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

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 in vivo. 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 in vivo 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.