High Dose Of Glutamate Research Articles

Isoliquiritigenin Protects Neuronal Cells against Glutamate Excitotoxicity.

It is considered that glutamate excitotoxicity may be a major factor in the pathological death of neurons and mediate the development of neurodegenerative diseases in humans. Here, we show that isoliquiritigenin (ILG) at a concentration of 0.5-5 µM protects primary neuroglial cell culture from glutamate-induced death (glutamate 100 µM). ILG (1 µM) prevented a sharp increase in [Ca2+]i and a decrease in mitochondrial potential (ΔΨm). With the background action of ILG (1-5 µM), there was an increase in oxygen consumption rate (OCR) in response to glutamate, as well as in reserve respiration. The neuroprotective effect of ILG (5 µM) was accompanied by an increase in non-mitochondrial respiration. The results show that ILG can protect cortical neurons from death by preventing the development of calcium deregulation and limiting mitochondrial dysfunction caused by a high dose of glutamate. We hypothesize that ILG will be useful in drug development for the prevention or treatment of neurodegenerative diseases accompanied by glutamate excitotoxicity.

Involvement of the periaqueductal gray in the descending antinociceptive effect induced by the central nucleus of amygdala.

Here we studied whether descending control of mechanical nociception by glutamate in the central nucleus of the amygdala (CeA) of healthy control animals is induced by amygdaloid NMDA receptors and relayed through the midbrain periaqueductal gray (PAG). Mechanical nociception in the hind paws was assessed in rats with chronic guide cannulae for glutamate administration in the right CeA and for inducing local anesthesia in the PAG. In a separate electrophysiological study, ON-like PAG neurons giving an excitatory response to noxious pinch of the tail were recorded in anesthetized rats following glutamate administration into the CeA. A high dose of glutamate (100 microg) in the CeA induced mechanical antinociception in the contra- but not ipsilateral hind limb. Antinociception was prevented by an NMDA receptor antagonist in the CeA or local anesthesia of the PAG. Discharge rate of ON-like PAG neurons was increased by a high dose of glutamate (100 microg) in the CeA and this increase was prevented by an NMDA receptor antagonist in the CeA. The results indicate that amygdaloid NMDA receptors in the CeA may induce contralaterally mechanical antinociception through a circuitry relaying in the PAG. Activation of ON-like PAG neurons is associated with the descending antinociceptive effect. Mechanisms and causality of this association still remain to be studied.

Neuronal Networks on Multielectrode Arrays as High Sensitive Biosensors of the Functional State of Nerve Cells

The dynamics of changes of spontaneous neuronal activity in primary hippocampal cell cultures developing on a multielectrode array (MEA) was studied using a multielectrode system. The intensification of bioelectrical activity, which depended on the duration of cultivation and stabilized by the second week in vitro, has been revealed. An increase in the concentration of glutamate (up to 2 μM) by addition into the incubation medium during the stabilization period resulted in a rapid and significant reorganization of the neuronal network activity pattern. On the other hand, inhibition and subsequent gradual recovery of the neuronal network activity immediately after the neurotransmitter addition at higher concentrations (50 or 100 μM) have been observed. At the same time, on some electrodes in the presence of high doses of glutamate (100 μM), a complete or partially irreversible suppression of the activity has been recorded. A significant reduction in spontaneous activity of the neuronal network, which was not accompanied by neuronal death, also occurred when copper ions (Cu2+) at a concentration of 10 μM were added into the incubation medium for 48 h. The obtained data demonstrate high biosensor sensitivity of the neuronal network cultured on the multielectrode array, which makes it possible to use it as an effective test system for studies of biologically active compounds.

Anthocyanins abrogate glutamate-induced AMPK activation, oxidative stress, neuroinflammation, and neurodegeneration in postnatal rat brain.

