Extracellular Glycine Concentrations Research Articles

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.

Glycine receptor regulation of neurokinin1 receptor function in rat dorsal horn neurones.

Activation of spinal neurokinin1 (NK1) receptors leads to increases in the extracellular concentration of glycine in the dorsal horn. We have investigated the role of the inhibitory glycine receptor as a regulator of NK1 receptor-mediated effects on dorsal horn neurones. Ionophoretic application of GR82334, a selective NK1 antagonist, did not alter dorsal horn neuronal activity evoked by cutaneous applications of mustard oil. However, in the presence of the glycine antagonists, strychnine or phenylbenzene-omega-phosphono-alpha-amino acid (PMBA), GR82334 displayed inhibitory properties. Therefore inhibitory glycine receptors may mask the contribution made by NK1 receptors to nociceptive processing. This is discussed with reference to the role of NK1 receptors during brief and long duration nociceptive transmission.

Potentiation of NMDA receptor-mediated responses by dynorphin at low extracellular glycine concentrations.

The effect of dynorphin A(1-13) on N-methyl-D-aspartate (NMDA)-activated currents was investigated in the presence of low extracellular glycine concentrations in Xenopus oocytes expressing recombinant heteromeric NMDA receptors and in cultured hippocampal neurons with the use of voltage-clamp techniques. At an extracellular added glycine concentration of 100 nM, dynorphin A(1-13) (10 microM) greatly increased the amplitude of NMDA-activated currents for all heteromeric subunit combinations tested; on average, the potentiation was: epsilon1/zeta1, 3,377 +/- 1,416% (mean +/- SE); epsilon2/zeta1, 1,897 +/- 893%; epsilon3/zeta1, 4,356 +/- 846%; and epsilon4/zeta1, 1,783 +/- 503%. Potentiation of NMDA-activated current by dynorphin A(1-13) was concentration dependent between 0.1 and 10 microM dynorphin A(1-13), with a half-maximal concentration value of 2.77 microM and an apparent Hill coefficient of 2.53, for epsilon2/zeta1 subunits at 100 nM added extracellular glycine. Percentage potentiation by dynorphin A(1-13) was maximal at the lowest glycine concentrations tested (0.01 and 0.1 microM), and decreased with increasing glycine concentration. No significant potentiation was observed at glycine concentrations > 0.1 microM for epsilon1/zeta1, epsilon2/zeta1, and epsilon4/zeta1 subunits, or at > 1 microM for epsilon3/zeta1 subunits. Potentiation of NMDA-activated currents by dynorphin A(1-13) was not inhibited by 1 microM of the kappa-opioid receptor antagonist nor-binaltorphimine, and potentiation was not observed with 10 microM of the kappa-opioid receptor agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzene-acetamide. Potentiation of NMDA-activated current by dynorphin A(1-13) was inhibited by the glycine antagonist kynurenic acid (50 microM). NMDA-activated current was also potentiated at low glycine concentrations by 10 microM dynorphin A(2-13) or (3-13), both of which have a glycine as the first amino acid, but not by 10 microM dynorphin A(4-13), which does not have glycine as an amino acid. In hippocampal neurons, 10 microM dynorphin A(1-13) or (2-13) potentiated steady-state NMDA-activated current in the absence of added extracellular glycine. The extracellular free glycine concentration, determined by high-performance liquid chromatography, was between 26 and 36 nM for the bathing solution in presence or absence of 10 microM dynorphin A(1-13), (2-13), (3-13), or (4-13), and did not differ significantly among these solutions. The observations are consistent with the potentiation of NMDA-activated current at low extracellular glycine concentrations resulting from an interaction of the glycine amino acids in dynorphin A(1-13) with the glycine coagonist site on the NMDA receptor. Because dynorphin A is an endogenous peptide that can be coreleased with glutamate at glutamatergic synapses, the potentiation of NMDA receptor-mediated responses could be an important physiological regulator of NMDA receptor function at these synapses.

