Abstracts

Rationale: GABAA inhibition powerfully controls emergent properties such as recurrent excitation and network synchronization, and is mediated by a Cl? conductance whose direction and functional effects depend on the Cl? gradient maintained by the electroneutral ion co-transporters NKCC1 and KCC2, which are driven by cation gradients generated by Na -K ATPases and alter [Cl-]i without net movement of charge across the membrane. NKCC1 inwardly transports Cl- and contributes to [Cl?]i in immature neurons, while KCC2 extrudes Cl- and contributes to the transition from depolarizing to hyperpolarizing GABA responses in maturing postnatal neurons. Given the importance of the Cl? gradient for GABAA inhibition and network excitability, it was of interest to determine if repeated seizures that define epilepsy and the progression of seizures and permanent network alterations induced by brief repeated seizures evoked by kindling alter expression of NKCC1 and KCC2. Western blotting and immunohistochemistry were used to quantify expression of NKCC1 and KCC2 in the hippocampus of kindled adult rats that experienced partial seizures (Class I-IV), 3-75 evoked secondary generalized (Class V) seizures, or spontaneous Class V seizures. Methods: Conventional western blot and immunohistochemical methods with antibodies to NKCC1 (T4 mouse monoclonal antihuman colonic T84 epithelial Na -K -Cl- co-transporter) or KCC2 (rabbit polyclonal anti-K -Cl- cotransporter) were used to assess expression in hippocampal extracts and brain sections at ~24hrs or > 3 months after the last evoked seizure. Results: NKCC1 expression increased in rats after induction of Class V seizures compared to control animals without seizures (p ? 0.05)or rats experiencing only partial seizures (Class I-IV), and increased to a maximum at ~75 Class V seizures. There were no further increases after >100 Class V seizures, a kindling stage associated with spontaneous seizures. Seizure-induced increases in NKCC1 expression were long-lasting and were noted as long as 3 months after 23-27 evoked Class V seizures. In contrast, KCC2 expression was not increased compared to controls except for a modest increase after 100 Class V seizures (p? 0.05). Immunohistochemical analysis revealed regional alterations of expression in the hippocampal subfields and the dentate gyrus. Conclusions: Repeated evoked secondary generalized seizures but not partial seizures induced robust long-lasting increases in expression of NKCC1 followed by modest increases in KCC2 expression at kindling stages associated with spontaneous seizures. The functional effects of the long-lasting chronic increase in NKCC1 prior to emergence of spontaneous seizures include Cl? loading that could influence E(Cl-), E(ipsp), or E(GABAa)as well as activity-dependent alterations in trans-membrane ionic gradients contributing to network synchronization and progressive adverse consequences of repeated seizures.