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Neuroscience

Analyzing Murine Schwann Cell Development Along Growing Axons

Published: November 21st, 2012

DOI:

10.3791/50016

1Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg , 2Department of Neuroanatomy, University of Heidelberg, 3FRIAS, University of Freiburg

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

1. Preparation of Collagen Gels

  1. Prepare a stock medium, containing 455 μl 10x MEM, 112 μl 7.5% NaHCO3, 50 μl glutamine and NaOH. The concentration and the amount of the NaOH depends on the rat- tail collagen preparation (see 1.2), the final volume of the stock medium being 1,000 μl.
  2. Prepare rat-tail collagen according to Ebendal (1). Store the Collagen solution at 4 °C. A degradation of the collagen solution could not be observed. After preparation of a ne.......

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Axonal growth is facilitated from SCG explants upon treatment with NGF (4) (movie scheme S1 Figure 1 scheme). This process is easily visible by any inverted microscope and can be followed by time-lapse imaging (movie S2). If a scientist is new to dissecting SCG from mouse embryos we strongly recommend a validation of the technique by a simple anti- tyrosine hydroxylase (TH) immunohistochemistry. TH is a common marker for catecholaminergic neurons (in this case sympathetic neurons) .......

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The development of the peripheral nervous system is an exciting process. When the development is completed, axons are ensheathed by SCs along the entire length, which can, in humans, often be over 100 cm. To this end the correct number of the required SCs has to be established during development and the SCs also have to move along extending axons to the periphery to ensure the complete axonal coverage. This holds true for myelinated but also for unmeylinated axons. In both cases all axons are in contact with SCs a.......

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We want to thank Urmas Arumae for sharing a collagen protocol and Jutta Fey and Ursula Hinz for excellent technical assistance. Furthermore we want to thank Christian F. Ackermann, Ulrike Engel and the Nikon Imaging Center at the University of Heidelberg and also Joachim Kirsch for kindly help for the video shoting. The work was partially funded through the Deutsche Forschungsgemeinschaft (SFB 592).

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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalogue Number Comments
10x MEM Gibco 21430
Sodium Bicarbonate (7.5%) Gibco 25080
Glutamine Gibco 25030
NaOH Merck 109137
NGF Roche 1058231 R&D#556-NG-100
Neurobasal Medium Gibco 21103
B27 Supplement Gibco 17504
antibiotics Gibco 15640
d-PBS Gibco 14040
insect needles FST 26002-20
syringe needle Braun BD # 300013
8 well chamber slide Lab tek 177402

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