Differential effects of nipecotic acid and 4,5,6,7-tetrahydroisoxazolo[4,5- c]pyridin-3-ol on extracellular γ-aminobutyrate levels in rat thalamus

Using the microdialysis technique and HPLC (high-performance liquid chromatography) determination of amino acids, the extracellular concentrations of γ-aminobutyrate (GABA), glutamate, aspartate and a number of other amino acids were determined in rat thalamus during infusion through the microdialysis tubing of the GABA transport inhibitors 4,5,6,7-tetrahydroisoxazolo[4,5- c]pyridin-3-ol (THPO) and nipecotic acid. Administration of 5.0 mM THPO led to a 200% increase in the extracellular GABA concentration. Simultaneous infusion of THPO and GABA (50 μM) increased the extracellular GABA concentration to 1200% of the basal level whereas GABA alone was found to increase the GABA level to 500%. If nipecotic acid (0.5 mM) was administered together with GABA (50 μM) the extracellular concentration of GABA was not increased further. While administration of GABA alone or GABA together with nipecotic acid had no effect on the extracellular levels of glutamate and aspartate it was found that GABA plus THPO increased the extracellular concentration of these amino acids. GABA administered alone or together with nipecotic acid or THPO led to relatively small but significant increases in the extracellular concentrations of the amino acids glycine, glutamine, serine and threonine. The results demonstrate that THPO, which preferentially inhibits glial GABA uptake and which is not a substrate for the GABA carriers, was more efficient increasing the extracellular concentration of GABA than nipocotic acid which is a substrate and an inhibitor of both neuronal and glial GABA uptake. This indicates that GABA uptake inhibitors that are not substrates for the carrier and which preferentially inhibit glial GABA uptake may constitute a group of drugs by which the efficacy of GABAergic neurotransmission may be enhanced.

High extracellular glycine does not potentiate N-methyl- d-aspartate-evoked depolarization in vivo

As N-methyl- d-aspartate receptor (NMDA) ionophore complexes have a distinct positive, allosteric regulatory site for glycine, it has been proposed that elevated extracellular glycine during or after cerebral ischaemia may induce excessive NMDA/glutamate receptor activation and, thereby, excitotoxicity. To test this hypothesis, we have perfused increasing concentrations of glycine, either alone or with co-application of NMDA, through a microdialysis probe implanted in the striatum of halothane anaesthetized rats. Changes in the extracellular field (DC) potential indicative of depolarization were recorded precisely at the site of drug application by an electrode incorporated within the dialysis fibre. Microdialysis application of up to 1 mM of glycine had no effect on the basal DC potential. Above 10 mM, glycine produced concentration-dependent depolarizations, but the amplitude of these responses remained very small (e.g. 0.52±0.05 mV for 100 mM glycine, n=10; i.e. around 30-fold smaller than that of a wave of spreading depression). Application of 200 μM NMDA via the microdialysis probe produced consistent short-lasting depolarizations (around 2.5 mV amplitude), but these were not potentiated by co-application of up to 100 mM glycine. These data do not support the view that increased extracellular concentrations of glycine, such as those observed in ischaemia, may be potentially excitotoxic. Nevertheless, as occupation of the glycine site coupled to the NMDA-receptor is required for NMDA/glutamate receptor activation, this site remains an attractive target for potential neuroprotective agents.

Glycine regulation of synaptic NMDA receptors in hippocampal neurons.

