Redistribution of glutamate and glutamine in slices of human neocortex exposed to combined hypoxia and glucose deprivation in vitro.

This study was undertaken to elucidate the roles of neurons and glial cells in the handling of glutamate and glutamine, a glutamate precursor, during cerebral ischemia. Slices (400-600 microns) from human neocortex obtained during surgery for epilepsy or brain tumors were incubated in artificial cerebrospinal fluid and subjected to 30 min of combined hypoxia and glucose deprivation (an in vitro model of brain ischemia). These slices, and control slices that had not been subjected to "ischemic" conditions, were then fixed and embedded. Ultrathin sections were processed according to a postembedding immunocytochemical method with polyclonal antibodies raised against glutamate or glutamine, followed by colloidal gold-labeled secondary antibodies. The gold particle densities over various tissue profiles were calculated from electron micrographs using a specially designed computer program. Combined hypoxia and glucose deprivation caused a reduced glutamate immunolabeling in neuronal somata, while that of glial processes increased. Following 1 h of recovery, the glutamate labeling of neuronal somata declined further to very low values, compared to control slices. The glutamate labeling of glial cells returned to normal levels following recovery. In axon terminals, no consistent change in the level of glutamate immunolabeling was observed. Immunolabeling of glutamine was low in both nerve terminals and neuronal somata in normal slices and was reduced to nondetectable levels in nerve terminals upon hypoxia and glucose deprivation. This treatment was also associated with a reduced glutamine immunolabeling in glial cells. Reversed glutamate uptake due to perturbations of the transmembrane ion concentrations and membrane potential probably contributes to the loss of neuronal glutamate under "ischemic" conditions. The increased glutamate labeling of glial cells under the same conditions can best be explained by assuming that glial cells resist a reversal of glutamate uptake, and that their ability to convert glutamate into glutamine is compromised due to the energy failure. The persistence of a nerve terminal pool of glutamate is compatible with recent biochemical data indicating that the exocytotic glutamate release is contingent on an adequate energy supply and therefore impeded during ischemia.

Calcium-dependent release of glutamate from human cerebral cortex.

The release of the excitatory amino acids glutamate and aspartate from human neocortex was investigated in vitro by utilizing brain tissue removed during anterior temporal lobectomies for tumor or epilepsy. Depolarization (50 mM K+) increased the glutamate release to 291% of control (809 pmol/mg/min) during blocked synaptic transmission and to 669% (1859 pmol/mg/min) when synaptic transmission was not blocked. Aspartate release increased to 141% (326 pmol/mg/min) and 178% (412 pmol/mg/min) respectively. The difference between release with and without blocked synaptic transmission was statistically significant only for glutamate (P less than 0.01). These data provides evidence for a Ca(2+)-dependent release of glutamate, supporting a possible role of this amino acid as a neurotransmitter in human neocortex.

Calcium dependent release of gamma-aminobutyric acid (GABA) from human cerebral cortex.

The release of the amino acids GABA, taurine, glycine, glutamine and leucine from human neocortex was investigated in vitro by utilizing brain tissue removed during 8 standard temporal lobectomies for epilepsy or tumor. Slices (0.5 mm thick) were cut from each biopsy and randomly placed in three different chambers. After 90 min preincubation, the three sets of slices were incubated for 60 s in wells containing, respectively, (A) regular ACSF (control), (B) ACSF with 50 mM K+ (to depolarize the cell membrane) and (C) ACSF with 50 mM K+, 0 mM Ca2+ and 4 mM Mg2+ (depolarization during blocked synaptic transmission). The content of amino acids in the wells was determined by high-performance liquid chromatography after pre-column derivatization of the amino acids with o-phthalaldehyde. Membrane depolarization (well B) increased the GABA release to 650% (620 pmol/mg) of control (well A, 95 pmol/mg). Blocking synaptic transmission (well C) reduced the evoked release by 50% (360 pmol/mg). The release of glycine, taurine, glutamine and leucine during membrane depolarization was not significantly different from the control values. The data provide evidence for a Ca(2+)-dependent release of GABA, supporting a possible role of this amino acid as a neurotransmitter in human neocortex.

Zinc and glycine do not modify low-magnesium-induced epileptiform activity in the immature neocortex in vitro.

Exposure of neocortical slices from immature rats to saline containing no added magnesium induced spontaneous epileptiform activity that consisted of bursts of low-amplitude isolated discharges lasting 50-90 sec, recurring every 90-300 sec. Bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-7-phosphonoheptanoic acid led to a rapid, reversible suppression of epileptiform activity, indicating involvement of NMDA receptors. Perfusion with zinc or glycine, putative modulators of the NMDA receptor, with suppressive and enhancing properties, respectively, had no effect on the frequency or duration of the epileptiform discharges. These results indicate that in the immature neocortex in vitro, application of zinc or glycine does not modulate NMDA receptor-mediated, low-magnesium-induced epileptiform discharges.

An experimental study of the effect of isoflurane on epileptiform bursts.

