Neuroprotective effect of N-acetyl-aspartyl-glutamate in combination with mild hypothermia in the endothelin-1 rat model of focal cerebral ischaemia

Previously we showed that treatment with mild hypothermia (34 degrees C for 2 h) after a focal cerebral infarct was neuroprotective by reducing apoptosis in the penumbra (cortex), but not in the core (striatum) of the infarct. In this study we examined whether administration of N-acetyl-aspartyl-glutamate (NAAG) in combination with mild hypothermia could improve striatal neuroprotection in the endothelin-1 rat model. NAAG (10 mg/kg i.p.) was injected under normothermic (37 degrees C) or mild hypothermic conditions, either 40 min before or 20 min after the insult. NAAG reduced caspase 3 immunoreactivity in the striatum, irrespective of the time of administration and brain temperature. This neuroprotective effect could be explained, at least partially, by decreased nitric oxide synthase activity in the striatum and was blocked by the group II metabotropic glutamate receptor antagonist, LY341495. Hypothermia applied together with NAAG reduced both cortical and striatal caspase 3 immunoreactivity, as well as the overall ischaemic damage in these areas. However, no pronounced improvement was seen in total damaged brain volume. Extracellular glutamate levels did not correlate with the observed protection, whatever treatment protocol was applied. We conclude that treatment with NAAG causes the same degree of neuroprotection as treatment with hypothermia. Combination of the two treatments, although reducing apoptosis, does not considerably improve ischaemic damage.     

Effect of the Non-NMDA Receptor Antagonist GYKI 52466 on the Microdialysate and Tissue Concentrations of Amino Acids Following Transient Forebrain Ischaemia

Abstract: The effect of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) on ischaemia-induced changes in the microdialysate and tissue concentrations of glutamate, aspartate, and γ-aminobutyric acid (GABA) was studied in rats. Twenty minutes of four-vessel occlusion resulted in a transient increase in microdialysate levels of glutamate, aspartate, and GABA in striatum, cortex, and hippocampus. Administration of GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min intravenously starting 20 min before onset of ischaemia) inhibited ischaemia-induced increases in microdialysate glutamate and GABA in striatum without affecting the increases in hippocampus or cortex. Twenty minutes of four-vessel occlusion resulted in immediate small decreases and larger delayed (72 h) decreases in tissue levels of glutamate and aspartate. Transient increases in tissue levels of GABA were shown in all three structures at the end of the ischaemic period. At 72 h, after the ischaemic period, significantly reduced GABA levels were observed in striatum and hippocampus. GYKI 52466, given under identical conditions as above, augmented the ischaemia-induced decrease in striatal tissue levels of glutamate and aspartate, without significantly affecting the decreases in hippocampus and cortex. Twenty minutes of ischaemia resulted in a large increase in microdialysate dopamine in striatum. GYKI 52466 failed to inhibit this increase. Kainic acid (500 μM infused through the probe for 20 min) caused increases in microdialysate glutamate and aspartate in the striatum. GYKI 52466 (10 mg/ kg bolus + 10 mg/kg/60 min) completely inhibited the kainic acid-induced glutamate release. In conclusion, the action of the non-NMDA antagonist, GYKI 52466, in the striatum is different from that in the cortex and hippocampus. The inhibition by GYKI 52466 of ischaemia-induced and kainate-induced increases in microdialysate glutamate concentration in the striatum may be related to the neuroprotection provided by GYKI 52466 in this region.     

Effects of Mild Hypothermia on the Release of Regional Glutamate and Glycine During Extended Transient Focal Cerebral Ischemia in Rats

