Live Imaging of Axonal Transport in the Adult Drosophila Central Nervous System

Live imaging of axons allows for the determination of motility and directionality of proteins or organelles. In Drosophila, axonal transport has been predominantly characterized in peripheral neurons, such as larval motor neurons and sensory neurons of the adult wing. As peripheral neurons and central nervous system (CNS) neurons are inherently different, we provide a method to live-image axonal transport of CNS neurons in the cervical connective using an upright or inverted microscope. The method involves dissecting and mounting an entire CNS in a glass bottom petri dish, which is suitable for imaging of nearly any axon in cervical connective. Here, we show an example for simultaneous imaging of both giant fiber axons, which are part of the fly's escape response circuitry, and due to their large diameter provide outstanding resolution.