Working with Xenopus Spinal Neurons in Live Cell Culture

Timothy M. Gomez, Dan Harrigan,* John Henley,^ and Estuardo Robles*

*Department of Anatomy Cell and Molecular Biology Training Program University of Wisconsin Medical School Madison, Wisconsin 53706

^Department of Molecular and Cell Biology University of California Berkeley, California 94720-3200

^Neuroscience Training Program University of Wisconsin Medical School Madison, Wisconsin 53706

I. Introduction II. Neuronal Labeling

A. Injection Sample Preparation

B. Blastomere Injection

C. Lipofection

D. Single Cell Injection

III. Culturing Xenopus Spinal Neurons

A. Dissociated Mixed Cultures

B. Neural-Enriched Cultures

C. Cocultures

D. Culture Substrata

IV. Live Cell Imaging and Manipulations

A. Imaging Chambers

B. Live Cell Imaging

C. Rapid Fixation and Staining

D. Ultraviolet Photolysis V. Summary

References

METHODS IN CELL BIOLOGY, VOL. 71 Copyright 2003, Elsevier Science (USA). All rights reserved. 0091-679X/03 $35.00

Neurons from the Xenopus spinal cord are highly versatile and easily manipulated, making them an ideal model system to answer questions regarding the cellular and molecular basis of early neural development and function. Xenopus has been a productive model system in studies ranging from axon growth and guidance to synaptic plasticity. Exogenous molecules, such as proteins, fluorescent tracers, and nucleic acids, can be injected into early blastomeres to load tracers in all neurons or into late blastomeres to target specifc classes of neurons based on established lineage maps. Xenopus spinal neurons also provide an excellent culture system, as neurons extend processes on a variety of substrata and develop at room temperature in minimal salt solutions. Live fluorescent neurons can be imaged for hours with fluorescence microscopy at room temperature in static cultures without neurotrophic support or serum. This highly reduced culture system minimizes variables that can confound interpretation of results. Cultures can be prepared at various stages of development as dissociated neurons or as spinal cord explants. Both excitatory and inhibitory neurons develop in culture, and synaptic contacts among neurons and between neurons and nonneuronal targets form naturally. The simple anatomy and rapid rostral-to-caudal development of the Xenopus spinal cord also make this an excellent in vivo model system to analyze axon guidance by identifiable classes of neurons. This chapter focuses on techniques that exploit both in vitro and in vivo qualities of this system.

I. Introduction

Xenopus laevis has proven to be an extremely productive embryological model system for over a century due in large part to the accessibility, size, rapid development, and ease in which large numbers of embryos are generated and manipulated at room temperature. Much of our understanding of the mechanisms of early vertebrate development comes from studies using Xenopus. Advantages offered by this organism continue to aid investigators using modern approaches, such as fluorescent fusion protein expression, electrophysiological recordings, and in vivo imaging. However, two disadvantages have made this system less popular than genetic model systems such as Drosophila and Zebrafish; Xenopus has a protracted generation time (1-2 years) and it is an allotetraploid (four copies of each chromosome). However, even these short comings are being challenged by the development of Xenopus transgenesis techniques based in sperm nuclear transplantation (Kroll and Amaya, 1996) and the use of X. tropicalis, the diploid cousin of X. laevis that has a 5-month generation time (Amaya et al., 1998). Xenopus transgenesis techniques have been described elsewhere (Offield et al., 2000; Sive et al., 2000) so are not discussed here. Instead this chapter focuses on techniques designed for the short-term modification of Xenopus neurons, which are well suited for the study of early developmental events.

The techniques described in this chapter take advantage of the ability to rapidly and efficiently label or operationally alter many neurons in a population and then examine those manipulations 24-36 h later in culture or in vivo. Particularly useful manipulations include the expression of fluorescent fusion proteins and dominantnegative (DN) or constitutively active (CA) mutant proteins. Our particular interest is in growth cone motility and axon guidance, thus the particular manipulations and assays described here reflect this interest, but our techniques are not limited to this focus. This chapter is divided into three sections. We begin with manipulations to label or modified neurons that are made to early embryos prior to culturing or in vivo imaging. The next section discusses several spinal cord culture techniques aimed at either isolating individual neurons or promoting interactions among neurons or between neurons and target cells in mixed cultures. The last section describes imaging and manipulating cells in culture.

II. Neuronal Labeling

This section describes labeling or functionally modifying neurons with molecules such as fluorescent tracers, physiological indicators, or mutant proteins using several alternative approaches at different stages of development. Molecules are introduced most easily into large numbers of neurons by the microinjection of individual precursor cells of cleavage-stage embryos. This approach is simple and appropriate for many types of reagents (Table 1), however, it can be problematic for molecules that alter some aspect of development prior to their intended function. For example, blastomere injection of DN or CA proteins or RNA/ DNA encoding mutant proteins can, in some cases, affect developmental events prior to neurogenesis or axon outgrowth. Control injections and titering the amount of injected material are often necessary to assure the specificity or localization of the molecules being tested. In instances where early expression non-specifically alters embyogenesis, methods for delayed introduction of molecules are presented.

Table I

Compounds Injected Effectively into Blastomere Stage Xenopus Embryos

Table I

Compounds Injected Effectively into Blastomere Stage Xenopus Embryos

Compound

Effective quantity (ng)

Reference

Fluorescent dextrans

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