BackgroundGlutamate-induced excitotoxicity, oxidative damage, and neuroinflammation are believed to play an important role in the development of a number of CNS disorders. We recently reported that a high dose of glutamate could induce AMPK-mediated neurodegeneration in the postnatal day 7 (PND7) rat brain. Yet, the mechanism of glutamate-induced oxidative stress and neuroinflammation in the postnatal brain is not well understood. Here, we report for the first time the mechanism of glutamate-induced oxidative damage, neuroinflammation, and neuroprotection by polyphenolic anthocyanins in PND7.MethodsPND7 rat brains, SH-SY5Y, and BV2 cells treated either alone with glutamate or in combination with anthocyanins and compound C were examined with Western blot and immunofluorescence techniques. Additionally, reactive oxygen species (ROS) assay and other ELISA kit assays were employed to know the therapeutic efficacy of anthocyanins against glutamate.ResultsA single injection of glutamate to developing rats significantly increased brain glutamate levels, activated and phosphorylated AMPK induction, and inhibited nuclear factor-E2-related factor 2 (Nrf2) after 2, 3, and 4 h in a time-dependent manner. In contrast, anthocyanin co-treatment significantly reduced glutamate-induced AMPK induction, ROS production, neuroinflammation, and neurodegeneration in the developing rat brain. Most importantly, anthocyanins increased glutathione (GSH and GSSG) levels and stimulated the endogenous antioxidant system, including Nrf2 and heme oxygenase-1 (HO-1), against glutamate-induced oxidative stress. Interestingly, blocking AMPK with compound C in young rats abolished glutamate-induced neurotoxicity. Similarly, all these experiments were replicated in SH-SY5Y cells by silencing AMPK with siRNA, which suggests that AMPK is the key mediator in glutamate-induced neurotoxicity.ConclusionsHere, we report for the first time that anthocyanins can potentially decrease glutamate-induced neurotoxicity in young rats. Our work demonstrates that glutamate is toxic to the developing rat brain and that anthocyanins can minimize the severity of glutamate-induced neurotoxicity in an AMPK-dependent manner.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0752-y) contains supplementary material, which is available to authorized users.

Vitamin C neuroprotection against dose-dependent glutamate-induced neurodegeneration in the postnatal brain.

Glutamate-induced excitotoxicity due to over-activation of glutamate receptors and associated energy depletion (phosphorylation and activation of AMPK) results in neuronal cell death in various neurological disorders. Restoration of energy balance during an excitotoxic insult is critical for neuronal survival. Ascorbic acid (vitamin C), an essential nutrient with well-known antioxidant potential, protects the brain from oxidative damage in various models of neurodegeneration. In this study, we reported the therapeutic efficacy of vitamin C in response to glutamate-induced excitation, resulting in energy depletion and apoptosis in the hippocampus of the developing rat brain. A single subcutaneous injection of glutamate at two different concentrations (5 and 10 mg/kg) in postnatal day 7 rat pups increased brain glutamate levels and increased the protein expression of neuronal apoptotic markers. Both doses of glutamate upregulated the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2, cytochrome-c release, caspase-3 activation and the expression of PARP-1. However, co-treatment of vitamin C (250 mg/kg) with glutamate decreased brain glutamate levels and reversed the changes induced by glutamate in the developing hippocampus. Interestingly, only a high dose of glutamate caused the phosphorylation and activation of AMPK and induced neuronal cell death, whereas a low dose of glutamate failed to mediate these effects. Vitamin C supplementation reduced the glutamate-induced phosphorylation of AMPK and attenuated neuronal cell death, as assessed morphologically by Fluoro Jade B in the hippocampal CA1 region of the developing brain. Taken together, our results indicated that glutamate in both concentrations is toxic to the immature rat brain, whereas vitamin C is pharmacologically effective against glutamate-induced neurodegeneration.

