Changes in Extracellular Concentrations of Glutamate, Aspartate, Glycine, Dopamine, Serotonin, and Dopamine Metabolites After Transient Global Ischemia in the Rabbit Brain

Although considerable evidence supports a role for excitatory amino acids in the pathogenesis of ischemic neuronal injury, few in vivo studies have examined the effect of increasing durations of ischemia on the extracellular concentrations of these agents. Recently, other neurotransmitters (e.g., glycine and dopamine) have been implicated in the mechanism of ischemic neuronal injury. Accordingly, this study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, glycine concentrations in the hippocampus, and dopamine, serotonin, and dopamine metabolites in the caudate nucleus with varying durations (5, 10, or 15 minutes) of transient global cerebral ischemia as evidence to support their pathogenetic roles. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. Glutamate and aspartate concentrations in the dialysate increased from baseline by 1-, 5-, and 13-fold and by 4-, 9-, and 31-fold, respectively, for the three ischemic durations. The concentrations returned to baseline rapidly after reperfusion. The peak concentrations of glutamate and aspartate were significantly higher with increasing ischemic duration. Dopamine concentrations increased by approximately 700-fold in response to all three ischemic durations and returned to baseline within 10 min of reperfusion. Glycine, in contrast, increased during ischemia by a mean of 4-fold, but remained elevated throughout the 80-min period of reperfusion. The final concentrations of glycine were significantly higher than baseline levels (p = 0.0002, Mann-Whitney test). That glutamate and aspartate concentrations in the hippocampus co-vary with the duration of global ischemia is taken as supportive evidence of their pathogenetic role in ischemic neuronal injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fulminant Hepatic Failure in Rats Induces Oxidative Stress Differentially in Cerebral Cortex,Cerebellum and Pons Medulla

Hepatic Encephalopathy (HE) is one of the most common complications of acute liver diseases and is known to have profound influence on the brain. Most of the studies, available from the literature are pertaining to whole brain homogenates or mitochondria. Since brain is highly heterogeneous with functions localized in specific areas, the present study was aimed to assess the oxidative stress in different regions of brain-cerebral cortex, cerebellum and pons medulla during acute HE. Acute liver failure was induced in 3-month old adult male Wistar rats by intraperitoneal injection of thioacetamide (300 mg/kg body weight for two days), a well known hepatotoxin. Oxidative stress conditions were assessed by free radical production, lipid peroxidation, nitric oxide levels, GSH/GSSG ratio and antioxidant enzyme machinery in three distinct structures of rat brain-cerebral cortex, cerebellum and pons medulla. Results of the present study indicate a significant increase in malondialdehyde (MDA) levels, reactive oxygen species (ROS), total nitric oxide levels [(NO) estimated by measuring (nitrites + nitrates)] and a decrease in GSH/GSSG ratio in all the regions of brain. There was also a marked decrease in the activity of the antioxidant enzymes-glutathione peroxidase, glutathione reductase and catalase while the super oxide dismutase activity (SOD) increased. However, the present study also revealed that pons medulla and cerebral cortex were more susceptible to oxidative stress than cerebellum. The increased vulnerability to oxidative stress in pons medulla could be due to the increased NO levels and increased activity of SOD and decreased glutathione peroxidase and glutathione reductase activities. In summary, the present study revealed that oxidative stress prevails in different cerebral regions analyzed during thioacetamide-induced acute liver failure with more pronounced effects on pons medulla and cerebral cortex. Murthy Ch.R.K—Deceased while in service.     

Effects of N-Acetyl Aspartate, Aspartate, and Glutamate on cAMP and cGMP Levels in Developing Rat Cerebral Cortex

N-Acetyl-aspartate (N-Ac-Asp) incubated with minced cerebral cortex caused a dose-dependent increase in the levels of cAMP and cGMP. This effect was followed during postnatal development. N-Ac-Asp elicits the greatest increase in cAMP in 5-day-old and in cGMP in 40-day-old rats. The levels of cyclic AMP were always higher than those of cGMP. We also studied the effects of L-aspartate (Asp) and L-glutamate (Glu) on the levels of cyclic nucleotides in the cerebral cortex minces of rats different ages, and observed that both amino acids produced the maximum increase in cAMP at 10 days, whereas in the case of cGMP the maximal effect of Asp occurs earlier than 20 days and of Glu after 40 days. In the adult rat, the N-Ac-Asp effect on cAMP was greater than that produced by either Asp or Glu, whereas the levels of cGMP were similarly affected by all three. The data show a peak response of cAMP and cGMP to N-Ac-Asp, Asp, and Glu during cortical maturation. Because this response varies with postnatal time, N-Ac-Asp, and Glu may act upon different receptor sites.     

