In vivo imaging of axonal transport in peripheral nerves of rodent forelimbs

Abstract

Axonal transport is the essential process by which neurons actively traffic a variety of cargoes between the cell soma and axon terminals. Accordingly, dysfunctional axonal transport is linked to many nervous system conditions. Therefore, being able to image and quantify this dynamic process in live neurons of animal disease models is beneficial for understanding neuropathology and testing new therapies at the preclinical level. As such, intravital approaches have been developed to assess cargo movement in the hindlimb sciatic nerves of live, anaesthetised mice. Here, we describe an adapted method for in vivo imaging of axonal transport in intact median and ulnar nerves of the rodent forelimb. Injection of a fluorescently labelled and non-toxic fragment of tetanus neurotoxin (HCT) into the mouse forepaw permits the identification of signalling endosomes in intact axons of median and ulnar nerves. Through immunofluorescent analysis of forelimb lumbrical muscles and median/ulnar nerves, we confirmed that HCT is taken up at motor nerve terminals and predominantly locates to motor axons. We then showed that the baseline trafficking of signalling endosomes is similar between the median/ulnar nerves and the sciatic nerve in adult wild-type mice. Importantly, this adapted method can be readily tailored for assessment of additional cargoes, such as mitochondria. By measuring transport in forelimb and hindlimb nerves, comparative anatomical and functional analyses can be performed in rodent disease models to aid our understanding of peripheral nerve disease pathogenesis and response to injury.

Boosting peripheral BDNF rescues impaired in vivo axonal transport in CMT2D mice

“…Given that neuromuscular pathology is comparatively mild or absent in CMT2D forelimbs, we adapted our in vivo imaging approach to perform intravital analysis of signaling endosome transport in median and ulnar nerves ( 46 ). To target muscles innervated by motor neurons within these nerves, we injected H C T-555 into the forepaw of wild-type and Gars C201R/+ mice at 3 months, representing an age at which axonal transport has been defective in CMT2D sciatic nerves for at least 2 months ( Figure 1, C and D ).…”

“…For imaging transport in sciatic nerves, H C T-555 was injected into the right lateral gastrocnemius and tibialis anterior muscles of isoflurane-anesthetized mice ( Figure 1, A and B ). Alternatively, H C T-555 was administered into the left forepaw (targeting lumbrical muscles, among others) for assessment of transport in median and ulnar nerves, as described ( 46 ). A 10 μL, 26 gauge Hamilton syringe (MilliporeSigma, 20779) or a pulled, glass micropipette (Drummond Scientific, 5-000-1001-X10) was used for intramuscular injections, whereas micropipettes alone were used for forepaw injections.…”

Fluorescence imaging of peripheral nerve function and structure

“…60 Katherine L. Gibbs group established a real-time imaging and detection method for individual axonal transport in live mouse nerve fibers using fluorescently labelled and non-toxic fragment of tetanus neurotoxin (HcT) and analyzed the transit rate of retrograde HcT transport in axons after intramuscular and footpad injections, providing a new tool to analyze the specific mechanism of axonal transport cargo. 61 Lang et al further improved the in vivo axonal transport imaging method for murine sensory and motor neurons by injecting fluorescently labeled HcT into mice's forepaws, allowing deeper research on the kinetics of transaxonal transport of cargoes such as mitochondria and increasing the understanding of peripheral nerve axonal transport and related diseases 62 [Fig. 3G].…”

The node of Ranvier influences thein vivoaxonal transport of mitochondria and signalling endosomes

“…We have previously determined that signalling endosome transport speeds are similar between motor axons innervating prototypical fast and slow muscles (Tosolini et al, 2022). Likewise, axonal transport speeds are comparable between hindlimb and forelimb peripheral nerves (Lang et al, 2023). In contrast, signalling endosome transport in MNs is faster than in sensory neurons innervating the same muscle (Sleigh et al, 2020a).…”