Live-Cell Superresolution Imaging of Retrograde Axonal Trafficking Using Pulse-Chase Labeling in Cultured Hippocampal Neurons

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2022 Jul 12

PMID 35819762

Authors

Tong Wang

Frederic A Meunier

Affiliations

Soon will be listed here.

Abstract

The entanglement of long axons found in cultured dissociated hippocampal neurons restricts the analysis of the machinery underlying directed axonal trafficking. Further, hippocampal neurons exhibit "en passant" presynapses that may confound the analysis of long-range retrograde axonal transport. To solve these issues, we and others have developed microfluid-based methods to specifically follow the fates of the retrograde axonal cargoes following pulse-chase labeling by super-resolution live-cell imaging, and automatically tracking their directed transport and analyzing their kinetical properties. These methods have allowed us to visualize the trafficking of fluorescently tagged signaling endosomes and autophagosomes derived from axonal terminals and resolve their localizations and movements with high spatial and temporal accuracy. In this chapter, we describe how to use a commercially available microfluidic device to enable the labeling and tracking of retrograde axonal carriers, including (1) how to culture and transfect rat hippocampal neurons in the microfluidic device; (2) how to perform pulse-chase to label specific populations of retrograde axonal carriers; and (3) how to conduct the automatic tracking and data analysis using open-source software.

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References

Taylor A, Blurton-Jones M, Rhee S, Cribbs D, Cotman C, Jeon N . A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods. 2005; 2(8):599-605. PMC: 1558906. DOI: 10.1038/nmeth777. View

Taylor A, Jeon N . Micro-scale and microfluidic devices for neurobiology. Curr Opin Neurobiol. 2010; 20(5):640-7. DOI: 10.1016/j.conb.2010.07.011. View

Wang J, Ren L, Li L, Liu W, Zhou J, Yu W . Microfluidics: a new cosset for neurobiology. Lab Chip. 2009; 9(5):644-52. DOI: 10.1039/b813495b. View

Nguyen Q, Son Y, Sanes J, Lichtman J . Nerve terminals form but fail to mature when postsynaptic differentiation is blocked: in vivo analysis using mammalian nerve-muscle chimeras. J Neurosci. 2000; 20(16):6077-86. PMC: 6772575. View

Grimm A, Winter N, Rattay T, Hartig F, Dammeier N, Auffenberg E . A look inside the nerve - Morphology of nerve fascicles in healthy controls and patients with polyneuropathy. Clin Neurophysiol. 2017; 128(12):2521-2526. DOI: 10.1016/j.clinph.2017.08.022. View

Wang T, Martin S, Nguyen T, Harper C, Gormal R, Martinez-Marmol R . Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB. Nat Commun. 2016; 7:12976. PMC: 5427517. DOI: 10.1038/ncomms12976. View

Restani L, Giribaldi F, Manich M, Bercsenyi K, Menendez G, Rossetto O . Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons. PLoS Pathog. 2013; 8(12):e1003087. PMC: 3531519. DOI: 10.1371/journal.ppat.1003087. View

Zweifel L, Kuruvilla R, Ginty D . Functions and mechanisms of retrograde neurotrophin signalling. Nat Rev Neurosci. 2005; 6(8):615-25. DOI: 10.1038/nrn1727. View

Harrington A, Ginty D . Long-distance retrograde neurotrophic factor signalling in neurons. Nat Rev Neurosci. 2013; 14(3):177-87. DOI: 10.1038/nrn3253. View

10.

Maday S, Twelvetrees A, Moughamian A, Holzbaur E . Axonal transport: cargo-specific mechanisms of motility and regulation. Neuron. 2014; 84(2):292-309. PMC: 4269290. DOI: 10.1016/j.neuron.2014.10.019. View

11.

Gowrishankar S, Yuan P, Wu Y, Schrag M, Paradise S, Grutzendler J . Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's disease amyloid plaques. Proc Natl Acad Sci U S A. 2015; 112(28):E3699-708. PMC: 4507205. DOI: 10.1073/pnas.1510329112. View

12.

Yue Z . Regulation of neuronal autophagy in axon: implication of autophagy in axonal function and dysfunction/degeneration. Autophagy. 2007; 3(2):139-41. DOI: 10.4161/auto.3602. View

13.

Farfel-Becker T, Roney J, Cheng X, Li S, Cuddy S, Sheng Z . Neuronal Soma-Derived Degradative Lysosomes Are Continuously Delivered to Distal Axons to Maintain Local Degradation Capacity. Cell Rep. 2019; 28(1):51-64.e4. PMC: 6696943. DOI: 10.1016/j.celrep.2019.06.013. View

14.

Lee S, Sato Y, Nixon R . Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer's-like axonal dystrophy. J Neurosci. 2011; 31(21):7817-30. PMC: 3351137. DOI: 10.1523/JNEUROSCI.6412-10.2011. View

15.

Zhou R, Han B, Xia C, Zhuang X . Membrane-associated periodic skeleton is a signaling platform for RTK transactivation in neurons. Science. 2019; 365(6456):929-934. PMC: 7063502. DOI: 10.1126/science.aaw5937. View

16.

Li H, Yang J, Tian C, Diao M, Wang Q, Zhao S . Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging. Nat Commun. 2020; 11(1):5699. PMC: 7659323. DOI: 10.1038/s41467-020-19510-5. View

17.

Joensuu M, Padmanabhan P, Durisic N, Bademosi A, Cooper-Williams E, Morrow I . Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles. J Cell Biol. 2016; 215(2):277-292. PMC: 5080683. DOI: 10.1083/jcb.201604001. View

18.

Bademosi A, Lauwers E, Padmanabhan P, Odierna L, Chai Y, Papadopulos A . In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters. Nat Commun. 2017; 8:13660. PMC: 5171881. DOI: 10.1038/ncomms13660. View

19.

Steketee M, Moysidis S, Jin X, Weinstein J, Pita-Thomas W, Raju H . Nanoparticle-mediated signaling endosome localization regulates growth cone motility and neurite growth. Proc Natl Acad Sci U S A. 2011; 108(47):19042-7. PMC: 3223462. DOI: 10.1073/pnas.1019624108. View

20.

Zhou B, Cai Q, Xie Y, Sheng Z . Snapin recruits dynein to BDNF-TrkB signaling endosomes for retrograde axonal transport and is essential for dendrite growth of cortical neurons. Cell Rep. 2012; 2(1):42-51. PMC: 3408618. DOI: 10.1016/j.celrep.2012.06.010. View