KCC2-dependent Steady-state Intracellular Chloride Concentration and pH in Cortical Layer 2/3 Neurons of Anesthetized and Awake Mice

Abstract

Neuronal intracellular Cl^- concentration ([Cl^-]_i) influences a wide range of processes such as neuronal inhibition, membrane potential dynamics, intracellular pH (pH_i) or cell volume. Up to date, neuronal [Cl^-]_i has predominantly been studied in model systems of reduced complexity. Here, we implemented the genetically encoded ratiometric Cl^- indicator Superclomeleon (SCLM) to estimate the steady-state [Cl^-]_i in cortical neurons from anesthetized and awake mice using 2-photon microscopy. Additionally, we implemented superecliptic pHluorin (SE-pHluorin) as a ratiometric sensor to estimate the intracellular steady-state pH (pH_i) of mouse cortical neurons <i>in vivo</i>. We estimated an average resting [Cl^-]_i of 6 ± 2 mM with no evidence of subcellular gradients in the proximal somato-dendritic domain and an average somatic pH_i of 7.1 ± 0.2. Neither [Cl^-]_i nor pH_i were affected by isoflurane anesthesia. We deleted the cation-Cl^- co-transporter KCC2 in single identified neurons of adult mice and found an increase of [Cl^-]_i to approximately 26 ± 8 mM, demonstrating that under <i>in vivo</i> conditions KCC2 produces low [Cl^-]_i in adult mouse neurons. In summary, neurons of the brain of awake adult mice exhibit a low and evenly distributed [Cl^-]_i in the proximal somato-dendritic compartment that is independent of anesthesia and requires KCC2 expression for its maintenance.