Expanding the Toolkit for <em>In Vivo</em> Imaging of Axonal Transport

Abstract

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative. This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.1. Pre-surgery preparation 1. Express H C T (H C T 441 , residues 875-1315) fused to an improved cysteine-rich tag in bacteria as a glutathione-S-transferase fusion protein as per 45 .

BDNF-dependent modulation of axonal transport is selectively impaired in ALS

“…Signalling endosomes were visualised in vivo by injecting the fluorescent atoxic binding fragment of tetanus neurotoxin (H C T-555), as previously described [ 49 , 50 ]. Briefly, H C T (residues 875–1315) fused to an improved cysteine-rich region was expressed in bacteria as a glutathione-S-transferase fusion protein [ 51 ], cleaved and subsequently labelled with AlexaFluor555 C 2 maleimide (Thermo Fisher Scientific, A-20346).…”

“…To investigate the influence of α-MN and skeletal muscle subtypes [10][11][12] on axonal transport dynamics in vivo, we labelled neurotrophin-containing signalling endosomes with a fluorescent atoxic tetanus neurotoxin binding fragment (H C T) [59,60] to assess axonal transport dynamics in sciatic nerves of live mice [49,50]. H C T is internalised into MNs upon binding to nidogens and polysialogangliosides at distal terminals [61], and is retrogradely transported in Rab7-positive signalling endosomes [44].…”

Bimodal regulation of axonal transport by the GDNF-RET signalling axis in healthy and diseased motor neurons

“…To independently assess the effect of RET inhibition on axonal transport, we tested the C3 inhibitor in vivo by measuring the rate of transport of signalling endosomes in the intact sciatic nerve of early symptomatic (∼P73) SOD1 G93A mice, a time point that corresponds with ∼20% loss of lumbar motor neurons 9 . Using a previously optimised experimental design 40,41 , HCT-555 and 25 ng BDNF were injected into the tibialis anterior and gastrocnemius muscles in the lower limb of anaesthetised female WT and SOD1 G93A mice. For in vivo delivery, we suspended C3 in 1 % methyl cellulose (MC), and injected it intraperitoneally at a final concentration of 10 mg/kg.…”

“…The in vivo axonal transport was performed as previously described 40,41 . Briefly, P73 female WT (non-transgenic) and SOD1 G93A mice were anesthetized using isoflurane (National Veterinary Services) and AlexaFluor555-conjugated HCT (13 μg) and BDNF or GDNF (25 ng) were injected intramuscularly into the exposed tibialis anterior and lateral gastrocnemius muscles of the right hind leg and the wounds then sutured (for GDNF experiments, only tibialis anterior was injected).…”

“…After 4+ h, mice were re-anesthetized using isoflurane and the sciatic nerve of the right leg was exposed. The anaesthetised mouse was placed on a heated stage in an environmental chamber and axonal transport was imaged in the intact sciatic nerve by time-lapse confocal microscopy as previously described 40,41,63 . An inverted Zeiss LSM 780 with 40x Plan-Apochromat oil immersion objective was used to locate the sciatic nerve and visualise axons containing HCT-555 labelled signalling endosomes.…”