Sustained depolarizing shift of the GABA reversal potential by glutamate receptor activation in hippocampal neurons

The inhibitory action of GABA is a consequence of a relatively hyperpolarized Cl− reversal potential (ECl), which results from the activity of K+–Cl− cotransporter (KCC2). In this study we investigated the effects of glutamate and glutamatergic synaptic activity on ECl. In dissociated culture of mature hippocampal neurons, the application of glutamate caused positive ECl shifts with two distinct temporal components. Following a large transient depolarizing state, the sustained depolarizing state (ECl-sustained) lasted more than 30 min. The ECl-sustained disappeared in the absence of external Ca2+ during glutamate application and was blocked by both AP5 and MK801, but not by nifedipine. The ECl-sustained was also induced by NMDA. The ECl-sustained was blocked by furosemide, a blocker of both KCC2 and NKCC1, but not bumetanide, a blocker of NKCC1. On the other hand, in immature neurons having less expression of KCC2, NMDA failed to induce the sustained depolarizing ECl shift. In organotypic slice cultured neurons, repetitive activation of glutamatergic afferents also generated a sustained depolarizing ECl shift. These results suggest that Ca2+ influx through NMDA receptors causes the down-regulation of KCC2 and gives rise to long lasting positive ECl shifts, which might contribute to hyperexcitability, LTP, and epileptiform discharges.