD patients. However, its very modest effect provides motivation for further research into new drugs in AD. We are the first to present a mathematical model of the NMDA receptor that allows the simulation of excitotoxicity and virtual memantine therapy.

Extracellular Glutamate Concentration Increases Linearly in Proportion to Decreases in Residual Cerebral Blood Flow After the Loss of Membrane Potential in a Rat Model of Ischemia.

Brain ischemia due to disruption of cerebral blood flow (CBF) results in increases in extracellular glutamate concentration and neuronal cell damage. However, the impact of CBF on glutamate dynamics after the loss of the membrane potential remains unknown. To determine this impact, we measured extracellular potential, CBF, and extracellular glutamate concentration in the parietal cortex in male Sprague-Dawley rats (n=21). CBF was reduced by bilateral occlusion of the common carotid arteries and exsanguination until loss of extracellular membrane potential was observed (low-flow group), or until CBF was further reduced by 5% to 10% of preischemia levels (severe-low-flow group). CBF was promptly restored 10 minutes after the loss of membrane potential. Histologic outcomes were evaluated 5 days later. Extracellular glutamate concentration in the low-flow group was significantly lower than that in the severe-low-flow group. Moreover, increases in extracellular glutamate concentration exhibited a linear relationship with decreases in CBF after the loss of membrane potential in the severe-low-flow group, and the percentage of damaged neurons exhibited a dose-response relationship with the extracellular glutamate concentration. The extracellular glutamate concentration required to cause 50% neuronal damage was estimated to be 387 μmol/L, at 8.7% of preischemia CBF. Regression analyses revealed that extracellular glutamate concentration increased by 21 μmol/L with each 1% decrease in residual CBF and that the percentage of damaged neurons increased by 2.6%. Our results indicate that residual CBF is an important factor that determines the extracellular glutamate concentration after the loss of membrane potential, and residual CBF would be one of the important determinants of neuronal cell prognosis.

GLT-1 mediates exercise-induced fatigue through modulation of glutamate and lactate in rats.

Glutamate has been implicated in exercise-induced fatigue, but the underlying mechanism is unknown. This study aimed to determine whether glutamate transporter-1 (GLT-1) has a key role in the regulation of exercise-induced fatigue. The expressions of GLT-1 and glial fibrillary acidic protein (GFAP) in the supplementary motor area of rats exposed to exhaustion were analyzed by immunohistochemistry. The expression of GLT-1 was further confirmed by Western blotting. The effects of GLT-1 on extracellular levels of glutamate and lactate and on exercise endurance were studied by microdialysis. GLT-1 expression was decreased in rats subjected to exercise-induced fatigue. Functional inhibition of GLT-1 using dihydrokainate and transcriptional inhibition of GLT-1 using antisense oligodeoxynucleotides led to a decrease in exercise endurance with a subsequent increase in extracellular glutamate concentration and decrease in extracellular lactate concentration. The expression of GLT-1 in the supplementary motor area is reduced after strenuous exercise, resulting in an increased extracellular glutamate concentration and decreased extracellular lactate level that may be responsible for the development of fatigue. GLT-1-mediated uptake of glutamate ameliorates exercise-induced fatigue in rat models.

Behavioral changes after nicotine challenge are associated with \u03b17 nicotinic acetylcholine receptor-stimulated glutamate release in the rat dorsal striatum

Neurochemical alterations associated with behavioral responses induced by re-exposure to nicotine have not been sufficiently characterized in the dorsal striatum. Herein, we report on changes in glutamate concentrations in the rat dorsal striatum associated with behavioral alterations after nicotine challenge. Nicotine challenge (0.4 mg/kg/day, subcutaneous) significantly increased extracellular glutamate concentrations up to the level observed with repeated nicotine administration. This increase occurred in parallel with an increase in behavioral changes in locomotor and rearing activities. In contrast, acute nicotine administration and nicotine withdrawal on days 1 and 6 did not alter glutamate levels or behavioral changes. Blockade of α7 nicotinic acetylcholine receptors (nAChRs) significantly decreased the nicotine challenge-induced increases in extracellular glutamate concentrations and locomotor and rearing activities. These findings suggest that behavioral changes in locomotor and rearing activities after re-exposure to nicotine are closely associated with hyperactivation of the glutamate response by stimulating α7 nAChRs in the rat dorsal striatum.