1. Although glycine has been identified as a required coagonist with glutamate at N-methyl-D-aspartate (NMDA) receptors, the understanding of glycine's role in excitatory synaptic neurotransmission is quite limited. In the present study, we used the whole cell patch-clamp technique to examine the ability of glycine to regulate current flow through synaptic NMDA receptors at excitatory synapses between cultured hippocampal neurons and in acutely isolated hippocampal slices. 2. These studies demonstrate that the glycine modulatory site on the synaptic NMDA receptor is not saturated under baseline conditions and that increased glycine concentrations can markedly increased NMDA-receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal neurons in both dissociated cell culture and in slice. Saturation of the maximal effect of glycine takes place at different concentrations for different cells in culture, suggesting the presence of heterogenous NMDA receptor subunit compositions. 3. Bath-applied glycine had no effect on the time course of EPSCs in either brain slice or culture, indicating that desensitization of the NMDA receptor is not prevented by glycine over the time course of an EPSC. 4. When extracellular glycine concentration is high, all miniature EPSCs recorded in the cultured hippocampal neurons contained NMDA components, indicating that segregation of non-NMDA receptors at individual synaptic boutons does not occur.

Effect of intrahippocampal dexamethasone on the levels of amino acid transmitters and neuronal excitability

Direct effect of type-II corticosteroid receptor agonist desamethasone on extracellular amino acid levels and neuronal excitability in the hippocampus was studied by simultaneous application of in vivo microdialysis and recording hippocampal evoked responses in freely moving male rats. Microdialysis probes and hippocampal recording electrodes were implanted to the CA1-CA3 regions of dorsal hippocampus. Local dexamethasone infusion via microdialysis resulted in a transient increase in glutamate level at 30 min, while glutamine decreased by 30–40% throughout the 180-min sampling period. Taurine increased by 50% and remained elevated up to 180 min. No significant changes were detected in extracellular concentration of asparagine, arginine, glycine, threonine, alanine and serine. In contrast, dexamethasone infusion to the striatum had no effect on the extracellular levels of amino acid transmitters. Effect of dexamethasone injected via microdialysis on the neural activity elicited by perforant path stimulation was a decrease in population spikes after 60 min starting dexamethasone infusion. Steroid effect on neural excitability was reversible. Our data indicate that local infusion of type-II receptor agonist dexamethasone has a complex effect in the hippocampus, starts with a change in extracellular glutamate and glutamine concentration and followed by a reduced synaptic excitability.

Effect of intrahippocampal dexamethasone on the levels of amino acid transmitters and neuronal excitability

Direct effect of type-II corticosteroid receptor agonist desamethasone on extracellular amino acid levels and neuronal excitability in the hippocampus was studied by simultaneous application of in vivo microdialysis and recording hippocampal evoked responses in freely moving male rats. Microdialysis probes and hippocampal recording electrodes were implanted to the CA1-CA3 regions of dorsal hippocampus. Local dexamethasone infusion via microdialysis resulted in a transient increase in glutamate level at 30 min, while glutamine decreased by 30–40% throughout the 180-min sampling period. Taurine increased by 50% and remained elevated up to 180 min. No significant changes were detected in extracellular concentration of asparagine, arginine, glycine, threonine, alanine and serine. In contrast, dexamethasone infusion to the striatum had no effect on the extracellular levels of amino acid transmitters. Effect of dexamethasone injected via microdialysis on the neural activity elicited by perforant path stimulation was a decrease in population spikes after 60 min starting dexamethasone infusion. Steroid effect on neural excitability was reversible. Our data indicate that local infusion of type-II receptor agonist dexamethasone has a complex effect in the hippocampus, starts with a change in extracellular glutamate and glutamine concentration and followed by a reduced synaptic excitability.

Effects of hypothermia, pentobarbital, and isoflurane on postdepolarization amino acid release during complete global cerebral ischemia.

