Live imaging of axonal transport in Drosophila pupal brain explants

Abstract

Axonal transport is essential for the initial growth, maintenance and synaptic plasticity of axons, and altered axonal transport has been observed in different models of neurodegenerative pathologies. Dissecting the mechanisms underlying axonal transport in developing or degenerating brains requires dynamic imaging of axonal cargo movement in living samples. Whereas methods exist to image axonal transport in Drosophila larval neurons, they are not suitable to follow this process during metamorphosis, when brains undergo extensive remodeling. Here we present a simple method that enables confocal imaging of both fast and slow axonal transport in Drosophila pupal brain explants. We describe how to prepare chambers adapted for live imaging, how to maintain brain explants under physiological conditions and how to monitor and quantitatively analyze the movement of fluorescently labeled cargoes. This protocol requires minimal equipment and is ideally suited for experiments that combine genetics, optogenetics and pharmacological approaches. The brains can be prepared for image acquisition in 1.5 h, and the protocol can be performed easily in any fly laboratory.

A conceptual view at microtubule plus end dynamics in neuronal axons

“…up to a century in humans, and continued MT turn-over is needed to prevent senescence through mechanical wear (Dumont et al, 2015) and remove irreversible posttranslational modifications (Janke and Kneussel, 2010). Accordingly, continued MT polymerisation has been demonstrated in adult Drosophila brains (Medioni et al, 2015, Nguyen et al, 2011, Rolls et al, 2007, Soares et al, 2014) and similar results can be expected in vertebrates. Thirdly, the MT volume of many axons seems to be a well-regulated property.…”

A conceptual view at microtubule plus end dynamics in neuronal axons

“…up to a century in humans, and continued MT turnover is needed to prevent senescence through mechanical wear (Dumont et al, 2015) and remove irreversible posttranslational modifications (Janke and Kneussel, 2010). Accordingly, continued MT polymerisation has been demonstrated in adult Drosophila brains (Medioni et al, 2015;Nguyen et al, 2011;Rolls et al, 2007;Soares et al, 2014) and similar results can be expected in vertebrates. Thirdly, the MT volume of many axons seems to be a well-regulated property.…”

CytoCensus: mapping cell identity and division in tissues and organs using machine learning

“…Elucidating the molecular basis of such developmental processes is not only essential for understanding basic neuroscience, but is also important for discovering new treatments for neurological diseases and cancer. Modern imaging approaches have proven indispensable in studying development in intact zebrafish (Danio rario) and Drosophila tissues (Barbosa & Ninkovic, 2016;Dray et al, 2015;Medioni et al, 2015;Rabinovich et al, 2015;Cabernard & Doe., 2013;Graeden & Sive, 2009). Tissue imaging approaches have also been combined with functional genetic screens, for example to discover neuroblast (NB) behaviour underlying defects in brain size or tumour formation (Berger et al, 2012;Homem & Knoblich, 2012;Neumüller et al, 2011).…”