Extracellular Levels Of Excitatory Amino Acids Research Articles

Berberine attenuates convulsing behavior and extracellular glutamate and aspartate changes in 4-aminopyridine treated rats.

Objective(s):K+ channel blocker 4-aminopyridine (4-AP) stimulates the release of glutamate from nerve terminals and induces seizures. Berberine as a potential herbal drug exerts several pharmacological actions on the central nervous system including anxiolytic, anticonvulsant, and neuroprotective properties. The present study aimed to investigate the effect of berberine on seizure onset and time course of the extracellular levels of excitatory amino acids (EAA), glutamate and aspartate, changes produced by 4-AP in rat hippocampus.Materials and Methods:The rats were given either saline or berberine (50, 100 and 200 mg/kg, IP) 40 min before administration of 4-AP (15 mg/kg, IP) and the onset of seizure was recorded. A group of rats also received diazepam (DZP, 15 mg/kg, IP) 20 min prior to 4-AP administration. Hippocampal extracellular levels of EAA were also measured using microdialysis assay. Analysis of the dialysate samples was performed by reversed-phase high performance liquid chromatography (HPLC) with precolumn derivatization with o-phthaldialdehyde and fluorescence detection.Results:Our findings suggest that berberine significantly delayed the seizure onset following 4-AP injection. There was a considerable increase in the extracellular glutamate and aspartate levels in 4-AP treated rats and 4-AP-evoked release of EAA was sharply reduced (about 4-5 fold especially at 20 min after 4-AP administration) in berberine treatment groups.Conclusion:The results of present study show that berberine attenuates 4-AP induced seizures by decreasing hippocampal aspartate and glutamate release in rats.

Involvement of metabotropic glutamate receptors in excitatory amino acid and GABA release following spinal cord injury in rat.

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations resulting in glutamate receptor-mediated excitotoxic events. The glutamate receptors include ionotropic (iGluRs) and metabotropic (mGluR) receptors. Of the three groups of mGluRs, group-I activation can initiate intracellular pathways that lead to further transmitter release. Groups II and III mGluRs function mainly as autoreceptors to regulate neurotransmitter release. In an effort to examine the role of mGluRs in the increase in EAAs following SCI, we administered AIDA, a potent group-I mGluR antagonist immediately after injury. To determine subtype specific roles of the group-I mGluRs, we evaluated EAA release following LY 367385 (mGluR1 antagonist) and MPEP (mGluR5 antagonist) administration. To evaluate group-II and -III mGluRs we administered APDC (group-II agonist) and L-AP4 (group-III agonist) immediately following injury; additionally, we initiated treatment with CPPG (group-II/-III antagonist) and LY 341495 (group-II antagonist) 5 min prior to injury. Subjects were adult male Sprague-Dawley rats (225-250 g), impact injured at T10 with an NYU impactor (12.5 mm drop). Agents were injected into the epicenter of injury, amino acids where collected by microdialysis fibers inserted 0.5 mm caudal from the edge of the impact region and quantified by HPLC. Treatment with AIDA significantly decreased extracellular EAA and GABA concentrations. MPEP reduced EAA concentrations without affecting GABA. Combining LY 367385 and MPEP resulted in a decrease in EAA and GABA concentrations greater than either agent alone. L-AP4 decreased EAA levels, while treatment with LY 341495 increased EAA levels. These results suggest that mGluRs play an important role in EAA toxicity following SCI.

Real‐time, two‐dimensional visualization of ischaemia‐induced glutamate release from hippocampal slices

The involvement of excitatory amino acid (EAA) toxicity in ischaemia-induced neuronal cell death has long been suggested. However, in the hippocampus, the brain site most vulnerable to ischaemia, the detailed spatial and temporal patterns of EAA release are not yet known. To address this issue, we have developed a novel strategy for the continuous, real-time, two-dimensional monitoring of EAA release from brain slices. As EAA detector, we used a cell line transformed with the N-methyl-D-aspartate (NMDA) receptor, which is exclusively activated by EAAs, leading to an increase in the intracellular Ca(2+) level. Combined with a calcium imaging technique, the use of this cell line allowed the temporal and regional analysis of EAA release from a brain slice placed directly on top of the clonal cells in a culture dish. Using this strategy, we demonstrated ischaemia-induced EAA release in rat hippocampal slices. Increased EAA release was seen initially in the CA1 region, about 3 min after the beginning of ischaemia, then in the CA3 region and dentate gyrus, and, finally, throughout the hippocampal slice. Regional differences in extracellular EAA levels were also seen, with more EAA being released from the CA1 region than from the middle dentate gyrus. The present results are especially interesting as neurons in the CA1 region are more vulnerable to ischaemia than those in the CA3 region and dentate gyrus.