Elevation of the Extracellular Concentrations of Glutamate and Aspartate in Rat Hippocampus During Transient Cerebral Ischemia Monitored by Intracerebral Microdialysis

Rats were implanted with 0.3-mm-diameter dialysis tubing through the hippocampus and subsequently perfused with Ringer's solution at a flow rate of 2 microliter/min. Samples of the perfusate representing the extracellular fluid were collected over 5-min periods and subsequently analyzed for contents of the amino acids glutamate, aspartate, glutamine, taurine, alanine, and serine. Samples were collected before, during, and after a 10-min period of transient complete cerebral ischemia. The extracellular contents of glutamate and aspartate were increased, respectively, eight- and threefold during the ischemic period; the taurine concentration also was increased 2.6-fold. During the same period the extracellular content of glutamine was significantly decreased (to 68% of the control value), whereas the concentrations of alanine and serine did not change significantly during the ischemic period. The concentrations of gamma-aminobutyric acid (GABA) were too low to be measured reliably. It is suggested that the large increase in the content of extracellular glutamate and aspartate in the hippocampus induced by the ischemia may be one of the causal factors in the damage to certain neurons observed after ischemia.     

Release of [3H]Dopamine from Striatal and Cerebral Cortical Slices from Rats with Thioacetamide-Induced Hepatic Encephalopathy: Different Responses to Stimulation by Potassium Ions and Agonists of Ionotropic Glutamate Receptors

The effects of depolarizing stimuli; high (50 mM) potassium ions and the glutamate receptor agonists N-methyl-D-aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) on the release of newly-loaded [³H]dopamine were studied in frontal cortical and striatal slices from control rats and from rats with acute hepatic encephalopathy induced with a hepatotoxin, thioacetamide. Hepatic encephalopathy enhanced the stimulatory effect of potassium ions by 20% in striatal slices and by 34% in frontal cortical slices. In striatal slices the stimulatory effects of N-methyl-D-aspartate and kainate were depressed in hepatic encephalopathy by 46% and 21%, respectively, which may be taken to reflect impaired modulation of striatal dopamine release by glutamate acting at N-methyl-D-aspartate or kainate receptors. In frontal cortical slices, the stimulatory effect of kainate was enhanced by 35% in hepatic encephalopathy but N-methyl-D-aspartate-stimulated release was not affected. The release evoked by 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate was not affected in hepatic encephalopathy in either brain region. Stimulation of dopamine release in the frontal cortex by depolarization or glutamate acting at kainate receptors could inhibit the activity of descending corticostriatal glutamatergic pathways, further impairing regulation of dopamine release by glutamate in the stratum.     

Specific [3H] Glutamate Binding in the Cerebral Cortex and Hippocampus of Rats During Development: Effect of Homocysteine-Induced Seizures

Specific [³H]glutamate binding to synaptic membranes from the cerebral cortex and hippocampus of 7-, 12- and 18-day-old rats was examined, both in control animals and during seizures induced by homocysteine. In the cerebral cortex a transient peak of glutamate binding was observed in 7-day-old group, whereas in the hippocampus it occurred in 12-day-old animals. Total specific [³H]glutamate binding was not influenced by preceding seizure activity in either of the age groups and both the studied regions. NMDA- and QA-sensitive glutamate bindings represent the highest portion of the total binding. Moreover, NMDA-sensitive binding in the cerebral cortex of 7-day-old rats is significantly higher as compared to the two more mature groups. The proportion of individual receptor subtypes on total binding in each age group was not influenced by preceding seizure activity. However, NMDA-sensitive binding in the hippocampus of 12-day-old rats, sacrificed during homocysteine-induced seizures, was significantly increased as compared to corresponding controls. In contrast to the effect of NMDA, AMPA, kainate and quisqualate which displaced to a different extent [³H]glutamate binding, homocysteine had no effect when added to membrane preparations. Similarly, [³H]CPP and [³H]AMPA bindings were not affected in the presence of homocysteine. It thus seems unlikely that homocysteine is an effective agonist for conventional ionotropic glutamate receptors. Its potential activity at some of the modulatory sites at the NMDA receptor channel complex or at metabotropic receptors has to be clarified in further experiments.     

