Chronic In Vivo Imaging of Ponto-Cerebellar Mossy Fibers Reveals Morphological Stability during Whisker Sensory Manipulation in the Adult Rat.

Daria Rylkova,Aidan R Crank,David J Linden

doi:10.1523/eneuro.0075-15.2015

Abstract

The cerebellum receives extensive disynaptic input from the neocortex via the basal pontine nuclei, the neurons of which send mossy fiber (MF) axons to the granule cell layer of the contralateral cerebellar hemisphere. Although this cortico-cerebellar circuit has been implicated in tasks such as sensory discrimination and motor learning, little is known about the potential role of MF morphological plasticity in the function of the cerebellar granule cell layer. To address this issue, we labeled MFs with EGFP via viral infection of the basal pons in adult rats and performed in vivo two-photon imaging of MFs in Crus I/II of the cerebellar hemisphere over a period of several weeks. Following the acquisition of baseline images, animals were housed in control, enriched, or deprived sensory environments. Morphological dynamics were assessed by tracing MF axons and their terminals, and by tracking the stability of filopodia arising from MF terminal rosettes. MF axons and terminals were found to be remarkably stable. Parameters derived neither from measurements of axonal arbor geometry nor from the morphology of individual rosettes and their filopodial extensions significantly changed under control conditions over 4 weeks of imaging. Increasing whisker stimulation by manipulating the sensory environment or decreasing such stimulation by whisker trimming also failed to alter MF structure. Our studies indicate that pontine MF axons projecting to Crus I/II in adult rats do not undergo significant structural rearrangements over the course of weeks, and that this stability is not altered by the sustained manipulation of whisker sensorimotor experience.

Highlights

While axons in the developing brain show dramatic growth and activity-driven structural plasticity (Witte et al, 1996; Ruthazer et al, 2003; Nishiyama and Linden, 2004; Huberman et al, 2008; Erzurumlu and Gaspar, 2012), the degree to which these processes persist in the adult brain is poorly understood
To examine the morphological dynamics of pontine mossy fiber (MF) in vivo, we labeled MF axons with EGFP mediated by associated virus (AAV) infection of the basal pons (Fig. 1c,d)
We investigated both the large-scale dynamics of the axonal arbor as well as the fine-scale morphology of the MF rosette over timescales ranging from minutes to weeks

Summary

Introduction

While axons in the developing brain show dramatic growth and activity-driven structural plasticity (Witte et al, 1996; Ruthazer et al, 2003; Nishiyama and Linden, 2004; Huberman et al, 2008; Erzurumlu and Gaspar, 2012), the degree to which these processes persist in the adult brain is poorly understood. The axons of layer 6 pyramidal cells, branching in layer 1 of barrel cortex, have highly dynamic boutons, with less than half remaining stable over the course of a month (De Paola et al, 2006). Axonal dynamics appear to be cell type specific since boutons on thalamocortical and layer 2/3 pyramidal cell axons innervating the same area of barrel cortex are highly stable over 1 month (De Paola et al, 2006). Axonal structure and bouton stability are much lower on transverse branches compared with ascending branches. While transverse branches fail to form conventional asymmetric synapses, ascending branches form conventional synapses with Purkinje cells as well as spillover synapses with interneurons (Nishiyama et al, 2007)

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