The present study is to determine the effect of mild hypothermia (MHT) on the release of glutamate and glycine in rats subjected to middle cerebral artery occlusion and reperfusion. The relationship between amino acid efflux and brain infarct volume was compared in different periods during MHT. Reversible middle cerebral artery occlusion was performed in Sprague-Dawley rats using a suture model. The rats were divided into four groups including (1) MHT during ischemia (MHTi), (2) MHT during reperfusion (MHTr), (3) MHT during ischemia and reperfusion (MHTi + r), and (4) a normothermic group (NT). Extracellular concentrations of glutamate and glycine in the cortex and striatum were monitored using in vivo microdialysis and analyzed using high-performance liquid chromatography. Morphometric measurements for infarct volume were performed using 2,3,5-triphenyltetrazolium chloride staining. The increase of glutamate and glycine in the ischemic cortex of the MHTi and MHTi + r rats during ischemic and reperfusion periods was significantly less than that of the NT rats (p < 0.05). However, there was no statistical difference among these groups in the peak of glutamate and glycine release in the striatum. Infarct volume paralleled the release of glutamate and glycine. The protective effect of MHTi and MHTi + r in reducing ischemia and reperfusion brain injury may be due to the attenuation of both glutamate and glycine release during ischemia and reperfusion.     

Neuroprotectant FK506 inhibits glutamate-induced apoptosis of astrocytes in vitro and in vivo

Neuron-astrocyte interactions are critical for signalling, energy metabolism, extracellular ion and glutamate homeostasis, volume regulation and neuroprotection in the CNS. Glutamate uptake by astrocytes may prevent excitotoxic glutamate elevation and determine neuronal survival. However, an excess of glutamate can cause the death of astrocytes. FK506, an inhibitor of calcineurin, and an immunosuppressive drug, is neuroprotective in animal models of neurologic diseases, including focal and global ischaemia. In the present work, we demonstrate that a single injection of FK506 60 min after a transient middle cerebral artery occlusion (MCAo) significantly decreases the number of terminal deoxynucleotidyl transferase nick-end labelling (TUNEL)-positive cells in the ischaemic cortex and striatum. Using 3-D confocal microscopy we found that, 24 h after MCAo, many TUNEL-positive cells in the ischaemic striatum and cortex are astrocytes. Furthermore, we demonstrate that exposure of cultured cortical astrocytes to 50-100 mM Glu for 24 h induces apoptotic alterations in nuclear morphology, DNA fragmentation, dissipation of mitochondrial transmembrane potential (DeltaPsi) and caspase activation. FK506 (1 muM) efficiently inhibits Glu-induced apoptosis of cultured astrocytes, DNA fragmentation and changes in mitochondrial DeltaPsi. Our findings suggest that modulation of glutamate-induced astrocyte death early after reperfusion may be a novel mechanism of FK506-mediated neuroprotection in ischaemia.     

D1 receptor‐mediated endogenous tPA upregulation contributes to blood‐brain barrier injury after acute ischaemic stroke

Blood‐brain barrier (BBB) integrity injury within the thrombolytic time window is becoming a critical target to reduce haemorrhage transformation (HT). We have previously reported that BBB damage was initially damaged in non‐infarcted striatum after acute ischaemia stroke. However, the underlying mechanism is not clear. Since acute ischaemic stroke could induce a significant increase of dopamine release in striatum, in current study, our aim is to investigate the role of dopamine receptor signal pathway in BBB integrity injury after acute ischaemia using rat middle cerebral artery occlusion model. Our data showed that 2‐h ischaemia induced a significant increase of endogenous tissue plasminogen activator (tPA) in BBB injury area and intra‐striatum infusion of tPA inhibitor neuroserpin, significantly alleviated 2‐h ischaemia‐induced BBB injury. In addition, intra‐striatum infusion of D1 receptor antagonist SCH23390 significantly decreased ischaemia‐induced upregulation of endogenous tPA, accompanied by decrease of BBB injury and occludin degradation. More important, inhibition of hypoxia‐inducible factor‐1 alpha with inhibitor YC‐1 significantly decreased 2‐h ischaemia‐induced endogenous tPA upregulation and BBB injury. Taken together, our data demonstrate that acute ischaemia disrupted BBB through activation of endogenous tPA via HIF‐1α upregulation, thus representing a new therapeutic target for protecting BBB after acute ischaemic stroke.     