Investigation on Acetylcholine,Aspartate, Glutamate and GABA Extracellular Levels from Ventral Hippocampus During Repeated Exploratory Activity in the Rat

The extracellular levels of aspartate, glutamate, -aminobutyric acid (GABA), and acetylcholine (ACh) were investigated by microdialysis, coupled with HPLC, in the ventral hippocampus of rats during two 30-min exploration periods. Motor activity was monitored. During exploration I, an increase in motor activity associated with a 315% increase in aspartate, 181% in glutamate, and 264% in ACh levels, occurred during the first 10 min. The increase in GABA level reached a maximum of 257% during the second 10 min. The neurotransmitter levels returned to basal values within 40 min. During exploration II, 1 h later, a smaller increase in neurotransmitter levels and motor activity was observed. In both explorations, the increase in neurotransmitter levels was completely abolished by 1 and 3 M TTX. A statistically significant relationship was found between neurotransmitter extracellular levels and motor activity, for aspartate and glutamate in exploration I, and for ACh in exploration I and II. In conclusion, exploratory activity is associated with or depends on the activation of neuronal systems in the ventral hippocampus releasing aspartate, glutamate, GABA, and ACh. The activation is dampened by habituation.     

Amphetamine Increases Extracellular Concentrations of Glutamate in the Prefrontal Cortex of the Awake Rat: A Microdialysis Study

Using microdialysis, the effect was investigated of intracerebral infusions of different doses of amphetamine (1.25, 2.5, 5, 10, and 20 g/l) on the extracellular concentrations of glutamate in the medial prefrontal cortex of the rat. Amphetamine produced a dose-related increase in extracellular concentrations of glutamate. At the highest dose, amphetamine increased extracellular glutamate by 445% of baseline as well as extracellular concentrations of taurine, and reduced extracellular concentrations of glutamine. Amphetamine did not modify other amino acids such as arginine. Increases in extracellular concentrations of glutamate and taurine were independent of calcium in the perfusion medium. This is the first study showing that amphetamine produces a calcium-independent increase in extracellular concentrations of glutamate and taurine in the medial prefrontal cortex of the rat.     

Evidence that Extracellular Concentrations of Dopamine Are Regulated by Noradrenergic Neurons in the Frontal Cortex of Rats

Abstract: Experiments were performed to confirm that noradrenergic terminals regulate extracellular concentrations of dopamine (DA) in the frontal cortex of rats. The effects of 20 mg/kg 1-[2-[bis(4-fluorphenyl)methoxy]-ethyl]-4-(3-phenylpropyl)piperazine (GBR 12909), a selective inhibitor of DA uptake, and 2.5 mg/kg desipramine (DMI) on the extracellular concentrations of DA in the frontal cortex and striatum were studied in rats given 6-hydroxydopamine (6 µg/µl) bilaterally into the locus coeruleus to destroy noradrenergic terminals. GBR 12909 increased dialysate DA similarly in the striatum of vehicle and 6-hydroxydopamine-treated rats, whereas in the frontal cortex it raised DA concentrations only in lesioned animals. DMI raised extracellular DA concentrations in the frontal cortex but not in the striatum of controls. The effect of DMI on cortical DA was abolished by the 6-hydroxydopamine lesion. GBR 12909, at a subcutaneous dose of 20 mg/kg, further increased cortical dialysate DA in rats given DMI intraperitoneally at 20 mg/kg or through the probe at 10⁻⁵ mol/L. The data support the hypothesis of an important regulation of the extracellular concentrations of DA in the frontal cortex by noradrenergic terminals.     