Effects of Ceftriaxone on Glial Glutamate Transporters in Wistar Rats Administered Sequential Ethanol and Methamphetamine.

Methamphetamine (METH) is one of the psychostimulants that is co-abused with ethanol. Repeated exposure to high dose of METH has been shown to cause increases in extracellular glutamate concentration. We have recently reported that ethanol exposure can also increase the extracellular glutamate concentration and downregulate the expression of glutamate transporter subtype 1 (GLT-1). GLT-1 is a glial transporter that regulates the majority of extracellular glutamate. A Wistar rat model of METH and ethanol co-abuse was used to examine the expression of GLT-1 as well as other glutamate transporters such as cystine/glutamate exchanger (xCT) and glutamate aspartate transporter (GLAST). We also examined the body temperature in rats administered METH, ethanol or both drugs. We further investigated the effects of ceftriaxone (CEF), a β-lactam antibiotic known to upregulate GLT-1, in this METH/ethanol co-abuse rat model. After 7 days of either ethanol (6 g/kg) or water oral gavage, Wistar rats received either saline or METH (10 mg/kg i.p. every 2 h × 4), followed by either saline or CEF (200 mg/kg) posttreatment. METH administered alone decreased GLT-1 expression in the nucleus accumbens (NAc) and prefrontal cortex (PFC) and increased body temperature, but did not reduce either xCT or GLAST expression in ethanol and water-pretreated rats. Interestingly, ethanol and METH were found to have an additive effect on the downregulation of GLT-1 expression in the NAc but not in the PFC. Moreover, ethanol alone caused GLT-1 downregulation in the NAc and elevated body temperature compared to control. Finally, CEF posttreatment significantly reversed METH-induced hyperthermia, restored GLT-1 expression, and increased xCT expression. These findings suggest the potential therapeutic role of CEF against METH- or ethanol/METH-induced hyperglutamatergic state and hyperthermia.

Collapsin Response Mediator Protein 2 (CRMP2) Interacts with N-Methyl-d-aspartate (NMDA) Receptor and Na+/Ca2+ Exchanger and Regulates Their Functional Activity

Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-d-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca(2+) dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, TAT-CBD3, but not CBD3 without TAT or TAT-scramble peptide, inhibited increases in cytosolic Ca(2+) mediated by the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. TAT-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, TAT-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca(2+) influx into neurons, was strongly suppressed by TAT-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to TAT-CBD3 exposure. Moreover, CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that TAT-CBD3 protects against glutamate-induced Ca(2+) dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of TAT-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.

Chronic ceftriaxone treatment rescues hippocampal memory deficit in AQP4 knockout mice via activation of GLT-1

Aquaporin-4 (AQP4) is the predominant water channel protein in the mammalian brain, and is mainly expressed in astrocytes. Besides its important role in water transport across the blood–brain barrier, our present study demonstrated that AQP4 deficiency impaired hippocampal long-term potentiation (LTP) and hippocampus-dependent memory formation, accompanied by the increase in extracellular glutamate concentration and N-methyl-d-aspartate (NMDA) receptor-mediated currents in hippocampal dentate gyrus (DG) region. The impairment of LTP and memory formation of AQP4 knockout (KO) mice was mediated by the downregulation of glutamate transporter-1 (GLT-1) expression/function, since it can be rescued by β-lactam antibiotic ceftriaxone (Cef), a potent GLT-1 stimulator. These results suggest that AQP4 functions as the modulator of synaptic plasticity and memory, and chronic Cef treatment rescues hippocampal memory deficit induced by AQP4 knockout.

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

Glutamate concentration at the synaptic level must be kept low in order to prevent excitotoxicity. Astrocytes play a key role in brain energetics, and also astrocytic glutamate transporters are responsible for the vast majority of glutamate uptake in CNS. Experiments with primary astrocytic cultures suggest that increased influx of glutamate cotransported with sodium at astrocytes favors its flux to the tricarboxylic acid cycle instead of the glutamate-glutamine cycle. Although metabolic coupling can be considered an emergent field of research with important recent discoveries, some basic aspects of glutamate metabolism still have not been characterized in brain tissue. Therefore, the aim of this study was to investigate whether the presence of extracellular glutamate is able to modulate the use of glutamate and glucose as energetic substrates. For this purpose, isolated hippocampi of mice were incubated with radiolabeled substrates, and CO2 radioactivity and extracellular lactate were measured. Our results point to a diminished oxidation of glucose with increasing extracellular glutamate concentration, glutamate presumably being the fuel, and might suggest that oxidation of glutamate could buffer excitotoxic conditions by high glutamate concentrations. In addition, these findings were reversed when glutamate uptake by astrocytes was impaired by the presence of (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-L-aspartic acid (TFB-TBOA). Taken together, our findings argue against the lactate shuttle theory, because glutamate did not cause any detectable increase in extracellular lactate content (or, presumably, in glycolysis), because the glutamate is being used as fuel instead of going to glutamine and back to neurons.