Comparison of ketamine stereoisomers on tissue metabolic activity in an in vitro model of global cerebral ischaemia

Ketamine (2-o-chlorophenenyl-2-methylaminocyclohexanone hydrochloride) is a dissociative general anaesthetic with neuroprotective properties. Since ketamine is optically active, we compared the neuroprotective efficacy of the (+)- or (-)-enantiomers in global cerebral ischaemia. Rat corticostriatal slices superfused with, or incubated in, artificial CSF at 34 degrees C were subjected to a brief ischaemic insult. Dopamine efflux was measured using fast cyclic voltammetry. Tissue metabolism was determined with 2,3,5-triphenyltetrazolium chloride staining, a marker of mitochondrial enzyme activity. In control slices, ischaemia caused rapid striatal dopamine release (to 122 microM over 18 s) after an initial delay of 149s. Racemic ketamine (100 micromol/l) significantly delayed (by 24%, P<0.05), slowed (by 63%, P<0.01) and reduced (by 27%, P<0.05) ischaemia-induced dopamine release. Ischaemia (10 min) also caused significant decreases in striatal (25%, P<0.01) and cortical (31%, P<0.001) metabolic activity, manifested as a drop in mean TTC staining intensity. Racemic ketamine and its (+)- and (-)-enantiomers (each 100 microM) attenuated the loss of metabolic activity in the striatum. However, in the cortex, only (+)-ketamine (100 microM) was significantly neuroprotective. We conclude that neuroprotection by ketamine in cerebral ischaemia is both region- and isomer-dependent.     

Neuroprotective effect of aspirin by inhibition of glutamate release after permanent focal cerebral ischaemia in rats

Aspirin reduces the size of infarcts after ischaemic stroke. Although this fact has been attributed to its anti-platelet actions, direct neuroprotective effects have also been reported. We have recently demonstrated that aspirin is neuroprotective by inhibiting glutamate release in 'in vitro' models of brain ischaemia, via an increase in ATP production. The present study was designed to determine whether the inhibition of glutamate release induced by aspirin might be protective in a whole-animal model of permanent focal brain ischaemia. Focal brain ischaemia was produced in male adult Fischer rats by occluding both the common carotid and middle cerebral arteries. Central and serum glutamate levels were determined at fixed intervals after occlusion. The animals were then killed and infarct volume was measured. Aspirin (30 mg/kg i.p. administered 2 h before the occlusion) produced a significant reduction in infarct volume, an effect that correlated with the inhibition caused by aspirin on ischaemia-induced increase in brain and serum glutamate concentrations after the onset of the ischaemia. Aspirin also inhibited ischaemia-induced decrease in brain ATP levels. Our present findings show a novel mechanism for the neuroprotective effects of aspirin, which takes place at concentrations in the anti-aggregant-analgesic range, useful in the management of patients with risk of ischaemic events.     

Metabolic Responses of the Dopaminergic System during Hypoxia in Newborn Brain

The purpose of this review is to describe the relationship between the dopamine and amino acid neurotransmitter systems and cortical oxygen pressure during different levels of cerebral hypoxia using newborn piglets as an animal model, adding new data from our laboratory. The extracellular dopamine increases as the oxygen pressure in the cortex decreases. The relationship between oxygen pressure and dopamine levels is the same whether the hypoxia is induced by reduced F_iO₂ (high-flow hypoxia) or by hypocapnia-induced cerebral vasoconstriction (low-flow hypoxia). Thus it appears that, particularly in mild hypoxia, the extracellular level of dopamine depends primarily on the oxygen concentration in the tissue with minimal influence of parameters such as blood flow and pH. There is no "oxygen reserve" in the brain of newborn piglets and the extracellular levels of dopamine in the striatum increase almost linearly with decrease in oxygen pressure, with even small decreases in oxygen pressure resulting in increased dopamine levels. In contrast, the changes in extracellular concentrations of the excitatory amino acids glutamate and aspartate are variable and transient. In a majority of 2- to 5 day-old piglets even very low oxygen pressures in the brain did not result in significant alterations in the extracellular levels of glutamate and aspartate. These changes in the dopaminergic system may contribute directly and indirectly to the neuronal damage that occurs during hypoxic/ischemic insult and reoxygenation in newborn brain, particularly in the striatum. A variety of mechanisms are discussed by which dopamine, in particular extracellular dopamine, can increase cellular toxicity.     