Hypothermia and anesthetics may protect the brain during ischemia by blocking the release of excitatory amino acids. The effects of hypothermia (28 degrees C), pentobarbital, and isoflurane on postischemic excitatory amino acid concentrations were compared. Rats were anesthetized with 0.8% halothane/50% N2O, vascular catheters were placed, and a glass microelectrode and microdialysis cannula were inserted into the cerebral cortex. Experimental groups were: (1) control, pericranial, t = 38 degrees C; (2) hypothermia, t = 28 degrees C; (3) pentobarbital, t = 38 degrees C; and (4) isoflurane, t = 38 degrees C. Halothane/N2O was continued in groups 1 and 2, whereas a deep burst-suppression or isoelectric electroencephalogram was achieved with the test drugs in groups 3 and 4. Cerebral metabolic rates were similar in groups 2, 3, and 4. After a baseline dialysis sample was collected, animals were killed with potassium chloride. The time to terminal depolarization was recorded, after which three consecutive 10-min dialysate samples were collected. Glutamate, aspartate, gamma-aminobutyric acid, and glycine concentrations were measured using high-performance liquid chromatography. Times to terminal depolarization were shorter in both pentobarbital and isoflurane groups than with hypothermia (103 +/- 15 and 127 +/- 10 vs. 195 +/- 20 s respectively, mean +/- SD). However, times to terminal depolarization in all three groups were longer than in control subjects (control = 70 +/- 9s). Postdepolarization concentrations of all compounds were lower in hypothermic animals (vs. normothermic control animals), but no reductions in glutamate, aspartate, or glycine concentrations were noted in pentobarbital or isoflurane groups. gamma-Aminobutyric acid concentrations were reduced by both anesthetics, but not to the same degree as with hypothermia. Pentobarbital and isoflurane prolonged the time to terminal depolarization, but did not influence the rate at which the extracellular concentrations of glutamate, aspartate, or glycine increased. By contrast, hypothermia reduced the release of all excitatory amino acids. These differences may explain the greater protective efficacy of hypothermia in the face of cerebral ischemia.

229 Selective formation of silent synapse on immature postsynaptic cells in coculture of hetero-aged neurons

The effects of 1-methyl-4-phenylpyridinium ion (MPP+) on N-methyl-d-aspartate (NMDA) receptor-channel complex were studied in rat hippocampal neurons using intracellular- and whole-cell voltage clamp-recording techniques. Intracellular recordings were made from CA1 pyramidal cells of rat hippocampal slices in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM) and picrotoxin (PTX; 50 μM) which block non-NMDA and GABAA receptors, respectively. Superfusion of MPP+ reversibly decreases the pharmacologically isolated NMDA receptor-mediated excitatory postsynaptic potential (EPSPNMDA) in a concentration-dependent manner. In other experiments, we observed that MPP+ attenuated NMDA-evoked whole-cell currents in a voltage- and use-dependent manner and was not dependent on the extracellular glycine or spermine concentration on neurons freshly dissociated from rat hippocampi CA1 region. These results suggest that MPP+, applied at micromolar concentrations, is a non-competitive NMDA receptor antagonist in rat hippocampal neurons.

Profound systemic hypothermia inhibits the release of neurotransmitter amino acids in spinal cord ischemia