Glutaminase Dysregulation in HIV-1-Infected Human Microglia Mediates Neurotoxicity: Relevant to HIV-1-Associated Neurocognitive Disorders

Microglia represent the main cellular targets of HIV-1 in the brain. Infected and/or activated microglia play a pathogenic role in HIV-associated neurocognitive disorders (HAND) by instigating primary dysfunction and subsequent death of neurons. Although microglia are known to secrete neurotoxins when infected with HIV-1, the detailed mechanism of neurotoxicity remains unclear. Using a human microglia primary culture system and macrophage-tropic HIV-1 strains, we have now demonstrated that HIV-1 infection of microglia resulted in a significant increase in extracellular glutamate concentrations and elevated levels of neurotoxicity. RNA and protein analysis revealed upregulation of the glutamate-generating enzyme glutaminase isoform glutaminase C in HIV-1-infected microglia. The clinical relevance of these findings was further corroborated with investigation of postmortem brain tissues. The glutaminase C levels in the brain tissues of HIV dementia individuals were significantly higher than HIV serum-negative control and correlated with elevated concentrations of glutamate. When glutaminase was subsequently inhibited by siRNA or by a small molecular inhibitor, the HIV-induced glutamate production and the neuronal loss was diminished. In conclusion, these findings support glutaminase as a potential component of the HAND pathogenic process as well as a novel therapeutic target in their treatment.

Effect of paliperidone and risperidone on extracellular glutamate in the prefrontal cortex of rats exposed to prenatal immune activation or MK-801

The NMDA glutamate hypofunction model of schizophrenia is based in part upon acute effects of NMDA receptor blockade in humans and rodents. Several laboratories have reported glutamate system abnormalities following prenatal exposure to immune challenge, a known environmental risk factor for schizophrenia. Here we report indices of NMDA glutamate receptor hypofunction following prenatal immune activation, as well as the effects of treatment during periadolescence with the atypical antipsychotic medications risperidone and paliperidone. Pregnant Sprague-Dawley rats were injected with polyinosinic:polycytidylic acid (poly I:C) or saline on gestational day 14. Male offspring were treated orally via drinking water with vehicle, risperidone (0.01 mg/kg/day), or paliperidone (0.01 mg/kg/day) between postnatal days 35 and 56 (periadolescence) and extracellular glutamate levels in the prefrontal cortex were determined by microdialysis at PD 56. Consistent with decreased NMDA receptor function, MK-801-induced increases in extracellular glutamate concentration were markedly blunted following prenatal immune activation. Further suggesting NMDA receptor hypofunction, prefrontal cortex basal extracellular glutamate was significantly elevated ( p < 0.05) in offspring of poly I:C treated dams. Pretreatment with low dose paliperidone or risperidone (0.01 mg/kg/day postnatal days 35–56) normalized prefrontal cortical basal extracellular glutamate ( p < 0.05 vs. poly I:C vehicle-treatment). Pretreatment with paliperidone and risperidone also prevented the acute MK-801-induced increase in extracellular glutamate. These observations demonstrate decreased NMDA receptor function and elevated extracellular glutamate, two key features of the NMDA glutamate receptor hypofunction model of schizophrenia, during periadolescence following prenatal immune activation. Treatment with the atypical antipsychotic medications paliperidone and risperidone normalized basal extracellular glutamate. Demonstration of glutamatergic abnormalities consistent with the NMDA glutamate receptor hypofunction model of schizophrenia as an early developmental consequence of prenatal immune action provides a model to identify novel early interventions targeting glutamatergic systems which play an important role in both positive and negative symptoms of schizophrenia.

