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

• Main Authors: Juan C. Boffi, Johannes Knabbe, Michaela Kaiser, Thomas Kuner
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2018-01-01
• Series: Frontiers in Cellular Neuroscience
• Subjects: neuronal; intracellular; chloride; pH; in vivo; ratiometric imaging
• Online Access: http://journal.frontiersin.org/article/10.3389/fncel.2018.00007/full

Neuronal intracellular Cl− concentration ([Cl−]i) influences a wide range of processes such as neuronal inhibition, membrane potential dynamics, intracellular pH (pHi) 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 (pHi) 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 pHi of 7.1 ± 0.2. Neither [Cl−]i nor pHi 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.