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

Area of Science:

• Neuroscience
• Developmental Biology
• Biochemistry

Background:

• Intraneuronal chloride concentrations ([Cl^-]_i) decrease during neural development.
• This developmental shift alters GABAergic neurotransmission from depolarizing to hyperpolarizing via GABA_A receptors.
• Early life experiences can significantly influence this critical developmental process.

Purpose of the Study:

• To evaluate the utility of the SuperClomeleon (SClm) fluorescent sensor for measuring [Cl^-]_i changes in brain slices.
• To monitor developmental changes in [Cl^-]_i in the hippocampus.
• To investigate the impact of early life stress (ELS) on [Cl^-]_i in the medial prefrontal cortex (mPFC).

Main Methods:

• Utilized SClm mice and two-photon microscopy in organotypic hippocampal slice cultures.
• Monitored developmental changes in [Cl^-]_i from day in vitro (DIV) 3 to DIV22.
• Assessed [Cl^-]_i in the mPFC of postnatal day 9 mouse pups subjected to ELS (nesting material restriction).

Main Results:

• Observed a significant decrease in [Cl^-]_i between DIV3 and DIV9, with further reductions by DIV22 in hippocampal cultures.
• ELS induced an increase in [Cl^-]_i in layer 2/3 mPFC neurons, suggesting a shift towards an immature chloride profile.
• While precise quantification of absolute chloride concentrations was challenging, the SClm sensor demonstrated sensitivity to physiological changes.

Conclusions:

• The SClm sensor is a valuable tool for assessing dynamic changes in neuronal chloride levels in brain slices.
• Developmental decrease in [Cl^-]_i is confirmed in hippocampal cultures.
• Early life stress disrupts normal developmental trajectory by increasing [Cl^-]_i in the mPFC.