Anders Lehmann

Ischemia-Induced Shift of Inhibitory and Excitatory Amino Acids from Intra- to Extracellular Compartments

Brain ischemia was induced for 10 or 30 min by clamping the common carotid arteries in rabbits whose vertebral arteries had previously been electrocauterized. EEG and tissue content of high energy phosphates were used to verify the ischemic state and to evaluate the degree of postischemic recovery. Extracellular levels and total contents of amino acids were followed in the hippocampus during ischemia and 4 h of recirculation. At the end of a 30-min ischemic period, GABA had increased 250 times, glutamate 160 times, and aspartate and taurine 30 times in the extracellular phase. The levels returned to normal within 30 min of reflow. A delayed increase of extracellular phosphoethanolamine and ethanolamine peaked after 1–2 h of reflow. Ten minutes of ischemia elicited considerably smaller but similar effects. With respect to total amino acids in the hippocampus, glutamate and aspartate decreased to 30–50% of control while GABA appeared unaffected after 4 h of reflow. Alanine, valine, phenylalanine, leucine, and isoleucine increased severalfold. The importance of toxic extracellular levels of excitatory amino acids, as well as of high extracellular levels of inhibitory amino acids, are considered in relation to the pathophysiology of neuronal cell loss during cerebral ischemia.

Effects of In Vivo Administration of Kainic Acid on the Extracellular Amino Acid Pool in the Rabbit Hippocampus

Abstract: The effect of local administration of kainic acid in the rabbit hippocampus was studied; the hippocampus was perfused continuously in the freely moving animal with an implanted 0.3‐mm dialysis fiber. The pattern of endogenous amino acids in the perfusate, reflecting extracellular amino acids, was monitored with liquid chromatography separation and fluorimetric detection of amino acid derivatives. Kainic acid was included in the perfusion medium for up to 70 min at 0.1–1.0 mM and, with time, induced epileptiform activity. Endogenous glutamic acid, taurine, and phosphoethanolamine levels were increased selectively at the lower perfusion concentrations of kainic acid. Long perfusion periods with higher concentrations increased the levels of virtually all amino acids. Perfusion of the hippocampus with depolarizing concentrations of potassium gave an amino acid response partly similar to that seen with kainic acid treatment. However, one notable difference between the two responses was that the extracellular concentration of glutamine, although not influenced by kainic acid, was significantly decreased after high potassium concentrations. These results confirm previous notions that kainic acid has a primarily excitatory effect, one manifestation of this effect being the release of glutamic acid.

The excitatory amino acid antagonist kynurenic acid administered after hypoxic-ischemia in neonatal rats offers neuroprotection

The neuroprotective effect of kynurenic acid, an unspecific antagonist of excitatory amino acid receptors, was evaluated in a model of hypoxic-ischemia in neonatal rats. One-week-old rats were subjected to ligation of the left carotid artery and exposure to 7.7% O2/92.3% N2 for 2 h. Kynurenic acid (300 mg/kg) was administered i.p. immediately after the period of hypoxic-ischemia in one group (n = 32) and compared with saline-treated (n = 27). After 2 weeks the rats were sacrificed and the brain damage evaluated by comparing the weight of the lesioned and unlesioned hemispheres. In rats receiving kynurenic acid the reduction in weight of the lesioned hemisphere was 25.4 +/- 3.3% as compared to 37.8 +/- 3.6% in saline-treated controls (P less than 0.001). The results suggest that excitatory amino acids are involved in the development of postischemic damage in the immature brain.

A role for taurine in the maintenance of homeostasis in the central nervous system during hyperexcitation

Employing the brain dialysis technique, we demonstrate that the aspartate congener N-methyl-D,L-aspartic acid (NMA) stimulates the release of endogenous taurine in vivo in a Ca2+-dependent manner. Furthermore, exogenous taurine (1-10 mM) inhibits the NMA-induced Ca2+ influx into intracellular compartments. This suggests that the extracellular taurine concentration may control Ca2+ movement and thereby provide a homeostatic mechanism in situations of excessive excitation.

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

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

Effects of status epilepticus on extracellular amino acids in the hippocampus

Extracellular amino acids were followed in the hippocampus during sustained seizures induced by systemic administration of kainic acid (KA) or bicuculline (BC). KA epilepsy was associated with marked increases in phosphoethanolamine (PEA) and taurine. Alanine and ethanolamine were moderately raised while other amino acids were unaffected. BC seizures encompassed a slightly different pattern of alterations. In contrast to KA seizures, BC epilepsy had no effect on taurine. Significant increments were observed for PEA and alanine while elevations of ethanolamine were subtle. In both types of seizures, glutamate and GABA remained unaffected extracellularly, probably due to efficient recapture mechanisms.