Brain injury after hypoxia-ischemia in newborn rats: relationship to extracellular levels of excitatory amino acids and cysteine

The aim of this study was to follow extracellular concentrations of excitatory amino acids (EAAs) and cysteine during neonatal hypoxia-ischemia (HI) and reflow and to relate these events to the extent of brain damage evaluated 6 h after the insult. Rat pups (PND 7–10) were subjected to unilateral ligation of the common carotid artery and exposed to hypoxia (7.7% O 2). Extracellular amino acids were sampled during HI and for 6 h of reperfusion with microdialysis and the levels were correlated with the extent of brain damage at the site of probe placement. The concentrations of glutamate, aspartate and cysteine increased transiently during HI (15×, 6× and 3×, respectively) in the extracellular space and returned to normal or remained slightly elevated during reperfusion. Changes of EAAs and cysteine were similar during HI in the infarcted, undamaged and border-zone regions. During reperfusion the concentrations of glutamate, aspartate and cysteine were higher in infarcted and border-zone areas compared to undamaged tissue. In neonatal rats, the extracellular levels of EAAs during HI do not correspond to the extent of brain injury whereas the EAA concentrations during reflow are related to the extent of infarction.

Involvement of the glutamatergic metabotropic receptors in the regulation of glutamate uptake and extracellular excitatory amino acid levels in the striatum of chloral hydrate-anesthetized rats

The microdialysis technique was used to assess in vivo the putative functional role of metabotropic excitatory amino acid receptors in regulating extracellular levels of the excitatory amino acids glutamate and aspartate in the striatum of chloral hydrate-anesthetized rats. Addition of the metabotropic glutamate receptor antagonist (+)-α-methyl-4-carboxyphenylglycine (MCPG) (10−3 or 4 × 10−3 M) in the dialysis probe did not modify the basal extracellular levels of glutamate and aspartate but induced a significant dose-dependent decrease in the KCl-elicited elevation of glutamate and aspartate extracellular levels. The effect of MCPG on glutamate extracellular concentration under K+ stimulation was reduced by the simultaneous superfusion of the metabotropic glutamate receptor agonist (2S,3S,4S)-α-(carboxycyclopropyl)glycine (L-CCG) (10−3 M) which had no significant effect when tested alone. In contrast, L-CCG alone significantly potentiated the KCl-elicited elevation of aspartate extracellular concentrations but failed to modify the MCPG effect on this amino acid concentration. In a parallel series of experiments, high-affinity glutamate uptake was measured ex vivo 20 min after an in vivo injection of 10 pmol of MCPG in the striatum. MCPG was found unable to modify the glutamate uptake rate. In vitro, MCPG (1–1000 μM) again had no effect on glutamate transport rate. These data suggest that metabotropic excitatory amino acid receptors (1) may act to increase the extracellular levels of glutamate and aspartate under depolarizing conditions, and (2) may not have a major role in the regulation of high affinity glutamate uptake under basal conditions. In addition, it can be assumed that the control of glutamate and aspartate extracellular levels may involve different metabotropic receptors.

Development of brain damage after neonatal hypoxia-ischemia: excitatory amino acids and cysteine.

