Abstracts

Rationale: Low intracellular chloride concentration ([Cl-]i) is an important determinant of the inhibitory postsynaptic GABAa–receptor signaling. Restoration of [Cl-]i equilibrium (ECl) after synaptic activity is achieved by the net regulated activities of the cation-Cl- co-transporters (CCC), such as the K+-Cl- co-transporter KCC2 (mediating Cl- efflux) and Na+-K+-2Cl- co-transporter NKCC1 (mediating Cl- influx). As secondary active co-transporters, CCCs derive their energy for transport of Cl- from the transmembrane gradients of K+ (for KCC2) or Na+ (for NKCC1). Acute seizures have been reported to rapidly increase [Cl-]i and extracellular potassium ([K+]o) that directly affect synaptic inhibition. Activity dependent increases in [Cl-]i shift the equilibrium potential of GABAa-R (EGABA) toward less negative value. Activity dependent increases in [K+]o may further shift EGABA due to the reduced electrochemical driving force for the KCC2 that less efficiently extrudes neuronal chloride at high levels of [K+]o. Consequently, depolarizing values of EGABA foster seizures and anticonvulsant resistance via failure of inhibition, and reduced chloride extrusion rates sustain seizure activity resulting in intermittent seizures and status epilepticus. Methods: Acute hippocampal slices from CLM-1 mice expressing chloride fluorophore Clomeleon were used as a model of traumatic brain injury. Non-invasive extracellular field potential recordings and two-photon fluorescence chloride imaging were used to monitor neuronal network activity and [Cl-]i. The effects of KCC2 inhibitor VU0463271 (0.1-1 µM) and putative KCC2 up-regulator CLP257 (1-10 µM) on neuronal chloride accumulation and extrusion rate during spontaneous ictal-like discharges (seizures), and frequency, duration and power of recurrent seizures were studied in a chronically epileptic hippocampal preparation in vitro. Results: Hippocampal slices were incubated for three-four weeks, during which a latent one week period was followed by spontaneous recurrent seizures (in 90% of slices) and electrical status epilepticus (20%). Status epilepticus (SE) was defined as continuous seizures for at least 5 min, or by 30 min of intermittent seizures without recovery between seizures. Under control conditions, recurrent seizures induced rapid (Cl-)i elevation in majority of pyramidal cells from 5-15 mM to 20-40 mM. Ictal-like epileptiform discharges and baseline intracellular Cl (ECl) recovered within 20-30 s. The VU0463271 KCC2 antagonist significantly increased the duration and power of ictal-like epileptiform discharges and corresponding Cl- transients in a dose-dependent manner. Resting (Cl-)i remained elevated during intermittent seizures and the decay time of chloride extrusion increased to 200-300 s, resulting in prolonged seizures and status epilepticus. Sodium channel blocker TTX in the presence of VU0463271 abolished recurrent seizures and recovered ECl within 200-300 s. In contrast, CLP257 improved (Cl-)i homeostasis and reduced the duration of recurrent seizures in a concentration-dependent manner. GABAA-R antagonist SR95531 (10 µM) as well as the NKCC1 and KCC2 blocker bumetanide (200 µM) prevented the acute anticonvulsant action of CLP257. Conclusions: Our results indicate that: 1) KCC2 transport activity efficiently recovers ECl during recurrent seizures and contributes to seizure termination; 2) inhibition of KCC2 transport activity delays chloride extrusion rate resulting in prolonged seizures and status epilepticus; 3) the anticonvulsant actions of CLP257 are at least partially mediated by enhanced KCC2 activity. Funding: NIH/NINDS RO1 NS040109