Ischemia-Induced Accumulation of Extracellular Amino Acids in Cerebral Cortex, White Matter, and Cerebrospinal Fluid

Abstract: In a global model of brain ischemia, accumulation of amino acids was studied in the extracellular space of the auditory cortex and the internal capsule using microdialysis, and in CSF of halothane anesthetized cats. In both brain regions, blood flow determined by hydrogen clearance decreased below 10 ml/100 g/min after extracranial multiple-vessel occlusion, and extracellular potassium activity ( K _e) measured in the dialysate increased significantly. A delayed rise in K _e was observed in CSF. In contrast, ischemic amino acid accumulation differed markedly between the two brain regions investigated. In cortex, transmitter amino acids glutamate, aspartate, and γ-aminobutyric acid (GABA) rose almost immediately after onset of ischemia, and increased 30-, 25-, and 250-fold, respectively, after 2 h of ischemia. The nontransmitter amino acids taurine, alanine, and serine increased 10-, seven-, and fourfold, respectively, whereas glutamine and essential amino acids (valine, phenylalanine, isoleucine, and leucine) increased only 1.5-fold. In the internal capsule, increases in amino acids, if any, were delayed and much smaller than in cortex. The largest alteration was a fivefold elevation of GABA. In CSF, changes in amino acids were small and comparable to those in the internal capsule. Our results demonstrate that ischemia-induced extracellular amino acid accumulation is a well localized phenomenon restricted to gray matter structures that possess release and reuptake systems for these substances. We assume that amino acids diffuse slowly into adjacent white matter structures, and into CSF.     

M1 Acetylcholine Receptor Stimulation Increases the Extracellular Concentrations of Glutamate and GABA in the Medial Prefrontal Cortex of the Rat

The present study was undertaken to examine the effects of different muscarinic receptor agonists on glutamate and GABA concentrations in the medial prefrontal cortex of the rat. In vivo perfusions were made in the conscious rat using a concentric push-pull cannulae system. Amino acid concentrations in samples were determined by HPLC with fluorometric detection. The intracortical perfusion of arecoline, a M1-M2 muscarinic receptor agonist, produced a significant increase in extracellular [GLU] and [GABA]. McN-A-343, a M1 muscarinic receptor agonist, but not the M2 muscarinic receptor agonist, oxotremorine, produced a significant increase in extracellular [GLU] and [GABA]. The effects of McN-A-343 on extracellular [GLU] and [GABA] were blocked by pirenzepine, a M1 muscarinic receptor antagonist. These results suggest that M1 muscarinic receptor stimulation increases the extracellular concentrations of GLU and GABA in the medial prefrontal cortex of the rat.     

Alteration in IGF-I Binding in the Cerebral Cortex and Cerebellum of Neonatal Rats During Protein-Calorie Malnutrition

Neonatal brain development in the rat is adversely affected by malnutrition. Alterations in tissue binding of IGF-I in the malnourished brain were tested in rat pups from mothers who were fed a 20% protein diet (C) or a 4% protein diet (M) starting from day 21 of gestation and continued throughout suckling. IGF-I binding in both cortex and cerebellum decreased progressively in C and M groups from day 6 to day 13. At day 9, 11, and 13, the binding was significantly greater (p < 0.02) in M compared to C groups. To investigate whether these changes might be related to the alteration in receptor activity, membranes were incubated with ¹²⁵I-IGF in the presence of excess insulin with or without unlabeled IGF-I. In the absence of insulin, specific IGF-I binding in the M group was increased by 41.8 ± 13.8% (mean ± SEM p < 0.05) relative to C group. Insulin produced a consistent but incomplete inhibition of binding in both C and M, of 75% and 67% respectively. In addition, the specific IGF-I binding in the presence of insulin was increased in M group by 70.2 ± 9.4% relative to C, p < 0.05. To characterize the nature of this binding, cerebral cortical membranes, from both groups, incubated with ¹²⁵I-IGF-I were cross-linked, and electro-phoresed on 6% and 10% SDS-PAGE gels under reducing conditions. Autoradiography of the 6% gel showed two specific bands at 115 kD and 240 kD, consistent with monomeric and dimeric forms of the IGF-I receptor, which were inhibited by excess insulin. In contrast, a 10% gel showed an additional band at 35 kD (IGF-binding protein) that was not inhibited by insulin. In both gels, membrane preparations from the M group showed a heightened intensity of the bands relative to C. The increase in binding protein relative to the receptor suggests a disequilibrium that may limit the availability of exogenous IGF-I to the tissues.     

