Summary: This study was undertaken to elucidate the roles of neurons and glial cells in the handling of gluta mate and glutamine, a glutamate precursor, during cere bral ischemia. Slices (400--6 00 fLm) 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 con trol slices that had not been subjected to "ischemic" con ditions, were then fixed and embedded. Ultrathin sec tions were processed according to a postembedding im munocytochemical method with polyclonal antibodies raised against glutamate or glutamine, followed by colloi dal 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 dep rivation caused a reduced glutamate immunolabeling in neuronal somata, while that of glial processes increased. Following 1 h of recovery, the glutamate labeling of neu ronal somata declined further to very low values, com pared to control slices. The glutamate labeling of glial cells returned to normal levels following recovery. InThe pathogenesis of ischemic brain damage is not fully understood, and a number of neurotoxic sub stances has been implicated (Krause et aI. , 1988; Siesjo, 1988;Ginsberg, 1990; Siesjo et aI. , 1990 503axon terminals, no consistent change in the level of glu tamate 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 dep rivation. This treatment was also associated with a re duced glutamine immunolabeling in glial cells. Reversed glutamate uptake due to perturbations of the transmem brane ion concentrations and membrane potential proba bly contributes to the loss of neuronal glutamate under "ischemic" conditions. The increased glutamate labeling of glial cells under the same conditions can best be ex plained by assuming that glial cells resist a reversal of glutamate uptake, and that their ability to convert gluta mate into glutamine is compromised due to the energy failure. The persistence of a nerve terminal pool of glu tamate is compatible with recent biochemical data indi cating that the exocytotic glutamate release is contingent on an adequate energy supply and therefore impeded dur ing ischemia.

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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.

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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.