Comparative Effect of Transient Global Ischemia on Extracellular Levels of Glutamate, Glycine, and γ-Aminobutyric Acid in Vulnerable and Nonvulnerable Brain Regions in the Rat

We evaluated whether regional differences in the magnitude of glutamate, gamma-aminobutyric acid (GABA), and glycine release could explain why some regions are vulnerable to ischemia whereas others are spared. By means of the microdialysis technique, the temporal profile of ischemia-induced changes in extracellular levels of glutamate, GABA, and glycine was compared in regions that demonstrate differing susceptibilities to a 10- and 20-min ischemic insult (dorsal hippocampus, anterior thalamus, somatosensory cortex, and dorsolateral striatum). The degree of ischemia (as established by local cerebral blood flow reduction) and the magnitude of histopathological neuronal damage were also evaluated in these regions. The blood flow reduction was severe and uniform in all regions; however, the histopathological outcome illustrated a different pattern. Whereas the CA1 sector of the hippocampus was severely damaged, the thalamus and cortex were relatively spared from both 10 and 20 min of ischemia. Striatal neurons were resistant to a 10-min insult but severely damaged after 20 min of ischemia. Ischemia-induced increase in glutamate and GABA content were of a similar magnitude and temporal profile in all four brain regions. A uniform increase in extracellular glycine levels was also observed in all four brain structures. The postischemic response, however, was different. Glycine levels remained twofold higher than baseline in the hippocampus but fell to baseline in the cortex and thalamus after both 10- and 20-min insults. In the striatum, glycine levels returned to baseline after 10 min of ischemia but remained relatively high after a 20-min insult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Post-ischaemic treatment with the cyclooxygenase-2 inhibitor nimesulide reduces blood-brain barrier disruption and leukocyte infiltration following transient focal cerebral ischaemia in rats

Several studies suggest that cyclooxygenase (COX)-2 plays a pivotal role in the progression of ischaemic brain damage. In the present study, we investigated the effects of selective inhibition of COX-2 with nimesulide (12 mg/kg) and selective inhibition of COX-1 with valeryl salicylate (VAS, 12-120 mg/kg) on prostaglandin E(2) (PGE(2)) levels, myeloperoxidase (MPO) activity, Evans blue (EB) extravasation and infarct volume in a standardized model of transient focal cerebral ischaemia in the rat. Post-ischaemic treatment with nimesulide markedly reduced the increase in PGE(2) levels in the ischaemic cerebral cortex 24 h after stroke and diminished infarct size by 48% with respect to vehicle-treated animals after 3 days of reperfusion. Furthermore, nimesulide significantly attenuated the blood-brain barrier (BBB) damage and leukocyte infiltration (as measured by EB leakage and MPO activity, respectively) seen at 48 h after the initial ischaemic episode. These studies provide the first experimental evidence that COX-2 inhibition with nimesulide is able to limit BBB disruption and leukocyte infiltration following transient focal cerebral ischaemia. Neuroprotection afforded by nimesulide is observed even when the treatment is delayed until 6 h after the onset of ischaemia, confirming a wide therapeutic window of COX-2 inhibitors in experimental stroke. On the contrary, selective inhibition of COX-1 with VAS had no significant effect on the evaluated parameters. These data suggest that COX-2 activity, but not COX-1 activity, contributes to the progression of focal ischaemic brain injury, and that the beneficial effects observed with non-selective COX inhibitors are probably associated to COX-2 rather than to COX-1 inhibition.     

Changes in regional levels of putative neurotransmitter amino acids in brain under unilateral forebrain ischemia

The levels of the neurotransmitter amino acids glutamate, aspartate, and GABA were determined in different brain regions during ischemia and post-ischemic recirculation periods using the unilateral carotid artery occlusion model of stroke in gerbils. The levels of glutamate, aspartate and GABA in ischemic hemisphere were increased significantly by 10 min of ischemia and later declined with time. Reperfusion for 30 min following 10 min. of ischemia further enhanced the levels of glutamate and aspartate. Increase in GABA levels were found during early periods of reperfusion. Regional variations in the changes of amino acids' levels were noticed following ischemia. Hippocampus showed the highest increase in glutamate levels followed by striatum and cerebral cortex. Aspartate levels in striatum and hippocampus increased during 10 min ischemia (46% and 30%) and recirculation (70% and 79%), whereas in cerebral cortex the levels were doubled only during recirculation. Ischemia induced elevations of GABA levels were observed in cerebral cortex (68%) and in hippocampus (30%), and the levels were normalized during recirculation. No changes in GABA levels were found in striatum. It is suggested that the large increase in the levels of excitatory neurotransmitter amino acids in brain regions specially in hippocampus during ischemia and recirculation may be one of the causal factors for ischemic brain damage.     

