Abstracts

RATIONALE: The action of GABA at ionotropic GABAA receptors (GABAARs) depends on the concentration of intracellular Cl- [Cl-]i. Elevated [Cl-]i in neonatal pyramidal neurons results in a depolarizing response to GABAAR activation. Low [Cl-]i in mature pyramidal neurons leads to a hyperpolarizing or shunting response. We examined the role of K+-Cl- coupled cotransport (KCC) in [Cl-]i homeostasis in neonatal and postnatal day 18-28 neocortical pyramidal neurons. METHODS: Brain slices (300-400 ?m)were prepared from postnatal day 3 and postnatal days 18-28 rats. Whole cell (WC) voltage clamp recordings were obtained from visually identified pyramidal neurons. GABA (250 ?M)was pressure-applied to the soma or to excised membrane patches. [Cl-]i was estimated from the reversal potential of GABAAR mediated currents using the Nernst equation. RESULTS: For three pipette Cl- concentrations (1, 20 ,and 40 mM), the calculated [Cl-]i for PN 18-28 neurons was significantly different from the [Cl-]i calculated from excised patches (p<0.03, n=41 WC, n=14 patches). With 1 mM pipette Cl-, [Cl-]i was 1.6 mM higher than in excised patches (n=11 WC; n=4 patches). Furosemide or lowered extracellular K+ ([K+]o) reduced [Cl-]i to values similar to excised patches (n=5). Elevated [K+]o increased [Cl-]i by 3.1 ? 1.1 mM (n=3). With 20 or 40 mM pipette Cl-, calculated [Cl-]i was significantly lower than the values obtained with excised patches. Elevated [K+]o or furosemide reduced this difference. No effect of [K+]o or furosemide on [Cl-]i was observed in neurons from PN 3 animals. CONCLUSIONS: These results provide evidence in support of a powerful, developmentally-regulated Cl- homeostatic mechanism in neocortical pyramidal neurons. KCC is the primary mechanism for both accumulation and extrusion of intracellular chloride. Under conditions of low [Cl-]i, KCC accumulates Cl-; when [Cl-]i is elevated, KCC extrudes Cl-. Since this mechanism is coupled to [K+]o, activity dependent increases in [K+]o are expected to raise [Cl-]i and possibly exacerbate hyperexcitability.