Abstracts

Rationale: Neuronal chloride concentration [Cl^-]_i is an important determinant of GABA_A receptor (GABA_AR)-mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCC) move Cl^- across the membrane. However, new evidence suggests CCCs are not the sole determinant of [Cl^-]_i. Methods: We tested the effects of specific NKCC1 and KCC2 blockade, osmotic challenge by mannitol, extracellular matrix disruption and cytotoxic alteration of volume constraints, induced by seizures, on neuronal [Cl^-]_i using two-photon imaging of brain slices from Clomeleon mice (genetically encoded ratiometric fluorophore sensitive to changes in [Cl^-]_i). Results: Our results demonstrated that cytoplasmic impermeant anions ([A]_i) and polyanionic extracellular matrix glycoproteins ([A]_o) constrain the local neuronal [Cl^-]. In healthy neurons, CCC inhibition had modest effects on [Cl^-]_i and neuronal volume, but substantial changes were produced by alterations of the balance between [A]_i and [A]_o. During seizures, there was a correlated increase of [Cl^-]_i and neuronal volume Conclusions: In physiological conditions, CCC are important elements of Cl^- homeostasis, but local impermeant anions determine the homeostatic set-point for [Cl^-], and hence, neuronal volume and the polarity of local GABA_AR signaling. Pathological conditions that alter [A], like traumatic brain injury, will lead to changes in [Cl^-]_i and may require different therapeutic approaches for seizure treatment.