Profound hypothermia induced with cardiopulmonary bypass has a protective effect on spinal cord function during operations on the thoracoabdominal aorta. The mechanism of this protection remains unknown. It has been proposed that the release of excitatory amino acids in the extracellular space plays a causal role in irreversible neuronal damage. We investigated the changes in extracellular neurotransmitter amino acid concentrations with the use of in vivo microdialysis in a swine model of spinal cord ischemia. All animals underwent left thoracotomy and right atrium-femoral artery cardiopulmonary bypass with additional aortic arch perfusion. Lumbar laminectomies were then done and microdialysis probes were inserted stereotactically in the anterior horn of the second and fourth segments of the lumbar spinal cord. The probes were perfused with artificial cerebrospinal fluid at a rate of 2 microliters/min and 15-minute samples were assayed by high-performance liquid chromatography. Group 1 animals (n = 6) underwent aortic clamping distal to the left subclavian artery and proximal to the renal arteries for 60 minutes at normothermia (37 degrees C) and group 2 animals (n = 5) were cooled to a rectal temperature of 20 degrees C before application of aortic clamps, maintained at this level during cardiopulmonary bypass until the aorta was unclamped, and then slowly rewarmed to 37 degrees C. Seven amino acids were studied, including two excitatory neurotransmitters (glutamate and aspartate) and five putative inhibitory neurotransmitters (glycine, gamma-aminobutyric acid, serine, adenosine, and taurine). Glutamate exhibited a threefold increase in extracellular concentration during normothermic ischemia compared with baseline values and remained elevated until 60 minutes after reperfusion. The increase in aspartate concentration was not significant. The extracellular concentrations of glycine and gamma-aminobutyric acid also increased significantly during ischemia and reperfusion. Hypothermia uniformly prevented the release of amino acids in the extracellular space. Glutamate levels remained significantly decreased even after rewarming to normothermia whereas glycine levels returned to baseline values. These results are consistent with a role for excitatory amino acids in the production of ischemic spinal cord injury and suggest that the mechanism of hypothermic protection may be related to decreased release of these amino acids in the ischemic spinal cord.

Intracerebral Human Microdialysis

In vivo microdialysis was introduced in 1982 as a technique to study cerebral neurochemistry in awake, freely moving animals. In small animals, bilateral carotid occlusion produces a 7- to 10-fold increase in extracellular glutamate concentrations. This rapidly falls with reperfusion. Increase in extracellular glutamate is currently believed to be a major factor in initiating neuronal injury. Glutamate antagonists are currently undergoing clinical trials in acute stroke. Human data on the extracellular levels of glutamate and other amino acids in the normal or ischemic brain are limited. In this communication we wish to report the extracellular concentrations of glutamate, serine, glutamine, glycine, taurine, alanine, and gamma-aminobutyric acid, as monitored by in vivo microdialysis, in the simulated ischemic model of the temporal lobe of the human brain. Intracerebral microdialysis was carried out in five patients who underwent resection of the temporal lobe for intractable epilepsy. Surgical excision leads to an acute (from partial to total, ie, from incomplete to complete) ischemic state of the resected brain. This was our model to study the changes in human extracellular fluid during acute focal ischemic conditions. Extracellular glutamate concentrations were 15 to 30 mumol/L in the preischemic samples. This increased to 380.69 +/- 42.14 mumol/L with partial (incomplete) ischemia and reached a peak of 1781.67 +/- 292.34 mumol/L (> 100-fold) with total isolation of the temporal pole (complete ischemia). The levels fell to 394.52 +/- 72.93 mumol/L 20 minutes after resection. Similar trends were observed with the onset of ischemia in the dialysate levels of serine, glutamine, glycine, alanine, taurine, and gamma-aminobutyric acid. Our results show that there is a significant increase in extracellular glutamate and other neurotransmitters with ischemia in the temporal lobe model of the human brain. This increase is of a higher magnitude than that in small animals.

Voltage- and use-dependent block by 1-methyl-4-phenylpyridinium ion (MPP +) of N-methyl- d-aspartate-activated currents in rat hippocampal neurons

The effects of 1-methyl-4-phenylpyridinium ion (MPP +) on N-methyl- d-aspartate (NMDA) receptor-channel complex were studied in rat hippocampal neurons using intracellular- and whole-cell voltage clamp-recording techniques. Intracellular recordings were made from CA1 pyramidal cells of rat hippocampal slices in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM) and picrotoxin (PTX; 50 μM) which block non-NMDA and GABA A receptors, respectively. Superfusion of MPP + reversibly decreases the pharmacologically isolated NMDA receptor-mediated excitatory postsynaptic potential (EPSPNMDA) in a concentration-dependent manner. In other experiments, we observed that MPP + attenuated NMDA-evoked whole-cell currents in a voltage- and use-dependent manner and was not dependent on the extracellular glycine or spermine concentration on neurons freshly dissociated from rat hippocampi CA1 region. These results suggest that MPP +, applied at micromolar concentrations, is a non-competitive NMDA receptor antagonist in rat hippocampal neurons.

