Measurement of intraneuronal transport in vivo in zebrafish larvae brain by tracking nanocrystal-labelled endosomes with fast non-linear microscopy

Motor proteins are responsible for the intracellular transport of critical cargoes such as organelles, vesicles, protein complexes and other materials within the cell, along the cytoskeleton. This transport is an essential molecular process participating in cell homeostasis, especially in neurons [1]. Axonal transport deficits are found in several neurological disorders and are a hallmark of neurodegenerative diseases [2]. Over the past few decades, much progress has been made in developing tools and methods to visualize and measure intracellular transport, by tracking the movement of molecular motors or the transported cargoes. In particular, fluorescent proteins (FP) fused to motors or cargo proteins [3] is largely used to investigate axonal transport, as it is a versatile tool with target specificity that is used in vivo in small organisms. These include Drosophila and Danio rerio (Zebrafish, Zf) larvae, which offers the advantage for optical microscopy of being transparent, and which are also amenable to genetic modifications. For instance, the motion of mitochondria was investigated in Zf larvae with genetically engineered fluorescent reporters [4] in the context of neurodegenerative disease [5] or axonal regeneration [6], using wide-field microscopy and more recently using a real-time 3D single particle tracking setup [7]. Such studies were also conducted in axons of motor-neurons of live mice, but these observations require a complex and invasive preparation consisting in surgically exposing the nerves. These various methods have reached a high degree of sophistication but to date the vast majority of axonal transport studies have only been conducted in sensory-motor neurons. Measurements in central nervous system neurons, where this transport also plays a key role, especially in disease conditions, are still lacking. Takihara et al [8] developed a low invasive surgical procedure allowing them to record by two-photon fluorescence microscopy bidirectional mitochondria motions in axons of retinal ganglion cells of a live mouse. Using two-photon microscopy imaging of the motor cortex, Knabbe et al [9] measured axonal transport properties of dense core vesicles labeled by a virally induced fluorescent reporter. While they addressed more complex systems, these live mouse observations [8,9] were carried out at moderate temporal resolutions of a maximum of one frame per second (corresponding to a dwell time of a few μs) with a spatial resolution of about 200 nm and for a maximum field-of-view size of 200 μm. Such a time resolution prevents the observation of transient events of short duration (