title: Multi-Modal Optical Imaging of the Cerebellum in Animals.
abstract: Thanks to their flexibility, optical techniques could be the key to explore anatomy, plasticity, and functionality of the cerebellum. As an example, an in vivo analysis of the dynamic remodeling of cerebellar axons by nonlinear microscopy can provide fundamental insights of the mechanism that promotes neuronal regeneration. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals. 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 vivo. Here, we show that single axonal branches can be dissected by laser axotomy, thus avoiding collateral damage to the adjacent dendrite and the formation of a persistent glial scar. Despite the very small denervated area, the injured axons consistently reshaped the connectivity with surrounding neurons and sprouted new branches through the intact surroundings. Correlative light and electron microscopy revealed that the sprouted branch contains large numbers of vesicles, with varicosities in the close vicinity of Purkinje dendrites. By using an RNA interference approach, we found that downregulating GAP-43 causes a significant increase in the turnover of presynaptic boutons and hampers the generation of reactive sprouts. Further, we report how nonlinear microscopy in combination with novel voltage sensitive dyes or transgenic mice allow optical registrations of action potential across a population of neurons opening promising prospective in understanding brain functionality. Finally, we describe novel implementations of light-sheet microscopy to resolve neuronal anatomy in whole cerebellum with cellular resolution. The understanding gained from these complementary optical methods may provide a deeper comprehension of the cerebellum.
authors.affiliation: European Laboratory for Non-Linear Spectroscopy, University of Florence, Via Nello Carrara 1, Sesto Fiorentino, 50019, Italy. allegra@lens.unifi.it.|National Research Council, National Institute of Optics, Largo Fermi 6, Florence, 50125, Italy. allegra@lens.unifi.it.
authors.forename: Anna Letizia
authors.initials: AL
authors.lastname: Allegra Mascaro
authors.affiliation: European Laboratory for Non-Linear Spectroscopy, University of Florence, Via Nello Carrara 1, Sesto Fiorentino, 50019, Italy.|National Research Council, National Institute of Optics, Largo Fermi 6, Florence, 50125, Italy.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Sacconi
authors.affiliation: European Laboratory for Non-Linear Spectroscopy, University of Florence, Via Nello Carrara 1, Sesto Fiorentino, 50019, Italy.|National Research Council, National Institute of Optics, Largo Fermi 6, Florence, 50125, Italy.
authors.forename: Ludovico
authors.initials: L
authors.lastname: Silvestri
authors.affiliation: Ctr. Interdisciplinaire de Microscopie Electronique, Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland.
authors.forename: Graham
authors.initials: G
authors.lastname: Knott
authors.affiliation: European Laboratory for Non-Linear Spectroscopy, University of Florence, Via Nello Carrara 1, Sesto Fiorentino, 50019, Italy.|National Research Council, National Institute of Optics, Largo Fermi 6, Florence, 50125, Italy.|Department of Physics and Astronomy, University of Florence, Via Sansone 1, Sesto Fiorentino, 50019, Italy.|International Center for Computational Neurophotonics (ICON) Foundation, Via Nello Carrara 1, Sesto Fiorentino, 50019, Italy.
authors.forename: Francesco S
authors.initials: FS
authors.lastname: Pavone
keywords: Axonal regeneration; Axotomy; Climbing fibers; In vivo; Laser nanodissection; Light sheet microscopy; Multi-photon imaging; Optical imaging; Plasticity; Purkinje cells; Random-access two-photon microscopy; Voltage-sensitive dyes