Analyzing Murine Schwann Cell Development Along Growing Axons

Summary

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

Abstract

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.

Protocol

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 new batch, estimate the NaOH concentration needed for the stock medium (see 1.1). Use the pH-indicator of the medium for titration of the amount and con....

Results

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) .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog 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