RuBi-Glutamate: two-photon and visible-light photoactivation of neurons and dendritic spines

ELODIE FINO; ROBERTO ARAYA; DARCY PETERKA; MARCELO SALIERNO; ROBERTO ETCHENIQUE; RAFAEL YUSTE

Chicago

Congreso; RuBi-Glutamate: two-photon and visible-light photoactivation of neurons and dendritic spines; 2009

SFN

Caged compounds are becoming widely used in neuroscience because they enable the optical manipulation of neuronal circuits. The most used caged-glutamate is the MNI-Glutamate. In spite of its utility, MNI-Glutamate needs to be applied to the tissue at relatively high (mM) concentrations for effective two-photon uncaging. Unfortunately, at these concentrations, MNI-glutamate, like many other caged compounds, is a very effective antagonist of GABAergic transmission. To help circumvent this problem, we have synthesized a novel caged-glutamate compound based on ruthenium photochemistry, the RuBi-Glutamate, and characterized his neurobiological applications. RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one- or two-photon excitation. In cortical pyramidal neurons, with laser beam multiplexing, two-photon uncaging of RuBi-Glutamate was able to reliably generate action potentials. It has high quantum efficiency and can be used at low concentrations (300 µM) for which we did not observe any effects on the spontaneous EPSCs of pyramidal cells. Nevertheless, we observed that 300 µM RuBi-Glutamate has a significant antagonist effect on endogenous GABAergic currents (close to 50%), but this effect is much smaller compared with that of MNI glutamate (~80% at 300 µM, ~100% at the effective concentration of 2.5 mM). We tested the use of two-photon uncaging of RuBi-Glutamate for activating dendritic spines or individual neurons. The two-photon uncaging on spines triggered reliable depolarizations in neurons, with physiological kinetics and a very good spatial resolution of the uncaging responses (~80% of reduction of the response when the laser beam is moved 1 μm away from the spine head). With laser beam multiplexing, RuBi-Glutamate can also be used to optically activate pyramidal neurons with single-cell precision. Indeed, with 300 µM RuBi-Glutamate, two-photon uncaging while targeting the soma induced 90% of action potential occurrence and this occurrence dropped to 5% when the targets are moved 5 μm away form the soma. Therefore, RuBi-Glutamate allows the optical activation of neuronal dendrites and circuits with visible and near-infrared light, which could make it an ideal tool for photoactivation experiments in vitro and in vivo.