A novel imaging protocol to dynamically study axonal transport in developing fly brains [with video]

How the transport of various molecules to neuronal processes (dendrites and axons) is regulated in vivo, in response to developmental signals, is currently largely unknown. Here, we describe a novel protocol enabling live-imaging of fluorescently-labeled molecules transported along axons in maturing Drosophila brains. This protocol can be combined with sophisticated genetic tools or drug treatments, and will be useful to researchers wishing to dynamically study the cellular mechanisms underlying axonal transport in both normal and pathological contexts.

HFSP Career Development Award holder Florence Besse and colleagues
authored on Fri, 29 May 2015

Neuronal cells exhibit long cellular extensions (dendrites and axons) specialized in the processing and transmission of neuronal information. These cellular processes are supplied with proteins, RNA, mitochondria, etc… via active transport of cargoes from cell bodies. How axonal transport is regulated inside brains undergoing developmental remodeling is still largely unclear. In this article, a novel live-imaging method developed to study this process dynamically in maturing Drosophila brains is described.

Figure: Developing Drosophila brains mounted in the imaging chamber. After dissection, pupal Drosophila brains are mounted in a drop of culture medium in the home-made imaging chamber and oriented under a fluorescent stereomicoscope (blue light) before starting live-imaging. The close-up shows remodeling Mushroom body gamma neurons marked with Green Fluorescent Protein (GFP).

This method relies on the dissection and mounting of brain explants in simple customized imaging chambers, on their culture in physiological conditions, and on highly-sensitive confocal live-imaging. It includes post-processing of 2D+t image sequences for quantitative analysis of axonal cargo trajectories. This protocol is versatile, and can easily be adapted to study the transport of various axonal cargoes and cytoskeletal elements in different neuronal populations and/or genetic contexts. We have successfully used it to monitor and characterize the transport of RNA/protein complexes in the axons of remodeling neurons during metamorphosis (Medioni et al., 2014).

The main advantages of this protocol are that it enables quantitative imaging of axonal cargoes in a genetically tractable model system, and can be combined with pharmacological treatments, and optogenetics…). This protocol will considerably improve out ability to analyze the cellular mechanisms underlying axonal transport in the context of central nervous system maturation or neurodegenerative disease models.

Video: Dynamic transport of ribonucleoprotein particles in the axons of intact Drosophila brains. Note that GFP-Imp, a conserved component of neuronal ribonucleoprotein granules, accumulates in particles that are dynamically transported to axons and undergo bi-directional motion. Brain explants from flies expressing GFP-Imp fusions in Mushroom Body γ neurons, and undergoing metamorphosis (28h After Puparium Formation pupae) were imaged using sensitive confocal microscopy. The imaged field corresponds to the proximal region of MB γ axon bundle. Scale bar: 5 μm.

Reference

Live-imaging of axonal transport in Drosophila pupal brain explants. (2015) Medioni C., Ephrussi A. and Besse, F. Nat Protoc. Apr;10(4):574-84. doi: 10.1038/nprot.2015.034.

Other references

Imp promotes axonal remodeling by regulating profilin mRNA during Drosophila brain development. (2014). Medioni, C., Ramialison M., Ephrussi, A. and Besse, F. Current Biol., Mar 31;24(7):793-800. doi: 10.1016/j.cub.2014.02.038.

Pubmed link