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

Sam de Kater1,2, Lotte J Herstel1, Corette J Wierenga1

  • 1Donders Institute for Brain, Cognition, and Behaviour, Radboud University, Nijmegen, the Netherlands.

Bio-Protocol
|March 14, 2025
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Summary

This study details a method to measure intracellular chloride levels in brain slices using the SuperClomeleon sensor. This technique aids research into neurodevelopmental disorders like autism spectrum disorder (ASD).

Keywords:
CalibrationChlorideCre-dependent AAVFRET sensorGABAOrganotypic hippocampal slicesSuperClomeleonTwo-photon microscopy

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Area of Science:

  • Neuroscience
  • Cell Biology
  • Biochemistry

Background:

  • Intracellular chloride concentration is vital for neurodevelopment, influencing GABAergic signaling.
  • Disruptions in chloride homeostasis are linked to neurodevelopmental disorders, such as autism spectrum disorder (ASD).
  • Advanced biosensors enable simultaneous measurement of chloride levels in multiple neurons.

Purpose of the Study:

  • To present an optimized protocol for using the SuperClomeleon (SClm) ratiometric chloride sensor.
  • To measure intracellular chloride concentration in mouse organotypic hippocampal slices.
  • To facilitate research on chloride homeostasis in neurodevelopmental contexts.

Main Methods:

  • Utilized the SuperClomeleon (SClm) ratiometric chloride sensor.
  • Employed two-photon microscopy to record SClm fluorescence responses.
  • Used virus-mediated transduction (adeno-associated viruses) for sensor delivery to specific neuronal types (pyramidal cells, GABAergic interneurons).
  • Described a calibration procedure for accurate chloride concentration determination.

Main Results:

  • Successfully calibrated and applied the SClm sensor in organotypic hippocampal slices.
  • Demonstrated effective expression of the SClm sensor in targeted hippocampal cell populations.
  • Established a reliable method for quantifying intracellular chloride levels.

Conclusions:

  • The described protocol provides a robust method for measuring intracellular chloride in brain slice models.
  • This technique is valuable for investigating the role of chloride homeostasis in neurodevelopment and disorders like ASD.
  • SuperClomeleon sensor application advances the study of neuronal signaling and chloride dynamics.