Study of Biophysical Parameters in Rubi-Gaba Uncaging using Non-Linear Photoactivation and Electrophysiology in Cerebellar Granule Cells

RuBi GABA is a photoactivatable molecule whose activity can be controlled by a pulse of light. These probe compounds are prepared via covalent appendage of a light-sensitive protecting group “the cage” (RuBi) to a signaling molecule (GABA). With the cage bound, the signaling molecule is unable to activate its receptor [Diana D., J Am Chem Soc., 2014]. One photon (UV-VIS light) and two-photon (700-900nm) absorption can be used to break the “cage” binding. The uncaged molecule becomes active and can bind on GABA A receptor site [Rial Verde E.M., Front Neural Circuits., 2008]. We described a novel approach to control the neurotransmitter release in time, space and amplitude of intensity. In particular, uncaging method and fluorescence microscopy coupled to patch clamp technique provide a useful approach to detect a selected biological target in a temporally and spatially confined way. We analyzed how the change of physical parameter such as uncaging distance, exposure time, laser power, linear and non-linear photoactivation influence the measurements and we have determined how these parameters change the modality and efficacy of the GABA release and consequently the GABA A response. Specifically, localization precision can be improved using advanced fluorescent optical methods, in particular, we used the super-resolved and non-linear fluorescence microscopy [Diaspro A. et al. QRB, 2006 ]. This allows exploring the release of caged GABA topically applied in situ at defined concentration and in a specific region of neuronal cells for mapping the localization and the functional distribution of GABA A receptors in cerebellar granule cells in vitro. Finally, we are able to explore the responses generated by specifics drugs in different regions of neuron.