Abstract
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 GABAA 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.
Highlights
Ruthenium–bipyridinetriphenylphosphine–GABA (RuBi–GABA) is a caged compound that allows studying the neuronal transmission in a specific region of a neuron
To verify that uncaged RuBi–GABA caused the activation of G ABAA receptors, we carefully checked that the measured currents, after the photoexcitation of the caged compound, returned to the initial value
To make sure that photolyzed RuBi–GABA directly acted on G ABAA receptors, experiments with the competitive antagonist at the GABA-binding site bicuculline were performed (Fig. 1). GABAA receptors were activated by 1 μM uncaged RuBi–GABA, but, after the administration of 20 μM bicuculline, that occupies the GABA site in the channel, no significant current variations were recorded
Summary
Ruthenium–bipyridinetriphenylphosphine–GABA (RuBi–GABA) is a caged compound that allows studying the neuronal transmission in a specific region of a neuron. Following linear—one-photon (1P)—and non-linear—multiphoton—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 GABAA 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. The photorelease of caged bioactive molecules is a powerful tool for studying molecular mechanisms because such molecules can be delivered at precise and controllable instants of time These probe compounds are prepared via covalent appendage of a light-sensitive protecting group, called “cage”, to a signaling molecule that is unable to activate its target until the bond is broken by light[5]. Uncaging by one-photon (1PE)[6,7,8,9] or two-photon (2PE)[7,8,10,11,12,13] excitation, coupled to patch-clamp, provide a useful technique to regulate the neurotransmitter release and to detect a selected biological target in a temporally and spatially confined way
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