Effect of hyperglycemia on peri-ischemic neurotransmitter levels in the rabbit hippocampus

This study examined the effect of preexisting hyperglycemia on the extracellular concentrations of glutamate and glycine in the rabbit hippocampus using in vivo microdialysis during brief episodes of transient global ischemia. Hyperglycemia has repeatedly been shown to exacerbate the neurologic injury produced by episodes of global cerebral ischemia. Under hypoxic conditions, glucose may be metabolized to glutamate, a known neurotoxin which has been implicated as a mediator of ischemic neuronal cell death. In this study, microdialysis probes were stereotacticly inserted into the dorsal hippocampus of anesthetized rabbits. Animals were randomized to receive an i.v. infusion of either saline or dextrose. Global cerebral ischemia was then produced by the combination of neck tourniquet inflation and the induction of systemic hypotension. Administration of dextrose had no effect of these basal levels of glutamate or glycine. During ischemia, glutamate and glycine concentrations increased several-fold when compared with baseline. However, hippocampal glutamate concentrations were lower in the dextrose-treated groups during the peri-ischemic period ( P = 0.02). Glycine concentrations were higher during the reperfusion period in the dextrose-treated animals when compared with saline controls ( P = 0.03). The increased concentration of extracellular glycine which was observed in the dextrose-treated animals may contribute to the neurologic injury which occurs during episodes of global ischemia. The results of this study suggest that hyperglycemia does not exert its detrimental effects by increasing the extracellular concentration of glutamate.

Effect of hypocapnia on extracellular glutamate and glycine concentrations during peri-ischemic period in the rabbit hippocampus.

Glutamate (GLU) is a neurotransmitter. Massive release of GLU and glycine (GLY) into the brain's extracellular space may be triggered by ischemia, and may result in acute neuronal lysis or delayed neuronal death. The aim of this study was to evaluate the possible relationship between hyperventilation and the level of GLU and GLY during brain ischemia. Rabbits were anesthetized with halothane and oxygen. Group 1 was allowed to hyperventilate (PaCO2 25-35 mmHg). PaCO2 was maintained throughout the study. Group 2 was a normal control group that maintained normocapnia. Two global cerebral ischemic episodes were produced. Microdialysate was collected during the peri-ischemic and reperfusion periods from the dorsal hippocampus. GLU and GLY concentrations were determined using high-performance liquid chromatography. In the control group, GLU and GLY were significantly elevated during each episode of ischemia; these levels returned to baseline within 10 minutes after reperfusion. In contrast, in the hyperventilation group GLU and GLY concentrations increased during ischemia, but they were not statistically significant. We were able to demonstrate that hypocapnia during periischemic period lowered extracellular GLU and GLY concentrations. These results can explain a part of the protective action of hypocapnia during cerebral ischemia.

Effects of Hypothermia or Anesthetics on Hippocampal Glutamate and Glycine Concentrations after Repeated Transient Global Cerebral Ischemia