Effects of Apomorphine on In Vivo Release of Dopamine and Its Metabolites in the Prefrontal Cortex and the Striatum, Studied by a Microdialysis Method

The effects of apomorphine (0.1-2.5 mg/kg) on release of endogenous dopamine and extracellular levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the prefrontal cortex and the striatum were examined in vivo by a microdialysis method. Apomorphine significantly reduced release of dopamine and the extracellular levels of dopamine metabolites, DOPAC and HVA, not only in the striatum, but also in the prefrontal cortex. These findings indicate that dopamine autoreceptors modulate in vivo release of dopamine in the prefrontal cortex.     

Glutamate Release in Experimental Ischaemia of the Retina: An Approach Using Microdialysis

A rabbit eye model of neural ischaemia is described that uses an increased pressure in the anterior eye chamber to block the capillary supply to the retina. A microdialysis probe placed very close to the retinal surface was used to monitor release of amino acids during ischaemia. A large (two- to threefold) increase in the release of glutamate and O-phosphoserine (twofold), but not of six other amino acids monitored, occurred during initial ischaemia. During reperfusion after release of intraocular pressure, much larger (five- to 10-fold) increases in the release of these amino acids were observed. Parallel ischaemic retinal tissue damage was observed. This damage was prevented by ketamine applied locally via a superfusion needle, suggesting that glutamate released during ischaemia, and particularly during reperfusion, was responsible for cell death.     

Release of Glutamate from Striatum of Freely Moving Rats by pros-Methylimidazoleacetic Acid

Abstract: The effect of pros -methylimidazoleacetic acid (p-MIAA) was measured on the release of glutamate and aspartate from cerebral cortex, hippocampus, and striatum of freely moving rats, and on the uptake of ¹⁴C by striatal slices incubated in the presence of l -[¹⁴C]-glutamate. Twenty-four hours after implantation of a dialysis fiber, striatum, hippocampus, or cerebral cortex spontaneously released both glutamate and aspartate in the micromolar range. p-MIAA (1 µ M to 1 m M ), added to the dialysis perfusate, elicited a concentration-dependent increase of glutamate release from striatum with a maximal increase of about threefold. This effect did not occur in hippocampus or cortex. In none of these regions did p-MIAA increase aspartate release significantly. The p-MIAA effect was not mimicked by its isomer tele -methylimidazoleacetic acid. p-MIAA did not influence the uptake of glutamate by striatal slices. The glutamate-releasing action of p-MIAA may affect striatal function and explain the positive correlation between levels of p-MIAA in CSF and the severity of Parkinson's disease.     

The kappa-opioid agonist, U-69593, decreases acute amphetamine-evoked behaviors and calcium-dependent dialysate levels of dopamine and glutamate in the ventral striatum

The effects of a kappa-opioid receptor agonist on acute amphetamine-induced behavioral activation and dialysate levels of dopamine and glutamate in the ventral striatum were investigated. Amphetamine (2.5 mg/kg i.p.) evoked a substantial increase in rearing, sniffing, and hole-poking behavior as well as dopamine and glutamate levels in the ventral striatum of awake rats. U-69593 (0.32 mg/kg s.c.) significantly decreased the amphetamine-evoked increase in behavior and dopamine and glutamate levels in the ventral striatum. Reverse dialysis of the selective kappa-opioid receptor antagonist, nor-binaltorphimine, into the ventral striatum antagonized the effects of U-69593 on amphetamine-induced behavior and dopamine and glutamate levels. Reverse dialysis of low calcium (0.1 mM) into the ventral striatum decreased basal dopamine, but not glutamate, dialysate levels by 91% 45 min after initiation of perfusion. Strikingly, 0.1 mM calcium perfusion significantly reduced the 2.5 mg/kg amphetamine-evoked increase in dopamine and glutamate levels in the ventral striatum, distinguishing a calcium-dependent and a calcium-independent component of release. U-69593 did not alter the calcium-independent component of amphetamine-evoked dopamine and glutamate levels. These data are consistent with the view that a transsynaptic mechanism augments the increase in dopamine and glutamate levels in the ventral striatum evoked by a moderately high dose of amphetamine and that stimulation of kappa-opioid receptors suppresses the calcium-dependent component of amphetamine's effects.     

