Monitoring Chloride Dynamics to Unveil Brain Physiology

Chloride ions (Cl⁻) are fundamental regulators of neuronal excitability and inhibitory signalling. Despite their central role in GABAergic transmission, the mechanisms controlling intracellular chloride concentration ([Cl⁻]ᵢ) and its temporal dynamics remain poorly understood. This thesis investigates how [Cl⁻]ᵢ varies across physiological and behavioural states, how these fluctuations are regulated, and their functional consequences for brain activity. During my PhD, I focused on improving the quantitative and dynamic measurement of [Cl⁻]ᵢ in vivo. I first employed LSSmClopHensor, a dual pH/Cl⁻ fluorescent sensor, to measure absolute changes in neuronal [Cl⁻]ᵢ under different conditions. I then contributed to the development and characterization of iClima, a genetically encoded chloride sensor optimized for stable and accurate measurements in vitro and in vivo. Compared with previous indicators, iClima shows reduced pH sensitivity within the physiological range and enhanced chloride affnity, enabling detection of subtle [Cl⁻]ᵢ fluctuations that were previously inaccessible. Using LSSmClopHensor combined with electrophysiology and behavioural paradigms, I dissected the mechanisms underlying diurnal [Cl⁻]ᵢ fluctuations and their coupling to sleep–wake history. Seizure susceptibility was found to follow a diurnal pattern, peaking when [Cl⁻]ᵢ is high, and lowering [Cl⁻]ᵢ prevented seizure occurrence, linking chloride dynamics to network excitability and epileptiform activity. iClima enabled chloride imaging in human cell lines, revealing circadian oscillations of [Cl⁻]ᵢ and suggesting that rhythmic ionic regulation is a conserved cellular feature. During epileptic events, iClima captured rapid chloride accumulation and subsequent recovery, providing unprecedented temporal resolution of chloride dynamics. Furthermore, iClima visualized physiological chloride responses in cortical pyramidal neurons following visual stimulation and movement, reflecting inhibitory synaptic activity—signals that were undetectable with previous sensors. This represents, to date, the first demonstration of this type. Together, these results demonstrate that chloride is not merely a passive ion but an active regulator of neuronal physiology. Understanding chloride dynamics is essential to comprehend how cells regulate their function and adapt to changing conditions. iClima represents a major advancement in optical tools for chloride imaging, offering the sensitivity and stability necessary to quantify fluctuations even at subcellular resolution. The next generation of optical tools will further elucidate how chloride shapes cellular signalling and the brain’s electrical and biochemical language.