Monitoring Changes in Intracellular Chloride Levels Using the FRET-Based SuperClomeleon Sensor in Organotypic Hippocampal Slices.

Abstract

The reduction in intracellular neuronal chloride concentration is a crucial event during neurodevelopment that shifts GABAergic signaling from depolarizing to hyperpolarizing. Alterations in chloride homeostasis are implicated in numerous neurodevelopmental disorders, including autism spectrum disorder (ASD). Recent advancements in biosensor technology allow the simultaneous determination of intracellular chloride concentration of multiple neurons. Here, we describe an optimized protocol for the use of the ratiometric chloride sensor SuperClomeleon (SClm) in organotypic hippocampal slices. We record chloride levels as fluorescence responses of the SClm sensor using two-photon microscopy. We discuss how the SClm sensor can be effectively delivered to specific cell types using virus-mediated transduction and describe the calibration procedure to determine the chloride concentration from SClm sensor responses. Key features • Calibration and use of the ratiometric chloride sensor SuperClomeleon to measure intracellular chloride levels in mouse organotypic hippocampal slices. • SuperClomeleon sensor expression in pyramidal cells or GABAergic interneurons using adeno-associated viruses.