Primary Neuron Culture for Nerve Growth and Axon Guidance Studies in Zebrafish (Danio rerio)

PLoS ONE, 2013 · DOI: 10.1371/journal.pone.0057539 · Published: March 4, 2013

Neurology Genetics Research Methodology & Design

Simple Explanation

This paper introduces a new method for growing zebrafish neurons in a lab setting, which allows scientists to study nerve growth and guidance. The method involves taking neurons from different parts of the zebrafish brain and spinal cord and growing them on special surfaces to see how they extend axons. Using this method, researchers can observe how nerve cells respond to different chemicals that either attract or repel them, similar to how nerves find their way during development. They also looked at the role of calcium, an important signaling molecule, in nerve growth. This new way of growing zebrafish neurons can help scientists better understand how the nervous system develops, how nerves regenerate after injury, and test new ideas about nerve growth in a controlled environment.

Study Duration

Not specified

Participants

Wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines

Evidence Level

In vitro study

Key Findings

1
Zebrafish neurons in culture extend axons with dynamic growth cones, exhibiting an average extension rate of 21.461.2 mm/hr for spinal cord neurons.
2
Axon extension and growth cone chemoattraction are stimulated by mammalian brain-derived neurotrophic factor (BDNF).
3
Elevation of cytoplasmic Ca2+ concentration is induced in the growth cone by BDNF application.

Research Summary

The study introduces a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons were dissociated and plated onto various substrates to optimize axon outgrowth. Functional assays demonstrated BDNF-dependent stimulation of outgrowth and growth cone chemoattraction, whereas myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca2+-imaging revealed local elevation of cytoplasmic Ca2+ concentration in the growth cone induced by BDNF application. The primary neuron culture model system may be useful for studies of neuronal development, chemotropic axon guidance, and mechanisms underlying inhibition of neural regeneration in vitro.

Practical Implications

Drug Discovery

The system can be used to screen drugs that promote or inhibit nerve growth.

Regeneration Research

The model can help define the mechanistic differences underlying robust versus limited regeneration in zebrafish and mammals.

Educational Tool

The relatively simple neuron culture system is amenable to introducing concepts of in vitro research in the classroom.

Study Limitations

1
The culture system generates a mixed population of neurons and other cell types.
2
The growth rate of neurons diminishes after 12 hours in culture.
3
The study relies on mammalian proteins to elicit outgrowth, which may not directly translate to zebrafish-specific mechanisms.

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