Epilepsy, Neurodegeneration, and Extracellular Glutamate in the Hippocampus of Awake and Anesthetized Rats Treated with Okadaic Acid

We have previously shown that the intrahippocampal microinjection of okadaic acid (OKA), a potent inhibitor of serine/threonine protein phosphatases, induces epileptic seizures, neuronal death, and the hyperphosphorylation of the NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor. We administered OKA by reverse microdialysis in the hippocampus of awake and halothane-anesthetized rats, with simultaneous collection of microdialysis fractions and recording of the EEG activity, and subsequent histological analysis. OKA produced intense behavioral and persistent EEG seizure activity in the awake rats but not in the anesthetized animals, and did not significantly alter the extracellular concentration of glutamate and aspartate detected in the microdialysis fractions. One day after the experiment a remarkable neurodegeneration of CA1 hippocampal region was observed in both the awake and the anesthetized rats. We conclude that the OKA-induced epilepsy cannot be ascribed to increased extracellular glutamate, but to an increased sensitivity of NMDA receptor. We propose that halothane protected against the epilepsy because it blocks NMDA receptor overactivation, and that the neurodegeneration of CA1 region is independent of this overactivation and due probably to alterations of cytoskeletal proteins consequent to the OKA-induced hyperphosphorylation.     

Reducing Ability of the Striatum and Cerebral Cortex in Rats following Acute Administration of Risperidone or Haloperidol: An Estimation by in Vivo Electron Paramagnetic Resonance Imaging

In vivo temporal electron paramagnetic resonance (EPR) imaging of the blood-brain barrier-permeable nitroxide radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (PCAM), in the brain of rats was conducted following acute administration of risperidone (RSP) or haloperidol (HPD). The half-life of the signal intensity of PCAM was obtained from a selected area in the temporal EPR images. The half-lives in the striatum and cerebral cortex for the RSP- or HPD-treated rats were significantly longer than for the control rats (p < 0.01). This finding indicates that the reducing abilities of the striatum and cerebral cortex decreased in the rats to which either RSP or HPD had been acutely administrated because the half-life of PCAM in the selected region of the brain reflects its reducing ability.     

Effects of Metabotropic Glutamate Receptor Stimulation on Cerebral O2 Consumption and Blood Flow During Focal Cerebral Ischemia in Rats

This investigation was performed to evaluate the effects of ACPD [(1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid], a metabotropic glutamate receptor agonist, on cerebral O₂ consumption during focal cerebral ischemia. Male Wistar rats were placed in control (n = 7) and ACPD (n = 7) groups under isoflurane anesthesia. Twenty minutes after middle cerebral artery (MCA) occlusion, gauze sponges with 10^–5 M ACPD or normal saline were placed on the ischemic cortex (IC) for a period of 40 min and were changed every 10 min. One hour after MCA occlusion, regional cerebral blood flow (rCBF) was determined using the C¹⁴-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined using microspectrophotometry. There were no statistical differences in vital signs, blood gases, and hemoglobin between the groups. In the control group, the cerebral blood flow and oxygen consumption of the IC were significantly lower than the contralateral cortex (rCBF: 45 ± 11 vs. 110 ± 11 ml/min/100 g, O₂ consumption: 2.9 ± 0.4 vs. 5.4 ± 1.1 ml O₂/min/100 g). ACPD did not change regional cerebral blood flow of the IC, but did significantly increase the oxygen extraction (7.8 ± 0.2 vs. 6.9 ± 0.3 ml O₂/100 ml) and oxygen consumption of the IC (4.3 ± 1.5 vs. 2.9 ± 0.4) compared to the control IC. Our data demonstrated that topical application of 10^–25 M ACPD to the ischemic area worsened cerebral O₂ balance. These data suggest that metabotropic glutamate receptors are not maximally activated during ischemia in the temporal cortex.     

