Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, UK.
Nat Protoc. 2016 Sep;11(9):1711-23. doi: 10.1038/nprot.2016.112. Epub 2016 Aug 18.
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.
人们对轴突货物运输与年龄相关的神经元功能障碍之间的联系越来越感兴趣。在成年动物的神经元中研究轴突运输需要活体或离体成像方法，这些方法在脊椎动物模型中既费力又昂贵。我们描述了使用半透明的果蝇翅膀中的感觉神经元来对轴突内货物运动进行成像的简单、非侵入性程序。一个关键方面是一种用于安装完整果蝇的方法，该方法允许对翅膀神经元中的运输进行详细成像。结合果蝇中现有的遗传工具，这是一个在动物的整个生命周期内研究轴突运输的可行系统，因此可以对货物动力学、神经元衰老和疾病之间的关系进行特征描述。用于成像的样品制备需要约 5 分钟，每个翅膀的运输通常拍摄 2-3 分钟。我们还记录了从获取的图像中定量运输参数的过程，并描述了如何调整该方案以研究衰老神经元中的其他细胞生物学过程。
