Abstract
Axonal transport, which is the process mediating the active shuttling of a variety cargoes from one end of an axon to the other, is essential for the development, function, and survival of neurons. Impairments in this dynamic process are linked to diverse nervous system diseases and advanced ageing. It is thus essential that we quantitatively study the kinetics of axonal transport to gain an improved understanding of neuropathology as well as the molecular and cellular mechanisms regulating cargo trafficking. One of the best ways to achieve this goal is by imaging individual, fluorescent cargoes in live systems and analyzing the kinetic properties of their progression along the axon. We have therefore developed an intravital technique to visualize different organelles, such as signaling endosomes and mitochondria, being actively transported in the axons of both motor and sensory neurons in live, anesthetized rodents. In this chapter, we provide step-by-step instructions on how to deliver specific organelle-targeting, fluorescent probes using several routes of administration to image individual cargoes being bidirectionally transported along axons within the exposed sciatic nerve. This method can provide detailed, physiologically relevant information on axonal transport, and is thus poised to elucidate mechanisms regulating this process in both health and disease.
Highlights
Axonal transport is the indispensable process whereby molecular motor proteins actively traverse the microtubule network of neurons to deliver a variety of different cargoes from one end of an axon to the other
Real-time tracking of individual cargoes in live axons has long been reported in various primary neuronal cultures and ex vivo tissue preparations; there is evidence to indicate that these experimental environments do not always fully replicate what is observed in intact neurons in vivo [5–9]
Neurotrophincontaining signaling endosomes are visualized by intramuscular injection of one of two fluorescently labeled probes: an atoxic binding fragment of tetanus neurotoxin (HCT) or an antibody against the extracellular domain of p75 neurotrophin receptor (p75NTR)
Summary
Axonal transport is the indispensable process whereby molecular motor proteins actively traverse the microtubule network of neurons to deliver a variety of different cargoes from one end of an axon to the other. Neurotrophincontaining signaling endosomes are visualized by intramuscular injection of one of two fluorescently labeled probes: an atoxic binding fragment of tetanus neurotoxin (HCT) or an antibody against the extracellular domain of p75 neurotrophin receptor (p75NTR) These probes are internalized at the neuromuscular junction and afferent nerve endings before being retrogradely transported within signaling endosomes toward the cell body of motor and sensory neurons of the sciatic nerve [16]. We outline an extension to the method that permits the visualization of additional cargo types (e.g., mitochondria) through injection of lipophilic dyes directly into the sciatic nerve [23], obviating the requirement for genetically encoded, fluorescent reporter strains (e.g., “MitoMice” [24]) This permits the simultaneous labeling of different cargoes with distinct fluorophores, which will be useful for developing a broader understanding of transport, as the trafficking of different cargoes is differentially regulated [25]. The method reported here will aid the assessment of axonal transport in rodent models of neurological disease, it can be used to determine the impact of aging and different drug treatments (e.g., chemotherapeutic molecules) on cargo trafficking, as well as improving our understanding of the basic mechanisms regulating the process
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