Effects of Endogenous Glutamate on Extracellular Concentrations of GABA, Dopamine, and Dopamine Metabolites in the Prefrontal Cortex of the Freely Moving Rat: Involvement of NMDA and AMPA/KA Receptors

Using microdialysis, interactions between endogenous glutamate, dopamine, and GABA were investigated in the medial prefrontal cortex of the freely moving rat. Interactions between glutamate and other neurotransmitters in the prefrontal cortex had already been studied using pharmacological agonists or antagonists of glutamate receptors. This research investigated whether glutamate itself, through the increase of its endogenous extracellular concentration, is able to modulate the extracellular concentrations of GABA and dopamine in the prefrontal cortex. Intracortical infusions of the selective glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) were used to increase the endogenous extracellular glutamate. PDC (0.5, 2, 8, 16 and 32 mM) produced a dose-related increase in dialysate glutamate in a range of 1–36 M. At the dose of 16 mM, PDC increased dialysate glutamate from 1.25 to 28 M. PDC also increased extracellular GABA and taurine, but not dopamine; and decreased extracellular concentrations of the dopamine metabolites DOPAC and HVA. NMDA and AMPA/KA receptor antagonists were used to investigate whether the increases of extracellular glutamate were responsible for the changes in the release of GABA, and dopamine metabolites. The NMDA antagonist had no effect on the increase of extracellular GABA, but blocked the decreases of extracellular DOPAC and HVA, produced by PDC. In contrast, the AMPA/KA antagonist blocked the increases of extracellular GABA without affecting the decreases of extracellular DOPAC and HVA produced by PDC. These results suggest that endogenous glutamate acts preferentially through NMDA receptors to decrease dopamine metabolism, and through AMPA/KA receptors to increase GABAergic activity in the medial prefrontal cortex of the awake rat.     

Effects of Branched-Chain L-Amino Acids, L-Phenylalanine, and L-Methionine on the Transport of L-Glutamine in Rat Brain Cortex In Vitro. Influence of Cations

Abstract: Uptake of L-glutamine (2 mM) by rat brain cortex slices against a concentration gradient is markedly inhibited (40%) by branched-chain Lamino acids (1 mM), L-phenylalanine (1 mM), or L-methionine (1 mM); that of L-asparagine (2 mM) is much less affected by these amino acids. Other amino acids investigated have little or no effect on cerebral L-glutamine uptake. The suppressions of L-glutamine uptake by the inhibitory amino acids are apparently blocked by high [K⁺], which itself has little or no effect on glutamine uptake. This abolition of suppression is partly explained by high [K⁺] retention of endogenous glutamine; in the absence of Ca²⁺ such retention disappears. The inhibitory amino acids (1 mM) also enhance the release of endogenous glutamine, exogenous glutamine with which slices have been loaded, or glutamine synthesized in the slices from exogenous glutamate. The enhanced release of endogenous glutamine is diminished by high [K⁺]. The suppression of glutamine uptake by the branched-chain amino acids is independent of the concentration of glutamine at low concentrations (0.25–0.5 mM), indicating non-competition, but is reduced with high concentration of glutamine. The inhibition by L-phenylalanine is noncompetitive. L-Glutamine (2 mM) exerts no inhibition of the cerebral uptakes of the branched-chain L-amino acids or Lphenylalanine (0.25–2 mM). The inhibitory amino acids are as active in suppressing L-glutamine uptake with immature rat brain slices as with adult, although the uptake, against a gradient, of L-glutamine in the infant rat brain is about one-half that in the adult. They are also just as inhibitory on the concentrative uptake of L-glutamine by a crude synaptosomal preparation derived from rat brain cortex. Such a nerve ending preparation takes up L-glutamine (0.25 mM), against a gradient, at about ninefold the rate at which it is taken up by cortex slices (for equal amounts of protein), and the uptake process is markedly suppressed by high [K⁺] in contrast to the effects of high [K⁺] with slices. The possible physiological and pathological consequences of the suppression of glutamine uptake are discussed.     

