Abstract
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.
Highlights
Neuronal [Cl−]i is of crucial importance for processes such as inhibitory neurotransmission, maintenance of the resting membrane potential, regulation of intracellular pH and cell volume (Kaila et al, 2014; Doyon et al, 2016)
This led to the development of a second generation of promising genetically encoded Cl− indicators with improved affinity and signal to noise ratio that could be used for in vivo applications, such as ClopHensor (Arosio et al, 2010) and Superclomeleon (SCLM; Grimley et al, 2013)
Our study reports non-invasive in vivo steady-state [Cl−]i and pHi estimations in layer 2/3 (L2/3) cortical neurons of anesthetized and awake mice
Summary
Neuronal [Cl−]i is of crucial importance for processes such as inhibitory neurotransmission, maintenance of the resting membrane potential, regulation of intracellular pH (pHi) and cell volume (Kaila et al, 2014; Doyon et al, 2016). The implementation of superecliptic (SE) pHluorin (Miesenböck et al, 1998) as an emission ratiometric pH indicator to monitor neuronal pHi has been reported in flies (Rossano et al, 2013) This suggests a strategy combining ratiometric SCLM and superecliptic pHluorin (SE-pHluorin) recordings as a promising alternative to study neuronal steady-state Cl− and pH in vivo using 2-photon microscopy. To gain insight into the factors affecting neuronal intracellular steady-state Cl− concentration, we compared [Cl−]i and pHi in anesthetized and awake mice as well as in single neurons lacking the cation-Cl− co-transporter KCC2 (Delpire, 2000; Ben-Ari, 2002; Kaila et al, 2014) These results and approach will contribute to future studies of the role of Cl− regulation in pathophysiological conditions such as epilepsy or schizophrenia in vivo (Kaila et al, 2014; Sullivan et al, 2015)
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