Roberto Etchenique

RuBi-Glutamate: Two-Photon and Visible-Light Photoactivation of Neurons and Dendritic spines.

We describe neurobiological applications of RuBi-Glutamate, a novel caged-glutamate compound based on ruthenium photochemistry. RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one- or two-photon excitation. It has high quantum efficiency and can be used at low concentrations, partly avoiding the blockade of GABAergic transmission present with other caged compounds. Two-photon uncaging of RuBi-Glutamate has a high spatial resolution and generates excitatory responses in individual dendritic spines with physiological kinetics. With laser beam multiplexing, two-photon RuBi-Glutamate uncaging can also be used to depolarize and fire pyramidal neurons with single-cell resolution. RuBi-Glutamate therefore enables the photoactivation of neuronal dendrites and circuits with visible or two-photon light sources, achieving single cell, or even single spine, precision.

Electrical Communication between Electrodes and Enzymes Mediated by Redox Hydrogels.

Different redox polymers based on poly(allylamine) with covalently attached ferrocene and pyridine groups that coordinate iron and ruthenium complexes were prepared, and hydrogels were obtained by cross-linking them with epichlorohydrin. Charge propagation from the underlying electrode, through the redox polymer and electrical communication with the enzyme FADH(2) of glucose oxidase, was studied by cyclic voltammetry and electrochemical impedance spectroscopy. The effects of electrolyte composition, concentration of enzyme and substrate, and electrode potential are reported. The role of different redox mediators covalently attached to the polymer backbone is discussed in terms of driving force and electrostatic barriers.

Photorelease of GABA with Visible Light Using an Inorganic Caging Group

We describe the selective photorelease of γ-amino butyric acid (GABA) with a novel caged-GABA compound that uses a ruthenium complex as photosensor. This compound (“RuBi-GABA”) can be excited with visible wavelengths, providing greater tissue penetration, less photo-toxicity, and faster photorelease kinetics than currently used UV light-sensitive caged compounds. Using pyramidal neurons from neocortical brain slices, we show that RuBi-GABA uncaging induces GABA-A receptor-mediated responses, has no detectable side effects on endogenous GABAergic and glutamatergic receptors and generates responses with kinetics and spatial resolution comparable to the best caged GABA compounds presently available. Finally, we illustrate two potential applications of RuBi-GABA uncaging: GABA receptor mapping, and optical silencing of neuronal firing.

A fast ruthenium polypyridine cage complex photoreleases glutamate with visible or IR light in one and two photon regimes

We introduce a new caged glutamate, based in a ruthenium bipyridyl core, that undergoes heterolytic cleavage after irradiation with visible light with wavelengths up to 532nm, yielding free glutamate in less than 50ns. Glutamate photorelease occurs also efficiently following two-photon (2P) excitation at 800nm, and has a functional cross section of 0.14GM.

A New Inorganic Photolabile Protecting Group for Highly Efficient Visible Light GABA Uncaging

Fil: Zayat, Leonardo Martin. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Quimica Inorganica, Analitica y Quimica Fisica; Argentina

Measurement of viscoelastic changes at electrodes modified with redox hydrogels with a quartz crystal device

A fast high-frequency ‘in situ’ transfer-function measurement with a quartz crystal microbalance (QCM) has been obtained by an ac voltage divider formed by the quartz crystal impedance (ZQ) and a measuring resistor (Rm). The frequency is swept by a voltage-controlled oscillator (VCO) and the ratio of output (Eo) and input (Ei) ac voltages gives the transfer function of the device. A non-linear fit of the transfer-function modulus vs. frequency around the natural resonance frequency of the system in the 10 MHz range was performed with a Butterworth–Van Dyke equivalent circuit. Mass and viscoelastic components were evaluated with the Sauerbrey–Kanazawa–Bruckenstein equations.The method was tested by varying the viscoelasticity of the quartz surface in sucrose solutions [i.e. by varying the quantity (ρLηL)1/2] and mass sensitivity was calibrated by silver deposition.Fast measurements in non-steady-state experiments were performed with redox hydrogels undergoing oxidation and reduction with anion and water exchange with the outside solution. Deconvolution of both rigid mass (Δm) and viscous resistance (R) was achieved and this explained the abnormal behaviour of the resonance-frequency shift (Δf) during cyclic voltammetry of ferrocene-modified poly(allylamine) hydrogels on a resonant electrochemical QCM.

Two-Photon Optical Interrogation of Individual Dendritic Spines with Caged Dopamine

We introduce a novel caged dopamine compound (RuBi-Dopa) based on ruthenium photochemistry. RuBi-Dopa has a high uncaging efficiency and can be released with visible (blue-green) and IR light in a two-photon regime. We combine two-photon photorelease of RuBi-Dopa with two-photon calcium imaging for an optical imaging and manipulation of dendritic spines in living brain slices, demonstrating that spines can express functional dopamine receptors. This novel compound allows mapping of functional dopamine receptors in living brain tissue with exquisite spatial resolution.

Ruthenium polypyridyl phototriggers: from beginnings to perspectives

Octahedral Ru(II) polypyridyl complexes constitute a superb platform to devise photoactive triggers capable of delivering entire molecules in a reliable, fast, efficient and clean way. Ruthenium coordination chemistry opens the way to caging a wide range of molecules, such as amino acids, nucleotides, neurotransmitters, fluorescent probes and genetic inducers. Contrary to other phototriggers, these Ru-based caged compounds are active with visible light, and can be photolysed even at 532 nm (green), enabling the use of simple and inexpensive equipment. These compounds are also active in the two-photon regime, a property that extends their scope to systems where IR light must be used to achieve high precision and penetrability. The state of the art and the future of ruthenium polypyridyl phototriggers are discussed, and several new applications are presented.

Simultaneous determination of the mechanical moduli and mass of thin layers using nonadditive quartz crystal acoustic impedance analysis

High frequency rheological properties of polymer films have been measured using a nonadditive quartz crystal resonator impedance approach. The method involves fast measurement of the quartz covered with the film in contact with solutions of different viscosities and the measurement of the equivalent impedance of the bare quartz resonator in contact with the same solutions. Although by using a standard quartz crystal acoustic impedance method it is not possible to determine more than two of the four film parameters (density, thickness, storage modulus G′, and loss modulus G″), we demonstrate that the interaction between the film under study and a semi-infinite layer of liquid gives the additional information needed to get the mechanical moduli G′ and G″ simultaneously with the mass or thickness of the film. An alternative approach to measure G′ and G″, which involves independent and very precise determination of the film thickness, is discussed. Experimental measurements of polystyrene using dioctyl phthal...

A ruthenium-rhodamine complex as an activatable fluorescent probe.

We describe the synthesis and characterization of a ruthenium-bipyridyl complex bearing a rhodamine-based fluorescent ligand. The complex is weakly fluorescent due to the quenching of rhodamine. Upon irradiation of the MLCT band it releases rhodamine in a fast and clean heterolytic reaction, increasing its fluorescence nearly 6-fold and making it the first visible-light activatable fluorophore based in transition metal chemistry. These properties and its lack of toxicity make it a good candidate for its use as a biologically friendly caged fluorescent probe. The use of this probe as a neuronal marker, and as a flow profiler in a thin, planar cavity and in a model flow injection analysis (FIA) is demonstrated.