High K+-induced Increase in Extracellular Glutamate in Zinc Deficiency and Endogenous Zinc Action

To understand the physiological significance of endogenous zinc release under excess excitation in the hippocampus, in the present study, high K + -induced increase in extracellular glutamate and endogenous zinc action against its increase were examined in young rats fed a zinc-deficient diet for 2 weeks. When the ventral hippocampus was perfused by 100 mM KCl, the extracellular concentration of glutamate was more increased in zinc-deficient rats than the control rats. Calcium orange signal in mossy fiber boutons was more increased in slices from zincdeficient rats after delivery of tetanic stimuli (100 Hz, 5 sec) to the dentate granule cell layer. The decrease in FM4-64 signal, which is a measurement of exocytosis, in mossy fiber boutons was also enhanced in slices from zinc-deficient rats. These results suggest that the abnormal increase in extracellular glutamate in the hippocampus induced with high K + in zinc deficiency is due to th ee nhancement of exocytosis associated with affected Ca 2+ mobilization. The expression of GLT-1, a glial glutamate transporter, in the hippocampus was higher in zincdeficient rats, suggesting that GLT-1 protein increased serves to maintain the basal concentration of extracellular glutamate, which was not different between the control and zinc-deficient rats. On the other hand, the increase in extracellular glutamate concentration induced with high K + was enhanced in the presence of 1 mM CaEDTA, a membrane-impermeable zinc chelator, in both the control and zinc-deficient rats. It is likely that zinc released from glutamatergic neurons serves to suppress glutamate release under excess excitation in the hippocampus.

Endothelin-1 reverses the histone deacetylase inhibitor-induced increase in glial glutamate transporter transcription without affecting histone acetylation levels

Astrocytes play a crucial role in maintaining glutamate homeostasis in the intact central nervous system. This function is profoundly impaired in the acutely and chronically diseased brain as evidenced by the decreased expression of the glial glutamate transporters, GLT-1/EAAT-2 and/or GLAST/EAAT-1, and a subsequent increase in extracellular glutamate concentrations which in turn induce excitotoxic neuronal cell death. We recently provided evidence that these disease-related disturbances of glial glutamate transport might be mediated in part by endothelins. This family of peptides not only massively reduces basal expression of GLT-1/EAAT-2 and GLAST/EAAT-1 in cultured astrocytes, but also completely overrides the exogenously induced expression of both glutamate transporter subtypes. The observed potency of this effect now prompted us to investigate whether endothelins inhibit glial glutamate transporter expression through an epigenetic, protein acetylation-dependent mechanism. We found that treating cultured astrocytes with the histone deacetylase inhibitor, trichostatin A, promotes transcription of both the GLT-1 and GLAST genes. These stimulatory influences were associated with an overall increase in acetylation of histones H3 and H4. The additional presence of endothelin-1 completely prevented the trichostatin A-induced increases in GLT-1- and GLAST expression without profoundly altering H3 and H4 acetylation levels. We conclude that endothelins inhibit glial glutamate transporter expression through an acetylation/deacetylation-independent process. We further suggest that modulating the activity of histone deacetylases represent an additional mechanism by which disease-associated disturbances of glial glutamate transporter expression are mediated.

Attenuation of abnormal glutamate release in zinc deficiency by zinc and Yokukansan

The mechanism of the abnormal increase in extracellular glutamate concentration in the hippocampus induced with 100 mM KCl in zinc deficiency is unknown. In the present study, the changes in glutamate release (exocytosis) and GLT-1, a glial glutamate transporter, expression were studied in young rats fed a zinc-deficient diet for 4 weeks. Exocytosis at mossy fiber boutons was enhanced as reported previously and GLT-1 protein was increased in the hippocampus. The enhanced exocytosis is thought to increase extracellular glutamate concentration. However, the basal concentration of extracellular glutamate in the hippocampus was not increased by zinc deficiency, suggesting that GLT-1 protein increased serves to maintain the basal concentration of extracellular glutamate. The enhanced exocytosis was attenuated in the presence of 100 μM ZnCl 2, which attenuated the abnormal increase in extracellular glutamate induced with high K + in zinc deficiency. The present study indicates that zinc attenuates abnormal glutamate release in zinc deficiency. The enhanced exocytosis was also attenuated in slices from zinc-deficient rats administered Yokukansan, a herbal medicine, in which the abnormal increase in extracellular glutamate induced with high K + was attenuated. It is likely that Yokukansan is useful for prevention or cure of abnormal glutamate release. The enhanced exocytosis in zinc deficiency is a possible mechanism on abnormal increase in extracellular glutamate in the hippocampus induced with high K +.

