Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

Summary

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

Abstract

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Introduction

Zebrafish have been used extensively to study development of the nervous system because of their optical transparence and ease of transgenesis, which when coupled, allow for spectacular imaging of dynamic cell behaviors in a living embryo. Additionally, because zebrafish and mammals share nearly all of the genes required for nervous system formation, cellular and molecular information collected in this model organism is directly relatable to other vertebrate species. Although incredibly powerful for neural developmental studies, the zebrafish and its unique attributes have the potential to also elucidate the mechanisms that maintain and rebuild the nervous system afte....

Protocol

1. Preparation and Mounting of Zebrafish Embryos for Ablation and Live Imaging

1. Prepare a stock of 0.8% low melt agarose in egg water. Aliquot into 13X 100 mm disposable culture tubes and store at 4 °C until needed.
2. Cross adult zebrafish containing stably integrated transgenes to fluorescently label motor neurons and glial cell types of interest. Collect zebrafish embryos in egg water and place in 28.5 °C incubator for correct staging later ³.
3. At approximately 24 hr post fertilization (hpf), remove egg water and add 0.002% 1-phenyl 2-thiourea (PTU) in egg water. Return embryos to the incubator.
4. Between 24 and....

Results

The assay described here can be used to assess the response of glial cells and other nerve-associated cell populations to axonal injury in vivo. Movie 1 shows an example of a nerve injury created using this method and the response of surrounding glial cells. This experiment was performed in Tg(nkx2.2a:megfp);Tg(olig2:dsred) zebrafish, in which perineurial glia express a membrane targeted EGFP and motor neurons express cytosolic DSRed. The injury was made along the rostral projection of .......

Discussion

The most critical steps of this experimental design are: 1) properly mounting larvae for injury and subsequent in vivo imaging and 2) calibrating the laser and selecting the correct power settings in order to create a clean nerve transection that results in minimal extra-tissue damage. To help ensure a successful axotomy for in vivo imaging and subsequent analysis, mount multiple larvae in either individual glass bottom dishes or in a glass bottom dish with dividers. After calibrating the laser, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phenylthiourea	Sigma	P7629-100G
Finquel Tricaine Methanesulfonate MS-222	Argent Chemical	C-FINQ-UE-100G
Low melting point agarose	Sigma	A9414-10G
Quad CELLview Cell Culture Dishes, Glass Bottom, Sterile, Greiner Bio One	VWR/Greiner	89125-444
Single well glass bottom Petri dishes 35 x 10 mm, 12 mm thick	Willco Wells	GWSt-3512
MicroPoint Laser System with all components	Andor Technology - purchased through Quorum Technologies, Inc.	2203-SYS
MicroPoint Laser Courmarin dye (435 nm)	Andor Technology	MP-27-435-DYE