Effects of Ischaemia on Neurotransmitter Release from the Isolated Retina

Abstract: The effects of "ischaemia" (glucose-free Krebs-bicarbonate medium gassed with N₂/CO₂ on the release of glutamate and other major neurotransmitters in the retina were examined using the isolated rat and rabbit retina. Amino acid transmitters, acetylcholine, and dopamine were measured by HPLC. The release of glutamate, aspartate, GABA, and glycine from ischaemic retinas was more than doubled after 30 min, and after 90 min of ischaemia the release of amino acids was ∼ 15–20-fold that of control values. Ischaemia also produced large increases in the release of dopamine from both the rat and especially the rabbit retina. In contrast, the release of acetylcholine from the rat retina was significantly decreased by ischaemia, although the release of choline was increased. Because the ischaemia-induced release of glutamate, aspartate, and GABA from the rat retina was completely Ca independent, and exposure of the retina to high K (50 m M ) did not stimulate amino acid release, it is concluded that the mechanisms underlying the ischaemia-induced release do not involve an initial release of K or an influx of calcium.     

Neuroprotective effects of MK 801 and hypothermia used alone and in combination in hypoxic-ischemic brain injury in neonatal rats.

Although accumulating evidence suggests that increased extracellular glutamate concentrations may play an important role in hypoxic-ischemic brain injury, dopamine and other catecholamines also seem to be involved. The N-methyl-D-aspartate receptor antagonist MK 801 and moderate hypothermia (32-34 degrees C) are each known to be neuroprotective, but their combined effect on the release and metabolism of neurotransmitters is unknown. Seven-day-old pups (n: 150) underwent right common carotid artery ligation to induce hemispheric ischemia, and were later subjected to 120 minutes of hypoxia with 8% O2 and 92% N2O. Half the rats (Group I, n: 74) were subjected to normothermic conditions throughout the hypoxic period. Moderate hypothermia (30-32 degrees C) was induced in the other pups (Group II, n: 76) immediately after artery occlusion, and was maintained throughout the hypoxic period. Prior to inducing hypoxia, half of the rats in each group (Groups IA and IIA) received vehicle solution (0.9% NaCI) and the other rats (Groups IB and IIB) received MK 801 (0.5 mg/kg) subcutaneously at 45 and 120 minutes after occlusion. Intracerebral temperature was recorded every 15 minutes after occlusion. Infarct area (n: 40) was calculated after staining with 2% 2,3,5 triphenyltetrazolium chloride. Neuronal damage (n: 42) was assessed by quantifying CA1-CA3 neuronal loss at five hippocampal levels. The amount of damage to the monoamine system of the corpus striatum was determined based on the dopamine and 3,4 dihydroxyphenylacetic acid levels in the corpus striatum in both hemispheres (n: 46), as measured by high-pressure liquid chromatography and compared with normal control pups' values (n: 10). The normothermia/saline-treated pups had significantly larger infarct areas than the MK 801 only, hypothermia only, or MK 801/hypothermia combination groups. Neuropathological examination and striatal tissue monoamine data also confirmed marked neuronal damage in this group. Although MK 801 treatment alone resulted in significantly smaller infarct area and less tissue damage than was observed in the normothermia/saline-treated group, the moderate hypothermia and the MK 801/hypothermia combination treatment groups both exhibited better neuronal protection, especially in the corpus striatum. The rats that received combined treatment also had a significantly lower mortality rate.     