Impairment of GABAergic neurotransmitter system in the amygdala of young rats after 4-week zinc deprivation

On the basis of the evidence that the excitability of hippocampal glutamatergic neurotransmitter system is enhanced by dietary zinc deficiency, the response of amygdalar neurotransmitter system was checked in young rats fed a zinc-deficient diet for 4 weeks. Extracellular zinc concentration in the amygdala, which was measured by the in vivo microdialysis, was almost the same as that in the hippocampus and decreased by zinc deficiency. Extracellular zinc concentration in the amygdala was increased both in the control and zinc-deficient rats by stimulation with 100 mM KCl, suggesting that the increase in extracellular zinc in the amygdala, as well as that in the hippocampus, is linked with neuronal depolarization. In amygdalar extracellular fluid, the basal glutamate concentration was not significantly different between the control and zinc-deficient rats and was increased to almost the same extent between them by stimulation with 100 mM KCl, unlike more increase in extracellular glutamate concentration in the hippocampus in zinc deficiency. On the other hand, the basal GABA concentration in the amygdalar extracellular fluid was significantly lower in zinc-deficient rats and was not increased both in the control and zinc-deficient rats by stimulation with 100 mM KCl. These results suggest that GABAergic neurotransmitter system is critically impaired in the amygdala of young rats after 4-week zinc deprivation.

Mitochondrial ATP-sensitive potassium channel activation protects cerebellar granule neurons from apoptosis induced by oxidative stress.

Mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels are present in the brain, and several reports have shown that mitoK(ATP) channel openers protect the brain against ischemic injury. However, the precise mechanisms of this protection are not well established. We hypothesized that mitoK(ATP) channel openers prevent apoptosis by preserving mitochondrial membrane potential. We investigated the effect of mitoK(ATP) channel openers on apoptosis induced by oxidative stress using cultured cerebellar granule neurons. The mitoK(ATP) channel opener diazoxide (100 micromol/L) significantly suppressed the number of cells with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive nuclei and the increase in caspase-3 activity induced by 20 micromol/L H2O2. Diazoxide and another opener, pinacidil, prevented the loss of mitochondrial inner membrane potential (Delta(Psi)m) induced by H2O2. These effects were abolished by 5-hydroxydecanoate (500 micromol/L), a mitoK(ATP) channel blocker. Cyclosporin A and bongkrekic acid, inhibitors of the mitochondrial permeability transition pore, also prevented Delta(Psi)m loss, confirming the involvement of the mitochondrial permeability transition in the apoptotic cascade in neurons. Furthermore, diazoxide prevented the increase in extracellular glutamate concentration induced by H2O2, but this effect was not attributable to activation of surface K(ATP) channels. MitoK(ATP) channel openers inhibited apoptosis by preserving mitochondrial inner membrane potential. These beneficial effects may suggest a possible new target for neuroprotection.

Protection of cortical neurons against oxygen-glucose deprivation and N-methyl- d-aspartate by DIDS and SITS

The Cl − channel blockers, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) or 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS) dose-dependently protected against oxygen-glucose deprivation in cultured rat cortical neurons. DIDS or SITS attenuated oxygen-glucose deprivation-induced increases in extracellular glutamate concentrations and intracellular Ca 2+. DIDS or SITS provided moderate protection against N-methyl- d-aspartate (NMDA) toxicity and decreased NMDA receptor-mediated increases in intracellular Ca 2+. Whole-cell NMDA receptor currents were attenuated 39±2% and 21±3% by 1 mM DIDS and SITS, respectively. Other Cl − channel blockers as equipotent as DIDS and SITS did not decrease oxygen-glucose deprivation- or NMDA-mediated neuronal Ca 2+ influx or toxicity. Neurotoxicity by exogenous glutamate was not prevented by SITS and was exacerbated by DIDS. Reductions in oxygen-glucose deprivation-induced increases in intracellular Ca 2+ levels underlie neuroprotection by DIDS and SITS. This was a reflection of lower extracellular [glutamate], direct inhibition of Ca 2+ influx through postsynaptic NMDA receptors, and possibly through other protective properties associated with DIDS and SITS.

Delayed anoxic depolarizations in hippocampal neurons of mice lacking the excitatory amino acid carrier 1.

