• biosensor
• calibrations
• diurnla
• variations

Neuronal Network consists of two different types of neurons: the excitatory and inhibitory systems. They are like two faces of the same coin that must be perfectly balanced to avoid several pathologies and diseases.
The inhibitory system is really fundamental in the modulation of the excitatory response, it influences the rhythms of the brain, synaptic plasticity and the flow of information in neuronal circuitry. The main inhibitory synaptic receptors are channels conducting chloride. These receptors are mostly activated by GABA , which is one of the most important inhibitory neurotransmitter.
The effect of GABA in the postsynaptic cell depends directly from the value of the equilibrium potential of chloride calculated with the Nernst equation, that includes the logarithmic ratio of the extracellular and intracellular chloride. When the equilibrium potential of chloride is lower (more negative) than the resting membrane potential, the opening of the -channel leads to an influx of the ion and an hyperpolarization of the cell. When the equilibrium potential is less negative than the resting membrane potential the result will be translated in an efflux of the ion that depolarizes the cell.
The intracellular concentration of chloride is regulated by two co-transporters : NKCC1 and KCC2 that are present at the level of the neuronal membrane. In the first period of development (<P12), there is an higher expression of NKCC1, that exploits the Na gradient to import Cl inside the cell. In this case GABA acts as an excitatory neurotransmitter, indeed the equilibrium potential is less negative with respect to the membrane potential, and chloride exits from the cell. The expression of KCC2 starts 12 days after birth and it allows the exit of Cl, using K gradient. (Y. Ben-Ari et al., 2007)
In this study we try to evaluate how intracellular concentration of chloride and pH can influence cortical circuitry. This has been possible by a genetically encoded fluorescent sensor, developed in our laboratory, called “ChlopHsensor”. It is formed by two fluorescent proteins a Cl and pH sensitive GFP mutant () and a red fluorescent protein LSSmKate2, that is Cl and pH insensitive but acts as a spectroscopic reference. The first aim of this thesis was performed different calibration experiments to improve the corrections applied during the custom-made analysis of our imaging. We try to investigate how concentration, non uniform imaging and scattering events could impair the quantification of our signal-noise ratio. After modifying our optical setup changing the red filter for the PMT (a photomultiplier which converts photons into electrical signal) we measured on cell cultures new coefficients for bleed through correction and ratio between green and red signal at []=0. Both are used for the quantification of chloride and pH.
We also have performed many experiments, using 2 YFP mice injected with an AAV for conditional YFP expression and for Cre recombinase expression in order to investigate how much scattering in the emission could affect the acquisition in function of depth of imaging. The second aim was investigate how chloride can influence cortical circuitry. Using a two-photon imaging technique, it has been possible to measure Cl and pH at cellular level in vivo.
Adult mice were electroporated in utero, transfecting the pyramidal neurons in the 2/3 layer of the visual cortex. We acquired at four different time of the day a spectral sequence using the same cells at different excitation wavelengths. The result shows that there is a circadian oscillation of the intracellular concentration of Cl but no variation in the pH. Chloride is higher at midnight and lower at noon, and there is a strong heterogeneity at any moment in time. We also used an inhibitor of NKCC1 called bumetanide. In this way, the drug blocks the transporter and the intracellular level of the ion decreases, we can observe an higher variation at midnight due to an higher concentration of Cl in that moment of the day.