MECHANISM OF EARLY ANOXIA-INDUCED SUPPRESSION OF THE GABA(A)-MEDIATED INHIBITORY POSTSYNAPTIC CURRENT

1. We investigated the mechanism of hypoxia-induced depression of gamma-aminobutyric acid-A (GABA(A))-mediated inhibitory postsynaptic currents (IPSCs) in CAI neurons of hippocampal slices from 21- to 28-day-old rats. Cells were examined by whole-cell patch-clamp recording and hypoxia was induced by switching perfusion of the slice from oxygenated artificial cerebral spinal fluid (ACSF) to ACSF saturated with 95% N-2-5% CO2. 2. Synaptic responses evoked by stimulation of the Schaffer collateral-commissural projection at a fixed holding potential (V-H=-60 mV) during anoxia revealed that the IPSC appeared more sensitive than the excitatory postsynaptic current to anoxia-induced depression. All subsequent studies examined the GABA,mediated IPSC synaptic responses in isolation by direct stimulation of GABA interneurons in the stratum radiatum in the presence of extracellular 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP) (20 mu M) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (50 mu M) to block glutamatergic currents and intracellular QX-314 (lidocaine N-ethyl bromide, 1 mM) to block GABA(A)-mediated currents. When studied in this manner (V-H=-60 mV) the GABA(A)-mediated IPSC appeared to change from an outward to inward current after exposure to anoxia. 3. The current-voltage relationship of GABA(A)-mediated IPSCs revealed that these changes resulted from a positive shift in the IPSC reversal potential without a significant change in the conductance. Thus under patch clamp apparent IPSC inhibition may result from a decrease in the extracellular concentration of chloride ions. Similar findings were observed with micropipettes that contained high intracellular chloride concentrations. 4. Miniature spontaneous IPSCs were examined in the presence of tetrodotoxin (1 mu M) with micropipettes containing high intracellular chloride concentrations. The miniature IPSCs (mIPSCs) appeared as spontaneous transient inward currents. Consistent with an anoxia-induced decrease in extracellular chloride, the mean amplitude of the mIPSCs increased after the onset of anoxia. A significant decrease in rise and decay time was also noted during anoxia. The frequency of the mIPSCs also increased by similar to 300%. 5. The resting input resistance of the cells was examined by measuring the current resulting from a 20-mV hyperpolarizing pulse. A significant reduction in resistance was observed 2 min after the onset of anoxia. This still occurred, although to a lesser degree, in the presence of glutamatergic blockers (20 mu M CPP plus 50 mu M CNQX). In the presence of both GABAergic (picrotoxin, 100 mu M) and glutamatergic blockers no significant reduction in resting input resistance was apparent after 2 min of anoxia. These results are consistent with increases in extracellular concentrations of GABA and possibly glutamate after anoxia observed by other investigators. 6. These data demonstrate that the early anoxia-induced depression of the GABA(A)-mediated IPSC, as measured by whole-cell patch clamp, is not likely to result from a change in GABA-activated conductance but from an alteration of its reversal potential.