In vivo micro-lesion of single dendrite with femtosecond laser pulses

Recently, two-photon microscopy has been used for high spatial resolution imaging of the intact neocortex in living rodents. In this work we used near-IR femtosecond laser pulses for a combination of two-photon microscopy and microdissection on fluorescently-labeled neuronal structures in living mice. Three-dimensional reconstructions of dendrites expressing the green fluorescence protein were made in the cortex of GFP-M and YFP-H transgenic mice. Afterwards, single dendrites were laser-dissected irradiating the structure with a high femtosecond laser energy dose. We report that laser dissection can be performed with micrometric precision and without any visible collateral damage of the surrounding neuronal structures. After laser irradiation, one part of the severed dendrite underwent degeneration and disappeared within 5 hours. Using a chronically implanted glass window, we performed long-term imaging in the area of the dissected dendrite. Images of the long-term morphological changes in the neuronal network after dendritic lesioning will be provided. Laser microdissection of selected structures of the neuronal branching in vivo represents a promising tool for neurobiological research.