• chloride imaging
• chloride sensors
• low gamma oscillation
• synaptic Inhibition

In the cortex, interneurons play a crucial regulatory role in controlling pyramidal cells, despite representing only a small percentage of the cortical neuronal population. To understand and measure the dynamics of cortical ionotropic GABAergic inhibition, mediated by chloride-permeable GABA-A receptors, it is essential to assess the spatiotemporal dynamics of chloride fluxes across the cell membrane and the resultant changes in intracellular chloride concentration.
This thesis presents the use of a novel genetically encoded ratiometric sensor, called iClima (improved Cl- imaging), to demonstrate in-vivo chloride dynamics after presentation of visual stimuli. Previous work in our lab has demonstrated the sensor’s ability to detect intracellular chloride concentration changes under pathological conditions, i.e., induced seizure episodes. In this work, we demonstrated the sensor’s ability to detect chloride changes under physiological conditions.
Using in-vivo two-photon imaging on awake head-fixed mice, we demonstrated the sensor's effectiveness in capturing real-time changes in chloride concentration in pyramidal cells of the primary visual cortex (V1) in response to visual stimuli, including steady light stimuli and drifting grating stimuli. Since the shift in chloride level at the physiological level is very small, we used a parameter (ΔRatio/Ratio) that indicates the percentage change in the intracellular chloride concentration rather than the exact measurement of its absolute value. We also compared the temporal dynamics of the onset and decay of the chloride transients during the active and rest periods of the mice. In addition, we performed electrophysiological recordings from V1 of anesthetized mice and observed that the dynamics of the chloride transients are related to the power of low gamma oscillations (25- 48 Hz), which is known to depend on GABA-A inhibition in the cortex.
This study is the first to demonstrate in-vivo imaging of chloride changes in response to non-pathological synaptic activity. Our findings indicate the potential of chloride imaging to detect physiological shifts in chloride concentrations, advancing our understanding of its role in neuronal network dynamics.