The neurotoxicity of ouabain, a sodium-potassium ATPase inhibitor, in the rat hippocampus

Intrahippocampal injection of 1 nmol ouabain, a sodium/potassium (Na+,K(+)-)ATPase inhibitor, produced a necrotic lesion within 4 days, characterised by a massive invasion by foaming macrophages. A lower dose of ouabain (0.1 nmol) produced a more discrete lesion of all groups of neuronal perikarya in the hippocampus, with only a minimal degree of glial infiltration. The neuronal perikaryal death produced in the subicular, CA1 and CA2 regions was only partially decreased by intraperitoneal injections of the anticonvulsants diazepam and MK-801; these drugs were without effect in the CA3 or hilar interneuronal regions. At neither dose of ouabain was there any indication of neuronal loss in brain regions outside the hippocampus, typically produced by prolonged seizure activity. It is suggested that ouabain has a two-fold action, a release of toxic acidic amino acids and a prolonged depolarization of neurons leading to osmolysis or calcium necrosis.

Effects of microdialysis-perfusion with anisoosmotic media on extracellular amino acids in the rat hippocampus and skeletal muscle

Abstract: Changes in the levels of amino acids have been implicated as being important in osmoregulation both within and outside the CNS. The present study addressed the question of whether changes in osmolality affect the extracellular concentration of amino acids in the rat hippocampus and femoral biceps muscle (FBM). Microdialysis probes were implanted in these tissues and perfused with standard physiological saline. Amino acid concentrations in the dialysate were determined with HPLC separation of o‐phthaldialdehyde derivatives and fluorescence detection. The osmolarity of the perfusion buffer was gradually decreased by reduction of the concentration of NaCl from 122 to 61 to 0 mM. In other experiments, the osmolarity was increased by elevation of the NaCl level from 122 to 183 to 244 mM or by addition of mannitol. Glutamate, aspartate, γ‐aminobutyrate, and alanine levels in dialysate from the hippocampus increased when the concentration of NaCl was decreased by 61 mM, and they were further elevated when NaCl was omitted. Taurine and phosphoethanolamine (PEA) levels were maximally elevated at the intermediary decrease of NaCl concentration, and glutamine in particular but also methionine and leucine were suppressed by perfusion with hypoosmolar medium. The amino acid response of the FBM differed substantially from that of the hippocampus. The aspartate content increased slightly, and there was a marginal transient increase in PEA level. Perfusion with media containing high concentrations of NaCl induced diminished dialysate levels of taurine, PEA, and glutamate, whereas levels of other amino acids were either unaffected or increased. Mannitol administration via the perfusion fluid led to reduced levels of taurine, PEA, glutamate, and aspartate. In contrast to the effects of high NaCl levels, hyperosmotic mannitol did not induce increases in level of any of the amino acids detected. The results suggest that taurine and PEA are involved in osmoregulation in the mammalian brain. From a quantitative viewpoint, taurine seems to be most important. Transmitter amino acids may also be involved in the maintenance of the volume of neural cells subjected to severe disturbances in osmotic equilibrium.

N‐Methylaspartate‐Evoked Liberation of Taurine and Phosphoethanolamine In Vivo: Site of Release

Abstract: The effect of N‐methyl‐d,l‐aspartic acid (NMA) on extracellular amino acids was studied in the rabbit hippocampus with the brain dialysis technique. Administration of 0.5 or 5 mM NMA caused a concentration‐dependent liberation of taurine and phosphoethanolamine (PEA). Taurine increased by 1,200% and PEA by 2,400% during perfusion with 5 mM NMA whereas most other amino acids rose by 20–100%. The effect of NMA appeared to be receptor‐mediated, as coperfusion with D‐2‐amino‐5‐phosphonovaleric acid curtailed the NMA response by some 90%. The NMA‐stimulated release of taurine and PEA was suppressed when Ca2+ was omitted and further inhibited when Co2+ was included in the perfusion medium. The effect of NMA was mimicked by the endogenous NMA agonist quinolinic acid and the partial NMA agonist d,l‐cis‐2,3‐piperidine dicarboxylic acid. Although the NMA‐evoked release of taurine and PEA was Ca2+‐dependent in vivo, NMA had no effect on Ca2+ accumulation in hippocampal synaptosomes. The previously reported NMA‐induced activation of dendritic Ca2+ spikes and the lack of effect on synaptosomal Ca2+ uptake suggest that taurine and PEA are released from sites other than nerve terminals, possibly from dendrosomatic sites. This notion was strengthened by the absence of an effect of NMA on the efflux of radiolabelled taurine from hippocampal synaptosomes. In contrast, high K+ stimulated synaptosomal uptake of Ca2+ and release of taurine.

The neurotoxicity of zinc in the rat hippocampus

Intrahippocampal injections of zinc chloride (5-10 nmol) caused a discrete lesion in the rat hippocampus, involving all neuronal perikarya. In addition to the necrosis, the lesion was also characterized by a decrease in staining of the neuropil, the presence of pyknotic neurons, and occasionally infarction. Pathological changes occurred within 8 h of an injection, and neuronal loss, as judged by the loss of Nissl staining, was complete within 24 h. On the other hand, the loss of acidophilic staining of the neurons was more gradual, as acid fuchsin staining was still present in neurons in the periphery of the damaged area 4 days later. In comparison with an excitotoxic lesion, glial infiltration into the damaged area was minimal, even up to 3 weeks later, suggesting that some glial cell toxicity also occurred.