Glutamate (mGluR-5) gene expression in brain regions of streptozotocin induced diabetic rats as a function of age: role in regulation of calcium release from the pancreatic islets in vitro

Metabotrophic glutamate receptors (mGluRs) modulate cellular activities involved in the processes of differentiation and degeneration. In this study, we have analysed the expression pattern of group-I metabotropic glutamate receptor (mGlu-5) in cerebral cortex, corpus striatum, brainstem and hippocampus of streptozotocin induced and insulin treated diabetic rats (D+I) as a function of age. Also, the functional role of glutamate receptors in intra cellular calcium release from the pancreatic islets was studied in vitro. The gene expression studies showed that mGlu-5 mRNA in the cerebral cortex increased siginficantly in 7 weeks old diabetic rats whereas decreased expression was observed in brainstem, corpus striatum and hippocampus when compared to control. 90 weeks old diabetic rats showed decreased expression in cerebral cortex, corpus striatum and hippocampus whereas in brainstem the expression increased significantly compared to their respective controls. In 7 weeks old D+I group, mGlu-5 mRNA expression was significantly decreased in cerebral cortex and corpus striatum whereas the expression increased significantly in brainstem and hippocampus. 90 weeks old D+I group showed an increased expression in cerebral cortex, while it was decreased significantly in corpus striatum, brainstem and hippocampus compared to their respective controls. In vitro studies showed that glutamate at lower concentration (10^-7 M) stimulated calcium release from the pancreatic islets. Our results suggest that mGlu-5 receptors have differential expression in brain regions of diabetes and D+I groups as a function of age. This will have clinical significance in management of degeneration in brain function and memory enhancement through glutamate receptors. Also, the regulatory role of glutamate receptors in calcium release has immense therapeutic application in insulin secretion and function.     

Chronic administration of valproic acid induces a decrease in rat striatal glutamate and taurine levels

Summary The effect of acute and chronic (10 days) administration of 200 mg/kg (i.p.) of valproic acid (VPA) on endogenous levels of aspartate, glutamate, alanine, glycine and taurine in the cerebral frontal cortex and corpus striatum of rats was studied. Quantification of the amino acid levels was performed by HPLC.Valproic acid (VPA) did not either induce changes on these neurotransmitters contents in corpus striatum after acute treatment. After chronic administration we found a decrease on the endogenous levels of glutamic acid (24%, p < 0.05) which was related to an increase (250%, p < 0.02) of the in vitro KCl evoked release of glutamate. We found decrements in taurine endogenous levels (22%, p < 0.05) which was not associated with an increase of its release.In cerebral frontal cortex there was not found any change neither under the acute nor under the chronic condition.Thus, it may be conclude that chronic treatment with VPA produces decreases on the endogenous levels of glutamate and taurine. However the relevance of this effect concerning it therapeutic action remains unclear.     

Evidence that glutaric acid reduces glutamate uptake by cerebral cortex of infant rats

The role of excitotoxicity in the cerebral damage of glutaryl-CoA dehydrogenase deficiency (GDD) is under intense debate. We therefore investigated the in vitro effect of glutaric (GA) and 3-hydroxyglutaric (3-OHGA) acids, which accumulate in GDD, on [(3)H]glutamate uptake by slices and synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Glutamate uptake was significantly decreased by high concentrations of GA in cortical slices of 7-day-old rats, but not in cerebral cortex from 15- and 30-day-old rats and in striatum from all studied ages. Furthermore, this effect was not due to cellular death and was prevented by N-acetylcysteine preadministration, suggesting the involvement of oxidative damage. In contrast, glutamate uptake by brain slices was not affected by 3-OHGA exposure. Immunoblot analysis revealed that GLAST transporters were more abundant in the cerebral cortex compared to the striatum of 7-day-old rats. Moreover, the simultaneous addition of GA and dihydrokainate (DHK), a specific inhibitor of GLT1, resulted in a significantly higher inhibition of [(3)H]glutamate uptake by cortical slices of 7-day-old rats than that induced by the sole presence of DHK. We also observed that both GA and 3-OHGA exposure did not alter the incorporation of glutamate into synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Finally, GA in vivo administration did not alter glutamate uptake into cortical slices from 7-day-old rats. Our findings may explain at least in part why cortical neurons are more vulnerable to damage at birth as evidenced by the frontotemporal cortical atrophy observed in newborns affected by GDD.