REDISTRIBUTION OF GLUTAMATE AND GLUTAMINE IN SLICES OF HUMAN NEOCORTEX EXPOSED TO COMBINED HYPOXIA AND GLUCOSE DEPRIVATION INVITRO

This study was undertaken to elucidate the roles of neurons and glial cells in the handling of glutamate and glutamine, a glutamate precurso r, during cerebral ischemia. Slices (400-600 mum) from human neocortex obtained during surgery for epilepsy or brain tumors were incubated i n artificial cerebrospinal fluid and subjected to 30 min of combined h ypoxia and glucose deprivation (an in vitro model of brain ischemia). These slices, and control slices that had not been subjected to ''isch emic'' conditions, were then fixed and embedded. Ultrathin sections we re processed according to a postembedding immunocytochemical method wi th polyclonal antibodies raised against glutamate or glutamine, follow ed by colloidal gold-labeled secondary antibodies. The gold particle d ensities over various tissue profiles were calculated from electron mi crographs using a specially designed computer program. Combined hypoxi a 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 fur ther to very low values, compared to control slices. The glutamate lab eling of glial cells returned to normal levels following recovery. In axon terminals, no consistent change in the level of glutamate immunol abeling was observed. Immunolabeling of glutamine was low in both nerv e terminals and neuronal somata in normal slices and was reduced to no ndetectable levels in nerve terminals upon hypoxia and glucose depriva tion. This treatment was also associated with a reduced glutamine immu nolabeling in glial cells. Reversed glutamate uptake due to perturbati ons of the transmembrane ion concentrations and membrane potential pro bably 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 re sist a reversal of glutamate uptake, and that their ability to convert glutamate into glutamine is compromised due to the energy failure. Th e persistence of a nerve terminal pool of glutamate is compatible with recent biochemical data indicating that the exocytotic glutamate rele ase is contingent on an adequate energy supply and therefore impeded d uring ischemia.