The aim of this study was to investigate the possible role of excitatory amino acids (EAAs) and cysteine in the development of brain damage after hypoxia-ischemia (HI) in neonates. In a rat model of neonatal HI, changes in extracellular (ec) amino acids in cerebral cortex were measured with microdialysis and correlated with the extent of brain damage at the site of probe placement. Extracellular concentrations of glutamate, aspartate and cysteine increased during HI and remained elevated during reperfusion. During HI the pattern of EAA changes was the same in the infarcted, undamaged and border zone regions. During reperfusion, however, the ec concentrations of glutamate, aspartate and cysteine were higher in infarcted and border zone areas compared to undamaged tissue. HI also produced a slight increase of tissue concentration of cysteine and decrease of tissue concentration of glutamate in parietal cortex of the HI hemisphere. The effect of cysteine on brain damage induced by HI and glutamate was also investigated. A subtoxic dose of cysteine potentiated glutamate toxicity in the arcuate nucleus and enhanced brain infarction after HI in neonatal rats. The results show that in neonatal HI the extracellular levels of EAAs during HI are not directly related to brain injury but the EAA levels during reflow predict the extent of infarction. Cysteine increases HI-induced brain injury and potentiates glutamate toxicity in neonatal rats. Speculatively, elevated level of cysteine during reperfusion may participate in the excitotoxic cascade leading to brain injury.

Opioid receptor antagonist nalmefene stereospecifically inhibits glutamate release during global cerebral ischemia

The opioid receptor antagonist nalmefene improves cellular bioenergetics and attenuates the reduction in tissue glutamate levels after global cerebral ischemia/reperfusion. The latter finding suggests that nalmefene might inhibit glutamate release during ischemia. To test this hypothesis, we used microdialysis techniques to examine the effect of nalmefene pretreatment on extracellular excitatory amino acid levels during global cerebral ischemia in rats. Saline, (−)-nalmefene (20, 100 or 500 μg/kg) or the inactive nalmefene enantiomer (+)-nalmefene (100 μg/kg) were given 15 min prior to induction of ischemia using a multi-vessel occlusion model. Pretreatment with (−)-nalmefene decreased peak dialysate glutamate in a dose-dependent fashion as compared to saline-treated controls, whereas (+)-nalmefene had no effect. These results suggest that opioid receptors may modulate glutamate release during ischemia and that inhibition of excitatory amino acid release may contribute to the protective actions of opioid receptor antagonists in cerebral ischemia.

Protective effects of serotonin reuptake inhibitors, citalopram and clomipramine, against hippocampal CA1 neuronal damage following transient ischemia in the gerbil

To clarify the role of serotonin in cerebral ischemia, we examined the effects of selective serotonin reuptake inhibitors, citalopram and clomipramine, on ischemic neuronal damage in the gerbil. Pretreatment with citalopram (40 mg/kg i.p.) and clomipramine (20 mg/kg i.p.) protected against neuronal destruction of hippocampal CA1 pyramidal cells following 5 min of forebrain ischemia. Furthermore, microdialysis assays showed that a striking increase in extracellular excitatory amino acid levels during ischemia was significantly inhibited by pretreatment with citalopram and clomipramine. However, citalopram (40 mg/kg i.p.) did not alter the extracellular amino acid concentrations in normal gerbils. Thus, serotonin reuptake inhibitors have a protective effect against ischemic neuronal damage. Furthermore, the present result suggests that the protective effect is mediated through prevention of the accumulation of extracellular excitatory amino acids during and after ischemia.

Intra- and extracellular changes of amino acids in the cerebral cortex of the neonatal rat during hypoxic-ischemia

Excitatory amino acids (EAAs) have been implicated to play a part in the development of hypoxic-ischemic brain injury in the neonate. The aim of the present study was to follow changes of intra- and extracellular (microdialysis) amino acids in the cerebral cortex in a model where cortical hypoxic-ischemic damage is produced consistently. Hypoxic-ischemia (unilateral ligation of the carotid artery + 2 h of exposure to 7.8% oxygen) caused a depletion of tissue ATP, phosphocreatine and glucose with a concomittant accumulation of AMP and lactic acid in cortical tissue. These changes were accompanied by a decrease of tissue aspartate and glutamine whereas the contents of γ-aminobutyric acid (GABA), phenylalanine, leucine, isoleucine, valine and alanine increased. In the extracellular fluid GABA, glutamate, aspartate, taurine, glycine and alanine all increased multi-fold during hypoxic-ischemia. Aspartate and glutamate returned to near initial levels 2 h after the end of the insult, whereas the elevation of glycine persisted during recovery. In conclusion, the high extracellular levels of EAAs and glycine may exert injurious effects during and after hypoxic-ischemia.