The effect of isoflurane on penicillin- and picrotoxin-induced epileptiform activity was tested using hippocampal slice preparations. Isoflurane reduced both the frequency of spontaneous epileptiform bursts and the number of population spikes within each burst in a dose-dependent manner. The last population spikes in the burst were most sensitive to the anesthetic, whereas the first 4-6 spikes were quite resistant and persisted until spontaneous activity was abolished at 3% isoflurane. Isoflurane increased the stimulus current required to evoke epileptiform bursts and shifted the relationship between stimulus current and population spike amplitude to the right. At 3% isoflurane, a dose that usually causes iso-electric EEG and abolishes all spontaneous epileptiform activity, responses could still be evoked, and then invariably had an epileptiform pattern. The maximum response was reduced compared to control and 1.5% isoflurane. With isoflurane there was a reduced tendency for activity to be transmitted from one region within the hippocampus to the other. This effect was also dose-dependent. However, transmitted activity always retained a typical epileptiform character, although the number of population spikes within a train to some extent decreased with increasing concentrations of isoflurane.

Changes in amino acid release and membrane potential during cerebral hypoxia and glucose deprivation.

Excessive release of glutamate is believed to play a major role in the susceptibility of neurons to ischaemia. Whether the glutamate release is the primary event or occurs in response to electrophysiologic alterations has not been clarified. In the present study, the amino acid release was therefore correlated to changes in electrophysiological parameters and energy status during conditions of low oxygen tension and varying glucose concentrations in rat hippocampal slices. Plain hypoxia failed to produce glutamate release. All neurons underwent, however, a slow depolarization causing most of the neurons to lose their membrane potential within 10 minutes. By restoring the membrane potential to resting level by current injection, the neurons could still be activated synaptically and respond to transmitter application. Following reoxygenation most of the cells regained their resting membrane potential, but showed reduced excitability. When the slices were exposed to hypoxia combined with glucose deprivation (simulated ischaemia), there was a pronounced increase in the glutamate release. This glutamate release was always preceded by a fast anoxic depolarization. Whereas hypoxia reduced the ATP content only to approximately 50%, ATP was depleted in slices exposed to simulated ischaemia. The results demonstrate that although the neurons lose their membrane potential completely during hypoxia, there is no glutamate release. A fast anoxic depolarization provoked by simulated ischaemia, however, is always followed by glutamate release, probably due to a more severe ATP depletion.

Failure of allopurinol to protect against cerebral injury when given after the start of hypoxia.

One cause of ischemic brain injury is free radical formation during recirculation. Allopurinol inhibits xanthine oxidase, an important source of free oxygen radicals. It is known that allopurinol pre-treatment has a protective action during cerebral ischemia. In the present study we exposed slices from the rat hippocampus to 9 minutes of hypoxia to test whether it is sufficient that allopurinol is present in the tissue at the time of reoxygenation. Forty-six slices loaded with allopurinol (10(-5) M) prior to reoxygenation (during hypoxia) were compared to 34 control slices. The response of the pyramidal cell population to orthodromic stimulation was reduced in both groups and there was not a significant difference between the two groups.

Amino-acid release from human cerebral cortex during simulated ischaemia in vitro.

The aim of the present study was to investigate the release of amino-acids in human cerebral cortex during membrane depolarization and simulated ischaemia (energy deprivation). Superfluous tissue from temporal Iobe resections for epilepsy was cut into 500 microns thick slices and incubated in vitro. Membrane depolarization with 50 mM K+ caused a release of glutamate, aspartate, GABA and glycine, but not glutamine or leucine. The release of glutamate and GABA was Ca(++)-dependent. Slices were exposed to simulated ischaemia (energy deprivation; ED) by combined glucose/oxygen deprivation. This caused a Ca(++)-independent release of glutamate, aspartate, GABA, glycine, and taurine which started after 8 min, peaked at the end or shortly after the 27 min period of ED, and returned to control levels within 11 min following termination of ED. Preloaded D-[3H]aspartate was released both during K(+)-stimulation and ED. Release of D-[3H]aspartate during ED was delayed compared to glutamate supporting an initial phase of synaptic glutamate release. Uptake of L-[3H]glutamate was increased during the period of glutamate release, suggesting passive diffusion across the cell membrane or enhanced transport efficacy in cellular elements with functioning uptake mechanisms.

Isoflurane increases the uptake of glutamate in synaptosomes from rat cerebral cortex.

We have studied the effect of increasing concentrations of isoflurane on high- and low-affinity uptake of L-glutamate using synaptosomes from rat cerebral cortex. In the high-affinity uptake range, 0.5% isoflurane had no effect on uptake velocity, while 1.5% and 3.0% isoflurane caused an increase in mean Vmax to 131 (SEM 54) and 210 (103)% of control, respectively. There was no significant change in the K(m) value. Vmax and K(m) values for low-affinity uptake of L-glutamate were unchanged by 1.5% isoflurane. These results provide evidence for an isoflurane-induced increase in high-affinity uptake of glutamate into presynaptic terminals. This effect may contribute to a reduction of transmitter in the synaptic cleft and thereby decreased excitatory synaptic transmission.