Using SuperClomeleon to Measure Changes in Intracellular Chloride during Development and after Early Life Stress

Abstract

AbstractIntraneuronal chloride concentrations ([Cl−]i) decrease during development resulting in a shift from depolarizing to hyperpolarizing GABA responses via chloride-permeable GABAAreceptors. This GABA shift plays a pivotal role in postnatal brain development, and can be strongly influenced by early life experience. Here, we assessed the applicability of the recently developed fluorescent SuperClomeleon (SClm) sensor to examine changes in [Cl−]iusing two-photon microscopy in brain slices. We used SClm mice of both sexes to monitor the developmental decrease in neuronal chloride levels in organotypic hippocampal cultures. We could discern a clear reduction in [Cl−]ibetween dayin vitro(DIV)3 and DIV9 (equivalent to the second postnatal weekin vivo) and a further decrease in some cells until DIV22. In addition, we assessed alterations in [Cl−]iin the medial prefrontal cortex (mPFC) of postnatal day (P)9 male SClm mouse pups after early life stress (ELS). ELS was induced by limiting nesting material between P2 and P9. ELS induced a shift toward higher (i.e., immature) chloride levels in layer 2/3 cells in the mPFC. Although conversion from SClm fluorescence to absolute chloride concentrations proved difficult, our study underscores that the SClm sensor is a powerful tool to measure physiological changes in [Cl−]iin brain slices.