The search for cerebroprotective pharmacologic interventions has been based on the assumption that reducing the cerebral metabolic rate may enhance the cerebral tolerance for ischemic episodes. Recently, evidence has accumulated implicating excitatory amino acids (e.g., glutamate) as mediators of ischemic brain injury. We investigated the effects of mild hypothermia (32 degrees C), pentobarbital, isoflurane, and propofol on hippocampal extracellular concentrations of glutamate and glycine after repeated global ischemia. New Zealand white rabbits were initially anesthetized with halothane in oxygen. Brain epidural temperature was reduced by external cooling in the hypothermia group to 32 degrees C (n = 5). A burst-suppressed electroencephalogram pattern was achieved in the other groups with isoflurane (n = 7), pentobarbital (n = 6), or propofol (n = 6). Halothane-anesthetized rabbits (1% inspired) served as the control group (n = 5). In all groups, two global cerebral ischemic episodes (each 7.5 min) were produced by a combination of neck tour niquet inflation and induction of systemic hypotension. Periischemic hippocampal glutamate and glycine concentrations were estimated using in vivo microdialysis and high-performance liquid chromatography (two-way analysis of variance, P < 0.05). Glutamate concentrations were similar in the five groups during the baseline period. Hypothermia (32 degrees C) was associated with significantly lower concentrations of glutamate during both the first and second ischemic periods when compared with all other groups. Although there were no differences in glycine concentrations among groups during the first ischemic episode, glycine concentrations were significantly lower in the hypothermic group compared with the control, isoflurane, and pentobarbital groups during the second episode of cerebral ischemia. Glycine concentrations also were lower in the propofol group when compared to the isoflurane and pentobarbital groups. Hypothermia (32 degrees C) attenuates ischemia-induced increases in both glutamate and glycine concentrations after repeated global cerebral ischemia. Propofol attenuated glycine increases in a manner similar to that of hypothermia but did not attenuate ischemia-induced glutamate increases. There were no differences in hippocampal glutamate or glycine concentrations for animals receiving isoflurane, halothane, or pentobarbital.

Selective reduction of N-methyl-D-aspartate-evoked responses by 1,3-di(2-tolyl)guanidine in mouse and rat cultured hippocampal pyramidal neurones.

1. The effects of 1,3-di(2-tolyl)guanidine (DTG) were examined on the responses of cultured hippocampal neurones to the excitatory amino acid analogues N-methyl-D-aspartate (NMDA), kainate, quisqualate and (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). 2. In rat hippocampal neurones loaded with the Ca(2+)-sensitive dye Fura-2, DTG (10-100 microM) produced a concentration-dependent depression of the NMDA-evoked rises in intracellular free calcium ([Ca2+]i), an effect that was not modified by changes in the extracellular glycine concentration. DTG (at 50 and 100 microM) also attenuated, although to a lesser extent, the rises in [Ca2+]i evoked by naturally-derived quisqualate. In contrast, 50 and 100 microM DTG did not depress responses evoked by kainate, AMPA and synthetic, glutamate-free (+)-quisqualate although on occasions DTG enhanced kainate- and AMPA-evoked rises in [Ca2+]i. 3. DTG attenuated NMDA-evoked currents recorded from mouse hippocampal neurones under whole-cell voltage-clamp with an IC50 (mean +/- s.e. mean) of 37 +/- 5 microM at a holding potential of -60 mV. The DTG block of NMDA-evoked responses was not competitive in nature and was not dependent on the extracellular glycine or spermine concentration. The block did, however, exhibit both voltage-, and use-, dependency. The steady-state current evoked by naturally-derived quisqualate was also attenuated by DTG whereas those evoked by kainate and AMPA were not. 4. We conclude that DTG, applied at micromolar concentrations, is a selective NMDA antagonist in cultured hippocampal neurones, the block exhibiting both Mg(2+)- and phencyclidine-like characteristics. Given the nanomolar affinity of DTG for sigma binding sites it is unlikely that the antagonism observed here is mediated by sigma-receptors, but the data emphasize the potential danger of ascribing the functional consequences of DTG administration solely to sigma receptor-mediated events.

Effect of S-emopamil, nimodipine, and mild hypothermia on hippocampal glutamate concentrations after repeated cerebral ischemia in rabbits.

