Laser Nanosurgery of Cerebellar Axons In Vivo
Authors: Allegra Mascaro A.L., Sacconi L., Pavone F.S.
Autors Affiliation: European Laboratory for Non-Linear Spectroscopy, University of Florence; National Institute of Optics, National Research Council; Department of Physics and Astronomy, University of Florence; International Center for Computational Neurophotonics (ICON Foundation)
Abstract: Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.
Journal/Review: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume: 89 Pages from: e51371-1 to: e51371-8
KeyWords: Neuroscience; Axonal labeling; Neuronal tracing; In vivo imaging; Two-photon microscopyDOI: 10.3791/51371Citations: 3data from “WEB OF SCIENCE” (of Thomson Reuters) are update at: 2022-01-23References taken from IsiWeb of Knowledge: (subscribers only)Connecting to view paper tab on IsiWeb: Click hereConnecting to view citations from IsiWeb: Click here