Abstracts

Rationale: [Cl^-] gradients maintained by the electroneutral ion co-transporters NKCC1 and KCC2 determine whether GABA has hyperpolarizing or depolarizing effects in both immature and adult neurons, thereby influencing GABA_A dependent control of neuronal and network properties contributing to seizures and epilepsy. NKCC1 is an inward directed Na -K -2Cl^- co-transporter that increases intracellular Cl^-. KCC2 is an outward directed K -Cl^- co-transporter that reduces intracellular Cl^- in normal [K ]_o, but reverses transport and imports Cl^- when [K ]_o increases, as occurs during seizures. Repeated Class V seizures but not partial seizures in kindled rats induce robust long-lasting increases in expression of NKCC1 followed by very modest increases in KCC2 after 100 Class V seizures, a kindling stage associated with emergence of spontaneous seizures (see Sutula et al., meeting abstracts). The goal of this study was to examine effects of the long-lasting seizure-induced increases of NKCC1 and KCC2 on resting membrane potential (RMP), reversal potential of the IPSP (E_ipsp), and spike (AP) threshold. Methods: Sharp electrode current-clamp recordings were obtained in granule cells of the dentate gyrus in hippocampal slices from normal and kindled rats that experienced repeated seizures evoked by perforant path stimulation. Monosynaptic IPSPs were evoked by a stimulus pulse applied near the recording electrode in 20 M DNQX and 50 M APV to block glutamate receptors. The peak amplitude of the IPSP was measured during constant DC pulses across a range of current intensities. IPSP amplitude was plotted as a function of the steady state membrane potential to determine the E_ipsp. E_ipsp was compared in slices obtained from rats that experienced only partial (Class I-IV) seizures, a range of 5-90 secondary generalized (Class V) seizures, and > 100 Class V seizures, a kindling stage associated with spontaneous seizures. Results: In 3.75 mM [K ]_o, there were no differences in RMP, E_ipsp, or AP between control and kindled rats. In 9.75 mM [K ]_o, the E_ipsp in slices from normal rats shifted from -73.3 mV to -54.0 mV, but in kindled rats with Class V seizures the E_ipsp shifted from -71.3 mV to only -61.4 mV (p< 0.001), a difference corresponding to the expected effects of increased NKCC1 expression and reversal of KCC2 regulated Cl^-- flux in [K ]_o = 9.75 mM. There were no differences in E_ipsp between normal and kindled rats with only partial seizures, or rats with < or > 100 Class V seizures. Conclusions: The results are consistent with Cl^- loading from seizure-induced increases in NKCC1 and reversal of Cl^- flux by KCC2 in elevated [K ]_o. While increases in intracellular Cl^- from seizure-induced increases in NKCC1 could contribute to excitatory effects of GABA in chronic epilepsy, the overall functional effects of seizure- induced alterations in co-transporters more likely dampen network excitability during interictal or ictal conditions when [K ]_o is elevated. Supported by NINDS RO1 25020.