Hypoxia leads to a rapid increase in vesicular release of glutamate. In addition, hypoxic glutamate release might be caused by reversed operation of neuronal glutamate transporters. An increase in extracellular glutamate concentration might be an important factor in generating anoxic depolarizations (AD) and subsequent neuronal damage. To study the AD and the vesicular release in hippocampal slices from CD1 wild-type mice and mice in which the neuronal glutamate transporter excitatory amino acid carrier 1 (EAAC1) had been knocked out, the authors performed recordings of field potentials and patch clamp recordings of CA1 pyramidal cells. Latency to anoxic depolarizations was enhanced in EAAC1-/- mice, whereas the hypoxia-induced increase in miniature excitatory postsynaptic current frequency occurred with similarly short latencies and to a similar extent in control and mutated animals. Additional block of glial glutamate uptake with TBOA (dl-threo-beta-benzyloxyaspartate), a nontransportable and potent inhibitor, dramatically reduced the latency to onset of AD and abolished the difference between wild-type mice and EAAC1-/- mice. The authors conclude that the neuronal glutamate transporter greatly influences the latency to generation of AD. Because ADs are not prevented in EAAC1-deficient mice, vesicular release mechanisms also seem to be involved. They become prominent when glial glutamate transport is blocked.

DNA microarray analysis of the contused spinal cord: effect of NMDA receptor inhibition.

Spinal cord injury (SCI)-induced neurodegeneration leads to irreversible and devastating motor and sensory dysfunction. Post-traumatic outcomes are determined by events occurring during the first 24 hours after SCI. An increase in extracellular glutamate concentration to neurotoxic levels is one of the earliest events after SCI. We used Affymetrix DNA oligonucleotide microarrays (with 1,322 DNA probes) analysis to measure gene expression in order to test the hypothesis that SCI-induced N-methyl-D-aspartate (NMDA) receptor activation triggers significant postinjury transcriptional changes. Here we report that SCI, 1 hour after trauma, induced change in mRNA levels of 165 genes and expression sequence tags (ESTs). SCI affected mRNA levels of those genes that regulate predominantly transcription factors, inflammation, cell survival, and membrane excitability. We also report that NMDA receptor inhibition (with -(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate [MK-801]) reversed the effect of SCI on about 50% of the SCI-affected mRNAs. Especially interesting is the finding that NMDA receptor activation participates in the up-regulation of inflammatory factors. Therefore, SCI-induced NMDA receptor activation is one of the dominant, early signals after trauma that leads to changes in mRNA levels of a number of genes relevant to recovery processes. The majority of MK-801 effects on the SCI-induced mRNA changes reported here are novel. Additionally, we found that the MK-801 treatment also changed the mRNA levels of 168 genes and ESTs that had not been affected by SCI alone, and that some of their gene products could have harmful effects on SCI outcome.

Selective and AMPA Receptor-Dependent Astrocyte Death Following Prolonged Blockade of Glutamate Reuptake in Rat Cerebellar Cultures

In this study we examined the effects of prolonged l-trans-pyrrolidine-2,4-dicarboxylate (PDC)-induced glutamate reuptake blockade on the viability of glial cells in cerebellar granule cell cultures. Immunofluorescence staining for the glial-specific intermediate filament protein, GFAP, revealed that the PDC- induced increase of extracellular glutamate concentration was accompanied by increased astrocyte death, while neurons and oligodendrocytes remained intact and viable. Astrocytic cell death was manifested as fragmentation of processes and cell bodies. The selective astrocyte death was completely prevented by the competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist, NBQX (10 μM), whereas MK-801 (10 μM), a noncompetitive blocker of N-methyl-d-aspartate receptors, gave only partial protection. Double staining for GFAP and the AMPA receptor subunits GluR2/3 showed that astrocytes had much higher immunoreactivity for GluR2/3 than neurons or oligodendrocytes, suggesting that the number of AMPA receptors is likely to be higher on astrocytes. Furthermore, we employed real-time RT-PCR to measure GluR1–4 subunit mRNA expression in control and PDC-exposed cultures. Following treatment with PDC, GluR1 and GluR4 mRNAs were reduced by 40% and GluR3 was reduced by 70% relative to control levels. In contrast, GluR2 expression was not affected by the PDC treatment, indicating that GluR3 was the dominant type of AMPA receptor subunit expressed on astrocytes. Our results show that astrocytes appear to be more vulnerable than neurons or oligodendrocytes to a gradual increase in the extracellular glutamate concentration, suggesting that astrocytes may be critically involved in the pathophysiology of slowly developing chronic neurodegenerative disorders.