Co-activation of synaptic and extrasynaptic NMDA receptors by neuronal insults determines cell death in acute brain slice

Overactivation of NMDA receptors is linked to cell death during neuronal insults. However the precise role of synaptic and extrasynaptic NMDA receptors remains to be further determined. In this study, we used the acute brain slice to examine the contributions of synaptic and extrasynaptic NMDA receptors to neuronal death. By activation of synaptic NMDA receptors with bath application of 100μM bicuculline in acute brain slices, we observed a significant up-regulation in activation of neuronal survival-related signaling (p-CREB, p-ERK1/2 and p-AKT), without an obvious increase of LDH release and neuronal death. Interestingly, activation of extrasynaptic NMDA receptors alone by high dose of glutamate (200μM) following blockade of synaptic NMDA receptors with co-application of 20μM MK801 and 100μM bicuculline, we failed to observe inhibition of neuronal survival signaling and neuronal damage. In contrast, co-activation of synaptic and extrasynaptic NMDA receptors by applying 200μM glutamate or oxygen–glucose deprivation (OGD) to acute brain slices for 30min, we observed a significant inhibition of CREB, ERK1/2 and AKT activation, an increase of LDH release and neuronal condensation. Together, co-activation of synaptic and extrasynaptic NMDA receptors by neuronal insults contributes to cell death in acute brain slice.

Role for NMDA receptors in visceral nociceptive transmission in the anterior cingulate cortex of viscerally hypersensitive rats.

We have identified colorectal distension (CRD)-responsive neurons in the anterior cingulate cortex (ACC) and demonstrated that persistence of a heightened visceral afferent nociceptive input to the ACC induces ACC sensitization. In the present study, we confirmed that rostral ACC neurons of sensitized rats [induced by chicken egg albumin (EA)] exhibit enhanced spike responses to CRD. Simultaneous in vivo recording and reverse microdialysis of single ACC neurons showed that a low dose of glutamate (50 microM) did not change basal ACC neuronal firing in normal rats but increased ACC neuronal firing in EA rats from 18 +/- 2 to 32 +/- 3.8 impulses/10 s. A high dose of glutamate (500 microM) produced 1.95-fold and a 4.27-fold increases of ACC neuronal firing in sham-treated rats and in EA rats, respectively, suggesting enhanced glutamatergic transmission in the ACC neurons of EA rats. Reverse microdialysis of the 3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainite receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) reduced basal and abolished CRD-induced ACC neuronal firing in normal rats. In contrast, microdialysis of N-methyl-d-aspartate (NMDA) receptor antagonist AP5 had no effect on ACC neuronal firing in normal rats. However, AP5 produced 86% inhibition of ACC neuronal firing evoked by 50 mmHg CRD in the EA rats. In conclusion, ACC nociceptive transmissions are mediated by glutamate AMPA receptors in the control rats. ACC responses to CRD are enhanced in viscerally hypersensitive rats. The enhancement of excitatory glutamatergic transmission in the ACC appears to mediate this response. Furthermore, NMDA receptors mediate ACC synaptic responses after the induction of visceral hypersensitivity.

Convulsant action of systemically administered glutamate and bicuculline methiodide in immature rats

Developmental changes of transport of drugs into the brain play an important role in ontogenetic neuropharmacology. Two convulsant drugs with different mechanisms of action (glutamate and bicuculline methiodide) were chosen to demonstrate these changes in developing rats. High dose of glutamate (4 g/kg i.p.) induced both minimal (predominantly clonic) and generalized tonic-clonic seizures in rat pups 7, 12, and 18 days old. In contrast, seizures were only exceptionally observed in 25 and 90 days old animals. Bicuculline methiodide was administered in a dose of 2 or 20 mg/kg i.p. The first sign of bicuculline methiodide action in all age groups was represented by automatisms, a symptomatology never seen after bicuculline hydrochloride administration. Minimal seizures were induced in 12-day-old and in a few 18-day-old and adult rats. Generalized seizures were common after the higher dose of bicuculline methiodide in 7- and 12-day-old rat pups, seldom in 18-day-old ones and never seen in 25-day-old and adult animals. Both glutamate and bicuculline methiodide enter the brain in immature rats but the mechanisms are probably different — glutamate is transported actively through the blood-brain barrier whereas no similar system is known for bicuculline methiodide.

