Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

Kristen N. Fantetti; Donna M. Fekete

doi:10.3791/3600

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels ...

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Neuroscience

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JoVE Journal Neuroscience

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

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11:08 min

December 20, 2011

DOI: 10.3791/3600-v

Kristen N. Fantetti¹, Donna M. Fekete¹

¹Department of Biological Sciences,Purdue University

Overview

This study demonstrates a method for dissecting and culturing chick statoacoustic ganglion and spinal cord explants. The explants are cultured in 3D collagen gels under serum-free conditions, allowing for the evaluation of neurite outgrowth in response to various growth factors.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Auditory Research

Background

Statoacoustic ganglion neurons are crucial for auditory function.
Understanding neurite outgrowth is essential for insights into neuron survival and guidance.
Growth factors play a significant role in neuronal development.
3D culture systems can mimic in vivo conditions for better experimental outcomes.

Purpose of Study

To evaluate the effects of growth factors on neurite outgrowth in cultured statoacoustic ganglion neurons.
To investigate the role of secreted ligands in neuron survival and axon guidance.
To develop a reliable method for studying auditory neuron behavior in vitro.

Methods Used

Dissection of statoacoustic ganglion and spinal cord from chick embryos.
Culturing explants in collagen gels under serum-free conditions.
Testing neurite responsiveness with growth factor-supplemented media.
Imaging explants with confocal microscopy after labeling with anti beta tubulin.

Main Results

Neurite outgrowth was observed in response to various growth factors.
Directional outgrowth was assessed using protein-coated beads.
Results indicated the importance of specific molecules for neuron survival.
The method provides a platform for further research in auditory neuron development.

Conclusions

The study successfully demonstrates a culture method for chick statoacoustic ganglion neurons.
Growth factors significantly influence neurite outgrowth and neuron survival.
This approach can enhance understanding of auditory system development.

Frequently Asked Questions

What are statoacoustic ganglion neurons?

Statoacoustic ganglion neurons are sensory neurons involved in hearing and balance, located in the inner ear.

Why is 3D culture important?

3D culture systems better mimic the natural environment of cells, leading to more accurate experimental results.

How do growth factors affect neurons?

Growth factors can promote neurite outgrowth, enhance survival, and influence the direction of neuronal growth.

What techniques are used to visualize neurites?

Confocal microscopy is used to image neurites after labeling with specific antibodies like anti beta tubulin.

What is the significance of this research?

This research helps identify key molecules for neuron survival and guidance, which is crucial for understanding auditory system development.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

The overall goal of the following experiment is to evaluate neurite outgrowth in stato acoustic ganglion neurons grown in culture when various growth factors are added to the media. This is achieved by first dissecting the stato acoustic ganglion and control spinal cords from the embryo to create X explants for culture. As a second step X explants are placed in collagen, either with soluble proteins to test for overall outgrowth or with protein coated beads to test for directional outgrowth.

Next X explants are labeled with anti beta tubulin and imaged with a confocal microscope. In order to visualize the neurites emerging from the ganglion results are obtained that show the effects that secreted ligands can exert on neuron survival and neurite outgrowth. This method can help to answer key questions in the field of auditory research, such as what molecules are important for otic neuron survival and axon guidance as sensory organs become innervated.

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