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Myelination of rodent hippocampal neurons in culture

Nature Protocols volume 7, pages 1774–1782 (2012)Cite this article

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Abstract

Axons of various hippocampal neurons are myelinated mainly postnatally, which is important for the proper function of neural circuits. Demyelination in the hippocampus has been observed in patients with multiple sclerosis, Alzheimer's disease or temporal lobe epilepsy. However, very little is known about the mechanisms and exact functions of the interaction between the myelin-making oligodendrocytes and the axons within the hippocampus. This is mainly attributable to the lack of a system suitable for molecular studies. We recently established a new myelin coculture from embryonic day (E) 18 rat embryos consisting of hippocampal neurons and oligodendrocytes, with which we identified a novel intra-axonal signaling pathway regulating the juxtaparanodal clustering of Kv1.2 channels. Here we describe the detailed protocol for this new coculture. It takes about 5 weeks to set up and use the system. This coculture is particularly useful for studying myelin-mediated regulation of ion channel trafficking and for understanding how neuronal excitability and synaptic transmission are regulated by myelination.

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Figure 1: Dissection procedure of rat brain from the E18 embryos for making the myelin coculture.
Figure 2: The timeline for making hippocampal neuron culture and coculture.
Figure 3: Maturation of oligodendrocytes cocultured with hippocampal neurons.
Figure 4: Myelination of hippocampal neurons expressing YFP.
Figure 5: Myelin segments of mature oligodendrocytes in the coculture.

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Acknowledgements

We thank J. Barry and Y. Gu for technical assistance, and the Campus Microscopy & Imaging facility at the Ohio State University and Research Core Services at Lerner Research Institute of Cleveland Clinic Foundation for technical assistance in transmission electron microscopy. This work was supported by a career transition fellowship award from the US National Multiple Sclerosis Society (grant TA3012A1) and a grant from the US National Institute of Neurological Disorders and Stroke/National Institutes of Health (R01NS062720) to C.G. All animal experiments have been conducted in accordance with the US National Institutes of Health Animal Use Guidelines.

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Authors and Affiliations

  1. Neuroscience Graduate Studies Program, The Ohio State University, Columbus, Ohio, USA

    Asa Gardner & Chen Gu

  2. Integrated Biomedical Graduate Program, The Ohio State University, Columbus, Ohio, USA

    Peter Jukkola & Chen Gu

  3. Department of Neuroscience, The Ohio State University, Columbus, Ohio, USA

    Chen Gu

Authors
  1. Asa Gardner
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  2. Peter Jukkola
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Contributions

A.G. and C.G. designed and performed the experiments. A.G., P.J. and C.G. wrote the manuscript. C.G. supervised the project.

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Correspondence to Chen Gu.

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Gardner, A., Jukkola, P. & Gu, C. Myelination of rodent hippocampal neurons in culture. Nat Protoc 7, 1774–1782 (2012). https://doi.org/10.1038/nprot.2012.100

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