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Improved method for efficient imaging of intracellular Cl(-) with Cl-Sensor using conventional fluorescence setup.

Perrine Friedel1, Piotr Bregestovski, Igor Medina

  • 1Inserm Unité 901 Marseille, France ; Aix-Marseille Université Marseille, France ; INMED Marseille, France.

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Summary

Researchers developed a new method to measure intracellular chloride concentration ([Cl(-)]i) in neurons using a modified Cl-Sensor. This technique overcomes previous limitations, enabling stable measurements for studying neurological disorders and screening drug targets like KCC2.

Keywords:
Cl-SensorKCC2fluorescent biosensorsintracellular chlorideneuronnon-invasive monitoringpatch clamp

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Area of Science:

  • Neuroscience
  • Cell Biology
  • Biophysics

Background:

  • Intracellular chloride concentration ([Cl(-)]i) is crucial for central nervous system function and implicated in neurological disorders.
  • Existing methods for measuring [Cl(-)]i using genetically encoded Cl-Sensors require specialized, ultra-sensitive equipment.
  • Previous attempts to use conventional microscopes with Cl-Sensors failed due to photobleaching of the YFPCl component.

Purpose of the Study:

  • To identify the cause of failure in previous Cl-Sensor recordings using conventional microscopes.
  • To develop a modified protocol for stable, long-lasting [Cl(-)]i measurements using Cl-Sensors with standard epifluorescence microscopes.
  • To validate the new protocol's sensitivity and utility in studying neuronal chloride dynamics and KCC2 activity.

Main Methods:

  • Analysis of Cl-Sensor component inactivation during excitation.
  • Modification of excitation light intensity and recording protocols for Cl-Sensor fluorescence.
  • Simultaneous imaging and patch clamp recording in cultured hippocampal neurons.
  • Assessment of sensitivity to detect changes in [Cl(-)]i.
  • Application for screening potassium-chloride co-transporter KCC2 activity.

Main Results:

  • Identified YFPCl inactivation as the cause of previous recording failures.
  • Developed a protocol with reduced excitation intensity (20-fold) enabling stable Cl-Sensor measurements.
  • Demonstrated detection of small [Cl(-)]i changes (as low as 2 mM) in mature neurons.
  • Validated the protocol's effectiveness for large-scale screening of KCC2 activity.

Conclusions:

  • The developed protocol overcomes limitations of previous Cl-Sensor applications, allowing [Cl(-)]i monitoring with conventional microscopes.
  • This method provides a sensitive and stable approach for studying neuronal chloride homeostasis.
  • The protocol is valuable for research into neurological disorders and the development of KCC2-targeted therapies.