3-Nitropropionic Acid Neurotoxicity in Organotypic Striatal and Corticostriatal Slice Cultures Is Dependent on Glucose and Glutamate

Mitochondrial inhibition by 3-nitropropionic acid (3-NPA) causes striatal degeneration reminiscent of Huntington's disease. We studied 3-NPA neurotoxicity and possible indirect excitotoxicity in organotypic striatal and corticostriatal slice cultures. Neurotoxicity was quantified by assay of lactate dehydrogenase in the medium and glutamic acid decarboxylase in tissue homogenates. 3-NPA toxicity (25–100 μM in 5 mM glucose, 24–48 h) appeared to be highly dependent on culture medium glucose levels. 3-NPA treatment caused also a dose-dependent lactate increase, reaching a maximum of threefold increase above control at 100 μM. Both a high dose of glutamate (5 mM) and glutamate uptake blockade by dl-threo-β-hydroxyaspartate potentiated 3-NPA neurotoxicity in corticostriatal slice cultures. Furthermore, striatum from corticostriatal cocultures was more sensitive to 3-NPA than striatum without cortex and tetrodotoxin, MK-801, and d-2-amino-5-phosphonopentanoic acid prevented or attenuated 3-NPA neurotoxicity, suggesting that membrane depolarization and/or neuronal activity of the glutamatergic corticostriatal pathway contributes to striatal pathology. The results indicate that in vivo characteristics of 3-NPA toxicity can be reproduced in organotypic corticostriatal slice cultures.

Media composition modulates excitatory amino acid-induced death of rat cerebellar granule cells.

This study examined the effects of maintaining cells in different media and the role of serum in glutamate and NMDA-induced neurotoxicity in rat cerebellar granule cells. Glutamate stimulated a concentration-dependent cell death with similar potency in cerebellar granule cells grown in BME and Neurobasal media without serum. However, the maximal cell death to glutamate and N-methyl-D-aspartate (NMDA) varied in the different media compositions. In the presence of serum, glutamate and NMDA-induced excitotoxicity was abolished, suggesting a factor(s) in serum which influences glutamate-receptor mediated death. The protective effect of serum could be overcome by chronic stimulation with high doses of glutamate. The glutamate-stimulated increase in intracellular calcium load was attenuated in the presence of serum, resulting from an elevated basal calcium level, suggesting an association between raised basal calcium and neuroprotection.

Role of amino acids in salivation and the localization of their receptors in the rat salivary gland.

The distribution of gamma-aminobutyric acid (GABA) receptor subunits such as GABAAR-gamma 1 and GABAAR-gamma 2, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type receptor subunits such as GluR-1, GluR-2/3 and GluR-4, and N-methyl-D-aspartic acid (NMDA) type subunits such as NR1 were investigated by immunocytochemistry. Furthermore, the roles of these amino acids, GABA and glutamate, on salivation were analyzed in the rat submandibular and sublingual glands. Some similarities were observed in the distribution patterns of GABAA type receptors and AMPA receptors. In the submandibular ganglion cells, collecting ducts and striated ducts, these subunits were expressed strongly; however, there were some differences in their expression patterns between the submandibular and sublingual gland acinar cells. Since these receptor subunits were expressed in the acinar cell bodies of the submandibular gland, they were not expressed in the acinar cells but were expressed in the myoepithelial cells in the sublingual gland. On the other hand, no NR1 expression was observed. To examine the roles of GABA and glutamate in salivation, the submandibular and sublingual glands were perfused partially with Ringer's solution via a facial artery to avoid systemic influence, and substrates were infused into the perfusion solution. No salivary secretion was evoked by GABA or glutamate infusion in the absence of electrical stimulation (2-3 V, 5 ms, 20 Hz). Salivary flow evoked by electrical stimulation of the chorda-lingual nerve caused significant inhibition by GABA (10(-6), 10(-5), 10(-4) and 10(-3) M) and the GABAAR agonist muscimol 10(-3) and 10(-6) M) (n = 6, P < 0.05). Such GABA-induced inhibition was antagonized by the GABAAR antagonists bicuculline (BCC; 10(-6) and 10(-3) M) and picrotoxin (PTX; 10(-6) and 10(-3) M). On the other hand, salivary flow evoked by electrical stimulation (8-10 V, 5 ms, 20 Hz) of the superior cervical ganglion (SCG) was not affected by GABA. While high doses of glutamate (10(-1) M) and NMDA (10(-1) M) showed no effects on salivary flow despite application of electrical stimulation, AMPA at a high concentration (10(-1) M) significantly inhibited salivary secretion (n = 6, P < 0.05). These studies revealed that inhibitory and excitatory amino acid receptors such as GABAA and AMPA type receptors are coexpressed in the rat salivary glands, and that GABA inhibits salivary secretion via GABAA receptors which may act with acetylcholine. However, the role of glutamate in salivation remains unclear despite the presence of AMPA type receptors. The present findings suggest that glutamate does not act alone but with other substances such as peptides and/or other amino acids.

