Abstracts

Current anticonvulsant therapies are ineffective for both neonatal seizures and intractable temporal lobe epilepsy. These disorders share a common trait: GABA is excitatory because principal neurons accumulate excess intracellular chloride (Cl[sub]i[/sub]), creating a driving force for Cl^- efflux. (Normally, GABA inhibits neurons by gating a net influx of Cl^- ions.) It has been suggested that NKCC1, an electroneutral Na⁺-K⁺-2Cl^- cotransporter expressed in neurons, is responsible for the accumulation of intracellular chloride that leads to GABA excitation. In the present study, we examined the kinetics and pharmacology of neuronal Cl^- transport in neonatal neurons to test whether these characteristics were consistent with NKCC1 activity. Hippocampal slices were prepared from male Sprague-Dawley rats age postnatal day 3 (P3) to P10. Whole-cell and gramicidin perforated patch recordings were performed in the CA1 pyramidal cell layer. GABA was picospritzed onto pyramidal cell dendrites to evoke Cl currents in bicarbonate-free media. The kinetics of Cl transport were estimated from the rate of recovery of Cl currents at various time intervals after a evoking an initial large, Cl-depleting current. In perinatal (P3-P10) rats, we found that the maximum velocity of inward Cl transport was 3.3 [plusmn] 1.1 mM/s. The average intracellular chloride concentration [Cl^-][sub]i[/sub] was 21.75 [plusmn] 0.50 mM in gramicidin recordings. Inward Cl transport was inhibited by intracellular Cl with a k[sub]i[/sub] of 18mM. Inward Cl transport was also inhibited by low micromolar bumetanide, a selective inhibitor of NKCC1. 1) Perinatal hippocampal neurons exhibit active inward Cl transport. 2) The kinetics and pharmacology of the active transport are consistent with NKCC1 activity 3) Because the k[sub]i[/sub] for NKCC1 is so close to the [Cl^-][sub]i[/sub], our data suggest that NKCC1 kinetics are an important determinant of [Cl^-][sub]i[/sub] and thus E[sub]GABA[/sub] in perinatal neurons. 4) Because the loop diuretic bumetanide (Bumex) inhibits NKCC1 activity at low micromolar concentrations, it represents a potentially powerful therapy for seizures occurring in the setting of Cl[sub]i[/sub] accumulation and GABA-mediated excitation, for example in the neonatal period. (Supported by a grant from the National Institutes of Health, National Institute of Neurological Disorders and Stroke.)