Depolarization-induced release of [H-3]D-aspartate from GABAergic neurons caused by reversal of glutamate transporters

Cultured neocortical neurons, which predominantly consist of GABAergic neur ons exhibit a pronounced stimulus-coupled GABA release. Since the cultures may contain a small population of glulamatergic neurons and the GABAergic n eurons have a high content of glutamate it was of interest to examine if gl utamate in addition to gamma-aminobutyric acid (GABA) could be released fro m these cultures. The neurons were preloaded with [H-3]D-aspartate and subs equently its release was followed during depolarization induced by a high p otassium concentration or the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepro pionic acid (AMPA) receptor agonists, AMPA and kainate. Depolarization of t he neurons with 55 mM potassium increased the release of [(3H)]D-aspartate by more than 10-fold. When the non-specific calcium-channel blockers cobalt or lanthanum were included in the stimulation buffer with potassium, the r elease of [H-3]D-aspartate was decreased by about 40%. These results indica ted that some of the released [H-3]D-aspartate might originate from a vesic ular pool. When AMPA was applied to the neurons, the release of [H-3]D-aspa rtate was increased 2-fold and could not be prevented or decreased by addit ion of cobalt. Since AMPA has a rapid desensitizing effect on AMPA receptor s, it was examined whether AMPA under non-desensitizing conditions was able to induce an increased release of [3H]D-aspartate as compared to the condi tions of applying AMPA alone. The desensitization of AMPA receptors was blo cked by 6-chloro-3,4-dihydro-3-(2-norbornen-5-yl)-2H-1,2,4-benzolhiadiazine -7-sulphonamide-1,1-dioxide (cyclothiazide). Under the non-desensitizing co nditions, the AMPA-induced release of [3H]D-aspartate was highly enhanced s howing about a 10-fold increase over basal release. Addition of cobalt or l anthanum did not decrease the amount of [H-3]D-aspartate released, indicati ng that the release originated from a cytoplasmic pool. Kainate, which indu ces an almost nondesensitizing effect on AMPA receptors, showed similar res ults as observed for AMPA under non-desensitizing conditions. The NMDA rece ptor antagonist (5R,10 S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohe pten-5,10-imine (MK-801) had only minor effects on the [H-3]D-aspartate rel ease induced by AMPA and kainate. Thus, the depolarization-induced release of [H-3]D-aspartate from cultured GABAergic neurons appears to be caused ma inly by reversal of the glutamate transporters. (C) 2000 Published by Elsev ier Science Ltd on behalf of ISDN. All rights reserved.