GABA A receptor-mediated inhibition depends on the maintenance of intracellular ClϪ concentration ([Cl Ϫ ] in ) at low levels. In neurons in the developing CNS, [Cl Ϫ ] in is elevated, E GABA is depolarizing, and GABA consequently is excitatory. Depolarizing GABAergic synaptic responses may be recapitulated in various neuropathological conditions, including epilepsy. In the present study, rat hippocampal dentate granule cells were recorded using gramicidin perforated patch techniques at varying times (1-60 d) after an epileptogenic injury, pilocarpine-induced status epilepticus (STEP). In normal, non-epileptic animals, these strongly inhibited dentate granule cells act as a gate, regulating hippocampal excitation, controlling seizure initiation and/or propagation. For 2 weeks after STEP, we found that E GABA was positively shifted in granule cells. This shift in E GABA altered synaptic integration, increased granule cell excitability, and resulted in compromised "gate" function of the dentate gyrus. E GABA recovered to control values at longer latencies post-STEP (2-8 weeks), when animals had developed epilepsy. During this period of shifted E GABA , expression of the Cl Ϫ extruding K ϩ /Cl Ϫ cotransporter, KCC2 was decreased. Application of the KCC2 blocker, furosemide, to control neurons mimicked E GABA shifts evident in granule cells post-STEP. Furthermore, post-STEP and furosemide effects interacted occlusively, both on E GABA in granule cells, and on gatekeeper function of the dentate gyrus. This suggests a shared mechanism, reduced KCC2 function. These findings demonstrate that decreased expression of KCC2 persists for weeks after an epileptogenic injury, reducing inhibitory efficacy and enhancing dentate granule cell excitability. This pathophysiological process may constitute a significant mechanism linking injury to the subsequent development of epilepsy.

GABA and glycine provide excitatory action during early development: they depolarize neurons and increase intracellular calcium concentration. As neurons mature, GABA and glycine become inhibitory. This switch from excitation to inhibition is thought to result from a shift of intracellular chloride concentration ([Cl-]i) from high to low, but in retina, measurements of [Cl-]i or chloride equilibrium potential (ECl) during development have not been made. Using the developing mouse retina, we systematically measured [Cl-]i in parallel with GABA's actions on calcium and chloride. In ganglion and amacrine cells, fura-2 imaging showed that before postnatal day (P) 6, exogenous GABA, acting via ionotropic GABA receptors, evoked calcium rise, which persisted in HCO3- -free buffer but was blocked with 0 extracellular calcium. After P6, GABA switched to inhibiting spontaneous calcium transients. Concomitant with this switch we observed the following: 6-methoxy-N-ethylquinolinium iodide (MEQ) chloride imaging showed that GABA caused an efflux of chloride before P6 and an influx afterward; gramicidin-perforated-patch recordings showed that the reversal potential for GABA decreased from -45 mV, near threshold for voltage-activated calcium channel, to -60 mV, near resting potential; MEQ imaging showed that [Cl-]i shifted steeply around P6 from 29 to 14 mM, corresponding to a decline of ECl from -39 to -58 mV. We also show that GABAergic amacrine cells became stratified by P4, potentially allowing GABA's excitatory action to shape circuit connectivity. Our results support the hypothesis that a shift from high [Cl-]i to low causes GABA to switch from excitatory to inhibitory.