Modulation of cone to horizontal cell transmission by calcium and pH in the fish retina.

The effects of small changes in the calcium and sodium concentrations and in the pH of superfusion medium on the membrane potential and light-evoked responses of cone horizontal cells in the goldfish retina were examined. Conventional intracellular recording, a bicarbonate-based superfusion medium, and a specially designed superfusion apparatus that reduced pressure wave disturbances were used. An increase in the extracellular calcium concentration, [Ca2+]o, from control levels (0.1 mM) to 1.0 mM hyperpolarized cone horizontal cells and reduced the magnitude of their light responses at all stimulus intensities. Addition of 20 mM NaCl to the 1.0 mM Ca2+ Ringer's solution reversed the hyperpolarizing effect of the 1.0 mM Ca2+ but addition of 20 mM choline, a monovalent cation that does not pass through cyclic GMP-activated channels, did not. Reduction of the superfusate pH from 7.6 to 7.2 by switching from a Ringer's solution gassed with 3% CO2 to one gassed with 10% CO2 hyperpolarized horizontal cells and reduced the magnitude of their light responses at all stimulus intensities for both 0.1 and 1.0 mM Ca2+ Ringer's solutions. An increase in pH to 8.2 by gassing the superfusate with 1% CO2 slightly depolarized the cells in 0.1 mM Ca2+ Ringer's solution but slightly hyperpolarized the cells in the 1.0 mM Ca2+ Ringer's solution. Following pharmacological isolation of the horizontal cells from synaptic input with high doses of glutamate (4-5 mM) and/or Co2+ (4 mM) treatment, no effect on horizontal cell membrane potential due to changes in pHo or [Ca2+]o was observed. These findings are discussed with respect to the cellular mechanisms and sites of action in the outer retina that are affected by changes in pHo and [Ca2+]o.

Development of glutamate and NMDA sensitivity of neurons within the cochlear nuclear complex of kittens

1. The effects of glutamate, N-methyl-D-aspartate (NMDA), and NMDA receptor antagonists, D-alpha-aminoadipate (D alpha AA) and 2-amino-5-phosphonovalerate (APV), microionophoretically applied onto neurons in the caudal divisions (posteroventral and dorsal subnuclei) of the cochlear nuclear complex (CN), were investigated during postnatal development in kittens with the use of extracellular techniques. From birth through postnatal day 7, microionophoretically applied glutamate elevated the acoustically evoked discharge rates of nearly 80% of neurons studied. Although fewer neurons were studied with the use of NMDA, approximately 65% of these responded to this glutamatergic agonist, and no developmental changes in the percentage of responsive neurons were observed. The actions of NMDA antagonists were studied in a relatively small number of neurons, and results support the supposition that glutamate, or a glutamate-like substance, acts as a CN neurotransmitter throughout postnatal life. 2. Approximately 64% of CN neurons encountered among neonatal animals were unresponsive to acoustic stimulation, even at the highest output levels available (> 120 dB SPL). That percentage declined monotonically during the next three postnatal days, such that approximately 23% of neurons encountered were unresponsive to acoustic stimulation on the third day. Essentially all encountered neurons were responsive to acoustic stimulation by the middle of the second postnatal week. Under conditions of simultaneous glutamate and acoustic stimulation, neurons in this general class of "acoustically unresponsive" neurons segregated into two groups. Glutamate increased spontaneous discharge rate in 45% of the neurons studied, however, these units remained acoustically unresponsive during combined sound and excitatory amino acid stimulation (group A1). In the second group (A2), 55% of the neurons that were acoustically unresponsive under control (sound alone) conditions responded to the acoustic component of the combined acoustic and glutamate stimulation (the experimental condition) in a frequency-dependent manner. A2 neurons exhibited temporal firing patterns characteristic of acoustically responsive neurons of corresponding age, suggesting that these neurons are functionally connected to the auditory periphery, whereas A1 neurons are not. 3. Dose-response curves were either sigmoidal or linear over the range that measurements were made, and maximum discharge rates evoked by high doses of glutamate in the youngest animals studied tended to be lower than those produced by acoustic stimulation alone in older animals. These results suggest that intrinsic properties contribute to the mechanism(s) that limits neuronal responsiveness. Average dose-response curve slopes were higher for neurons recorded from older animals, also indicating that intrinsic properties regulating dynamic response range are acquired postnatally in the kitten CN.(ABSTRACT TRUNCATED AT 400 WORDS)