Extracellular levels of brain aspartate, glutamate and GABA during an inhibitory avoidance response in the rat

The extracellular levels of aspartate, glutamate and GABA were measured by microdialysis, coupled with an HPLC method, in rat prefrontal cortex (mPFC) and ventral hippocampus (VH) before and during the performance of a step-down inhibitory task. The basal levels of glutamate were about 50% higher than those of aspartate, and GABA levels were about 20-folds smaller than those of the excitatory amino acids. There were no significant differences in the basal levels of any of the three amino acids between the two brain regions. The extracellular levels of aspartate increased during acquisition and recall trials in both VH and mPFC, whereas those of glutamate increased in the VH during acquisition only. A significant increase in GABA levels was also detected during acquisition but only in the mPFC. The neuronal origin of the increased extracellular levels of aspartate, glutamate and GABA was demonstrated by administering tetrodotoxin directly into the mPFC or VH by reverse dialysis. These findings, together with previous evidence from our and other laboratories, indicate a differential release of aspartate and glutamate from excitatory neurons during the performance of behavioral responses, and therefore, distinct roles for the two excitatory amino acids should be envisaged.     

Reduction of Glutamate Uptake into Cerebral Cortex of Developing Rats by the Branched-Chain Alpha-Keto Acids Accumulating in Maple Syrup Urine Disease

In the current study we investigated the effect of the branched-chain alpha-keto acids (BCKA) co-ketoisocaproic (KIC), alpha-keto-beta-methylvaleric (KMV), and alpha-ketoisovaleric (KIV) acids, which accumulate in maple syrup urine disease (MSUD), on the in vitro uptake of [3H]glutamate by cerebral cortical slices from rats aged 9, 21, and 60 days of life. We initially observed that glutamate uptake into cerebral cortex of 9- and 21-day-old rats was significantly higher, as compared to that of 60-day-old rats. Furthermore, KIC inhibited this uptake by tissue slices at all ages studied, whereas KMV and KIV produced the same effect only in cortical slices of 21- and 60-day-old rats. Kinetic assays showed that KIC significantly inhibited glutamate uptake in the presence of high glutamate concentrations (50 microM and greater). We also verified that the reduction of glutamate uptake was not due to cellular death, as evidenced by tetrazolium salt and lactate dehydrogenase viability tests of cortical slices in the presence of the BCKA. It is therefore presumed that the reduced glutamate uptake caused by the BCKA accumulating in MSUD may lead to higher extracellular glutamate levels and potentially to excitotoxicity, which may contribute to the neurological dysfunction of the affected individuals.     

Influence of Perfusate Glucose Concentration on Dialysate Lactate, Pyruvate, Aspartate, and Glutamate Levels Under Basal and Hypoxic Conditions: A Microdialysis Study in Rat Brain

Abstract: The aim of this study was to evaluate the influence of perfusion media with different glucose concentrations on dialysate levels of lactate, pyruvate, aspartate (Asp), and glutamate (Glu) under basal and hypoxic conditions in rat brain neocortex. Intracerebral microdialysis was performed with the rat under general anesthesia using bilateral probes (o.d. 0.3 mm; membrane length, 2 mm) perfused with artificial CSF containing 0.0 and 3.0 m M glucose, respectively. Basal dialysate levels were obtained 2 h after probe implantation in artificially ventilated animals. Dialysate levels of glucose were also measured for the two different perfusion fluids. The mean absolute extracellular concentration of glucose was estimated by a modification of the no-net-flux method to be 3.3 mmol/L, corresponding to an average in vivo recovery of 6% for glucose. Hypoxia was induced by lowering the inspired oxygen concentration to 3%. Hypoxia caused a disturbance of cortical electrical activity, evidenced by slower frequency and lower amplitudes on the electroencephalogram compared with prehypoxic conditions. This was associated with significant elevations of lactate, Asp, and Glu levels. There were no statistically significant differences in dialysate metabolite levels between the two perfusion fluids, during either normal or hypoxic conditions. We conclude that microdialysis with glucose-free perfusion fluid does not drain brain extracellular glucose in anesthetized rats to the extent that the dialysate lactate, pyruvate, Asp, and Glu levels during basal or hypoxic conditions are altered.