A simple method for imaging axonal transport in aging neurons using the adult Drosophila wing

Abstract

There is growing interest in the link between axonal cargo transport and age-associated neuronal dysfunction. The study of axonal transport in neurons of adult animals requires intravital or ex vivo imaging approaches, which are laborious and expensive in vertebrate models. We describe simple, noninvasive procedures for imaging cargo motility within axons using sensory neurons of the translucent Drosophila wing. A key aspect is a method for mounting the intact fly that allows detailed imaging of transport in wing neurons. Coupled with existing genetic tools in Drosophila, this is a tractable system for studying axonal transport over the life span of an animal and thus for characterization of the relationship between cargo dynamics, neuronal aging and disease. Preparation of a sample for imaging takes ∼5 min, with transport typically filmed for 2-3 min per wing. We also document procedures for the quantification of transport parameters from the acquired images and describe how the protocol can be adapted to study other cell biological processes in aging neurons.

Oxidative Stress Orchestrates Cell Polarity to Promote Embryonic Wound Healing

“…The localization of DCF fluorescence in tubular structures inside the wound (Figures 1B,1D,and 1F) suggested that ROS production upon wounding could be occurring in mitochondria. To establish the source of ROS upon wounding, we injected H2DCFDA into embryos expressing mCherry:mito, a mitochondrial marker (Vagnoni and Bullock, 2016). We quantified a significant colocalization between the mitochondrial marker and DCF (maximum correlation coefficient inside the wound of 0.6 ± 0.1; p < 2 3 10 À65 ; Figure 1F and Video S1).…”

Mitostasis in Neurons: Maintaining Mitochondria in an Extended Cellular Architecture

“…Thus, how neuronal mitochondria behave in vivo has also required attention. The last decade has seen substantial progress to address this question, based on experimental paradigms that allow monitoring mitochondrial dynamics in vivo in the peripheral and central nervous system of a wide range of species including nematodes (Fatouros et al, 2012; Rawson et al, 2014; Williams et al, 2013), fruit flies (Babic et al, 2015; Pilling et al, 2006; Vagnoni and Bullock, 2016; Wang and Schwarz, 2009a), zebrafish (O’Donnell et al, 2013; Plucinska et al, 2012), and mice (Chandrasekaran et al, 2006; Misgeld et al, 2007). Reassuringly, the basic tenets of mitochondrial dynamics derived from in vitro work have been confirmed in vivo.…”

From two to three dimensions: The importance of the third dimension for evaluating the limits to neuronal miniaturization in insects

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