Antiischemic and metabolic effects of glutamate during pacing in patients with stable angina pectoris secondary to either coronary artery disease or syndrome X

The effects of glutamate on anginal threshold, cardiac metabolism and hemodynamics were studied in 11 patients with stable angina pectoris, positive stress test results, and pacing-induced myocardial lactate release due to coronary artery disease (CAD) (n = 9) or syndrome X (n = 2). Data were obtained before, during and after 2 identical periods of coronary sinus pacing, the second being preceded by an intravenous injection of monosodium glutamate 1.2 (n = 7) or 2.5 (n = 4) mg/kg body weight. After glutamate administration, pacing time to onset of angina increased from mean ± standard deviation 103 ± 53 to 166 ± 71 seconds (p < 0.01) and ST-segment depression after pacing decreased from 2.3 ± 1.0 to 1.6 ± 1.1 mm (p < 0.01). Arterial glutamate concentration increased 60% (p < 0.01) after the low dose and 150% (p < 0.01) after the high dose of glutamate. Regardless of dose, myocardial glutamate uptake increased by 25% (p < 0.01). Pacing-induced cardiac release of lactate diminished 50% (p < 0.05), whereas the releases of xanthine and hypoxanthine were unchanged by glutamate. Arterial free fatty acids decreased 20% (p < 0.01). Circulating levels and cardiac exchanges of alanine, glucose and citrate were unchanged. Glutamate did not influence heart rate, arterial blood pressure, coronary blood flow, coronary vascular resistance or myocardial oxygen consumption. One patient complained of short-lasting burning sensations after receiving the high glutamate dose. In conclusion, augmented provision of glutamate enhances pacing tolerance in stable angina, presumably by a metabolic improvement of cardiac energy production during ischemia.

Alteration of medullary respiratory unit discharge by iontophoretic application of putative neurotransmitters.

1 Cats with midcollicular decerebration were vagotomized, paralyzed and artificially ventilated. Phrenic nerve activity was recorded as an index of central respiratory rhythm. Medullary respiratory neurones and non-respiratory cells located in approximation to the ventral respiratory nucleus were tested for their responsiveness to iontophoretically applied gamma-aminobutyric acid (GABA), acetylcholine (ACh) and glutamate. 2 GABA tended to inhibit, whereas ACh and glutamate excited activity both of respiratory and non-respiratory units. Some phase-spanning respiratory unit activities were converted to phasic discharge patterns linked to either inspiration or expiration concomitant with application of low GABA doses. Appropriate applications of GABA also resulted in a complete cessation of the respiratory or non-respiratory neuronal activities. 3 While application of ACh or glutamate induced continuous firing in phasic, phase-spanning respiratory neurones, the periodic discharge patterns of inspiratory or expiratory units was not altered by ACh or, in many instances, by glutamate. Only at high doses of glutamate was the phasic discharge of some inspiratory or expiratory units converted to tonic activity. 4 These observations suggest that strong inhibitory processes serve to maintain the phasic firing pattern of respiratory units. These data also support the concept that active-inhibitory phase-switching mechanisms serve to define respiratory rhythmicity.