We sought to determine the effects of two different calcium channel antagonists, S-emopamil and nimodipine, on hippocampal glutamate and glycine release and to compare their effects to those of mild hypothermia. New Zealand White rabbits were subjected to two 7.5-minute episodes of global cerebral ischemia at a 1-hour interval produced by neck tourniquet inflation (20 psi) combined with hypotension during halothane anesthesia. Hippocampal extracellular concentrations of glutamate and glycine were monitored using in vivo microdialysis. Animals were randomized to receive either S-emopamil (1 mg.kg-1 bolus, 0.1 mg.kg-1.min-1 infusion), nimodipine (10 micrograms.kg-1 bolus, 1 microgram.kg-1.min-1 infusion), hypothermia (32 degrees C), or saline (control) before ischemia. Drug infusion and hypothermia were continued throughout the study periods. In all four groups, both ischemic episodes resulted in significant elevations of hippocampal extracellular concentrations of glutamate and glycine (baseline vs peak, P < .01 in all groups). However, glutamate levels were significantly lower in the S-emopamil (P = .0001) and hypothermia (P = .0003) groups when compared with the control group after the onset of the first ischemic episode through 1 hour after the second ischemic episode. There was no significant difference between the four groups in the concentrations of glycine. There was no significant difference between the peak concentrations of glutamate or glycine after each ischemic period. These results suggest that preischemic administration of S-emopamil, but not nimodipine, attenuates the increase in hippocampal extracellular concentrations of glutamate in the peri-ischemic period in this model, and that this effect is also observed when mild hypothermia is instituted before ischemia. Decreased concentrations of glutamate after ischemic episodes may be a possible mechanism for the observed neuroprotective properties of S-emopamil and mild hypothermia (32 degrees C).

Haloperidol interacts with the strychnine-insensitive glycine site at the NMDA receptor in cultured mouse hippocampal neurones

N-Methyl-D-aspartate (NMDA)-evoked responses in voltage-clamped hippocampal neurones in culture were reversibly, but not completely, attenuated on superfusion with micromolar concentrations (0.1–100 μM) of haloperidol with an IC 50 (± S.E.M.) value of 1.9 ± 0.2 μM (n = 7). In contrast, kainate- and (RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-evoked responses were relatively unaffected on application of 50 μM haloperidol. The NMDA receptor antagonist action of haloperidol was neither competitive in nature nor voltage-dependent but was reduced upon elevation of the extracellular concentration of glycine. Furthermore, in the absence of added glycine haloperidol (at 0.1 μM) frequently potentiated NMDA-evoked responses. Haloperidol thus appears to be a partial agonist for the strychnine-insensitive glycine site associated with the NMDA receptor-channel complex.

AMPA and NMDA receptor antagonists do not decrease hippocampal glutamate concentrations during transient global ischemia.

Increased extracellular concentrations of glutamate during episodes of cerebral ischemia may be due in part to a positive glutaminergic feedback loop. We evaluated the effect of selective AMPA or NMDA receptor antagonists on hippocampal extracellular concentrations of excitatory amino acids during ischemia and reperfusion. Thirteen New Zealand white rabbits were subjected to 10 min of global cerebral ischemia produced by neck tourniquet inflation (20 psi) combined with systemic hypotension during halothane (1-1.5%) anesthesia. Hippocampal extracellular concentrations of glutamate, aspartate, and glycine were monitored using in vivo microdialysis. NBQX (a selective AMPA receptor antagonist), MK801 (a noncompetitive NMDA receptor antagonist), or 5% dextrose was administered starting 1 h before ischemia. The NBQX group (n = 4) received 5 mg.kg-1 of NBQX intravenously (dissolved in 5% dextrose) over 5 min followed by an infusion of 5 mg.kg-1.h-1. The 5% dextrose group (n = 4) received an equivalent volume of 5% dextrose. The peak concentrations of glutamate, aspartate, and glycine in the early reperfusion period were 5-8-fold, 9-10-fold, and 4-5-fold higher than preischemic values, respectively. There were no significant differences, however, among the three groups in the concentrations of glutamate, aspartate, or glycine at any time during the study. These results do not support the existence of a positive feedback loop for glutamate mediated via AMPA or NMDA autoreceptors in the hippocampus during transient global ischemia or reperfusion.