Precise 3D modulation of electro-optical parameters during neurotransmitter uncaging experiments with neurons in vitro

• Published in: Scientific reports 2020-08, Vol.10 (1), p.13380-13380, Article 13380
• Main Authors: Cozzolino, Marco, Bazzurro, Virginia, Gatta, Elena, Bianchini, Paolo, Angeli, Elena, Robello, Mauro, Diaspro, Alberto
• Format: Article
• Language: English
• Subjects: 631/378; 631/57; 639/624; Animals; Binding sites; Cells, Cultured; Cerebellum; Cerebellum - cytology; Controlled release; Experiments; gamma-Aminobutyric Acid - metabolism; Humanities and Social Sciences; Lasers; Light; Localization; Microscopy, Confocal; Molecular Imaging - methods; multidisciplinary; Neurons; Neurons - metabolism; Neurons - physiology; Neurotransmitter Agents - metabolism; Patch-Clamp Techniques; Photoactivation; Power; Rats, Sprague-Dawley; Receptors, GABA-A - metabolism; Ruthenium; Science; Science (multidisciplinary); Synaptic Transmission - physiology; γ-Aminobutyric acid A receptors
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-70217-5

Ruthenium–bipyridinetriphenylphosphine–GABA (RuBi–GABA) is a caged compound that allows studying the neuronal transmission in a specific region of a neuron. The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is bound to a caged group that blocks the interaction of the neurotransmitter with its receptor site. Following linear—one-photon (1P)—and non-linear—multi-photon—absorption of light, the covalent bond of the caged molecule is broken, and GABA is released. Such a controlled release in time and space allows investigating the interaction with its receptor in four dimensions (X,Y,Z,t). Taking advantage of this strategy, we succeeded in addressing the modulation of GABA A in rat cerebellar neurons by coupling the photoactivation process, by confocal or two-photon excitation microscopy, with the electrophysiological technique of the patch-clamp in the whole-cell configuration. Key parameters have been comprehensively investigated and correlated in a temporally and spatially confined way, namely: photoactivation laser power, time of exposure, and distance of the uncaging point from the cell of interest along the X, Y, Z spatial coordinates. The goal of studying specific biological events as a function of controlled physical parameters has been achieved.