A conditional transgenic reporter of presynaptic terminals reveals novel features of the mouse corticospinal tract

Pasquale D’Acunzo,Vincenzo Zimarino,Mattia Ferro,Antonio Malgaroli,G Giacomo Consalez,Aurora Badaloni,Maddalena Ripamonti

doi:10.3389/fnana.2013.00050

Pasquale D’Acunzo, Vincenzo Zimarino + Show 5 more

Open Access

https://doi.org/10.3389/fnana.2013.00050

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Abstract

In many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), synaptic alterations precede the demise of the neuronal cell, making synapses a useful vantage point from which to monitor the onset and progression of clinical signs and pathological changes. While murine models of ALS display many features in common with the clinical picture observed in patients, corticospinal tract (CST) involvement is usually less severe in mice than the picture observed in humans. In this paper we describe the characterization of a new conditional transgenic line obtained by targeted integration of a GFP-VAMP2 fusion gene into the Rosa26 locus, and devised to permit the detection of genetically defined presynaptic terminals in wild type mice and murine models of neural disorders. This reporter molecule is selectively enriched in presynaptic boutons, significantly reducing the background signal produced by fibers of passage. The specific features of this reporter line allow us to strongly support the view that murine CST terminals give rise to very few direct contacts with spinal motor neurons. Moreover, the evidence described here reveals the existence of previously uncharacterized, putative direct connections between CST presynaptic boutons and Renshaw neurons in the spinal cord. These results constitute a proof of concept for the potential application of this indicator line to morphological analyses of wild type and diseased synapses.

Highlights

Numerous murine models of neurodegenerative diseases exist and, in many of them, synaptic alterations predate the demise of the neuronal cell body and can be used as a predictor of disease onset and progression
We have developed a new Cre-inducible presynaptic reporter consisting of the presynaptic protein VAMP2 fused N-terminally to EGFP
The chimeric molecule was constructed as follows: starting from a 2 Kb rat Vamp2 cDNA (Elferink et al, 1989), the CDS was modified in order to generate an N-term fusion to EGFP and a C-term fusion to a spacer peptide derived from the ectodomain of TfR (Grote et al, 1995) followed by a Myc tag

Summary

Introduction

Numerous murine models of neurodegenerative diseases exist and, in many of them, synaptic alterations predate the demise of the neuronal cell body and can be used as a predictor of disease onset and progression (reviewed in Conforti et al, 2007). Depending on the nature of the selected molecule, these substances can travel in vivo from the cell soma to the axon terminal or vice versa, and are visualized thanks to (immuno) histochemical techniques (Zaborszky et al, 2006). While these approaches guarantee cellular resolution, sensitivity and stability, several pitfalls in their use remain. The analysis of murine models of neurodegenerative disorders would benefit from the availability of presynaptic terminal markers, transgenic (Tg) reporters permitting the selective detection of genetically defined subsets of synaptic boutons. While other reporters already exist, they are usually non-selective, making it difficult to distinguish between presynaptic compartments and axons in transit through a given territory

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