Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Primary support cultures of hippocampal and substantia nigra neurons

Nature Protocols volume 4, pages 78–85 (2009)Cite this article

Abstract

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. Their use is limited, as culturing at low density is often not possible or is dependent on sophisticated methods. Here we present a novel method for culturing embryonic (E16.5) murine hippocampal neurons, using a spatially separated ring of cortical neurons for neurotrophic support. This method allows long-term cultures at a very low cell density, and therefore, the study of single embryo preparations and isolated neurons. This method has been adopted for neurons from the substantia nigra (E16.5), with support from a ring of striatal neurons.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Dissection of substantia nigra and striatum from embryonic E16.5 brains.
Figure 2: Stepwise procedure for preparing ring support cultures.
Figure 3: Collecting brains from E16.5 embryos.
Figure 4: Dissection of hippocampus and cortex from embryonic E16.5 brains.
Figure 5: Long-term survival of hippocampal neurons depends on cortical support ring.
Figure 6: Neuronal maturation of hippocampal neurons in low-density support cultures.
Figure 7: Tyrosine hydroxylase (TH)-positive neurons in striatal support cultures.

Similar content being viewed by others

References

  1. Banker, G. & Goslin, K. Culturing Nerve Cells, (MIT Press, Cambridge, 1998).

    Google Scholar 

  2. Plachta, N. et al. Identification of a lectin causing the degeneration of neuronal processes using engineered embryonic stem cells. Nat. Neurosci. 10, 712–719 (2007).

    Article  CAS  Google Scholar 

  3. Bibel, M., Richter, J., Lacroix, E. & Barde, Y.A. Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells. Nat. Protoc. 2, 1034–1043 (2007).

    Article  CAS  Google Scholar 

  4. Lee, H.Y. et al. Instructive role of Wnt/beta-catenin in sensory fate specification in neural crest stem cells. Science 303, 1020–1023 (2004).

    Article  CAS  Google Scholar 

  5. Kleber, M. et al. Neural crest stem cell maintenance by combinatorial Wnt and BMP signaling. J. Cell Biol. 169, 309–320 (2005).

    Article  CAS  Google Scholar 

  6. Ackermann, M. & Matus, A. Activity-induced targeting of profilin and stabilization of dendritic spine morphology. Nat. Neurosci. 6, 1194–1200 (2003).

    Article  CAS  Google Scholar 

  7. Fischer, M., Kaech, S., Knutti, D. & Matus, A. Rapid actin-based plasticity in dendritic spines. Neuron 20, 847–854 (1998).

    Article  CAS  Google Scholar 

  8. Calabrese, B. & Halpain, S. Essential role for the PKC target MARCKS in maintaining dendritic spine morphology. Neuron 48, 77–90 (2005).

    Article  CAS  Google Scholar 

  9. Minamide, L.S., Striegl, A.M., Boyle, J.A., Meberg, P.J. & Bamburg, J.R. Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that disrupt distal neurite function. Nat. Cell Biol. 2, 628–636 (2000).

    Article  CAS  Google Scholar 

  10. Banker, G.A. & Cowan, W.M. Rat hippocampal neurons in dispersed cell culture. Brain Res. 126, 397–342 (1977).

    Article  CAS  Google Scholar 

  11. Raineteau, O., Rietschin, L., Gradwohl, G., Guillemot, F. & Gahwiler, B.H. Neurogenesis in hippocampal slice cultures. Mol. Cell Neurosci. 26, 241–250 (2004).

    Article  CAS  Google Scholar 

  12. Banker, G.A. Trophic interactions between astroglial cells and hippocampal neurons in culture. Science 209, 809–810 (1980).

    Article  CAS  Google Scholar 

  13. Dotti, C.G., Sullivan, C.A. & Banker, G.A. The establishment of polarity by hippocampal neurons in culture. J. Neurosci. 8, 1454–1468 (1988).

    Article  CAS  Google Scholar 

  14. Kaech, S. & Banker, G. Culturing hippocampal neurons. Nat. Protoc. 1, 2406–2415 (2006).

    Article  CAS  Google Scholar 

  15. Ittner, L.M. et al. Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia. PNAS 105, 15997–16002 (2008).

    Article  CAS  Google Scholar 

  16. Kurosinski, P., Guggisberg, M. & Gotz, J. Alzheimer's and Parkinson's disease—overlapping or synergistic pathologies? Trends Mol. Med. 8, 3–5 (2002).

    Article  CAS  Google Scholar 

  17. Rayport, S. et al. Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology. J. Neurosci. 12, 4264–4280 (1992).

    Article  CAS  Google Scholar 

  18. Cardozo, D.L. Midbrain dopaminergic neurons from postnatal rat in long-term primary culture. Neuroscience 56, 409–421 (1993).

    Article  CAS  Google Scholar 

  19. Shi, W.X. & Rayport, S. GABA synapses formed in vitro by local axon collaterals of nucleus accumbens neurons. J. Neurosci. 14, 4548–4560 (1994).

    Article  CAS  Google Scholar 

  20. Sulzer, D. et al. Dopamine neurons make glutamatergic synapses in vitro . J. Neurosci. 18, 4588–4602 (1998).

    Article  CAS  Google Scholar 

  21. Congar, P., Bergevin, A. & Trudeau, L.E. D2 receptors inhibit the secretory process downstream from calcium influx in dopaminergic neurons: implication of K+ channels. J. Neurophysiol. 87, 1046–1056 (2002).

    Article  CAS  Google Scholar 

  22. Gille, G. et al. Pergolide protects dopaminergic neurons in primary culture under stress conditions. J. Neural Transm. 109, 633–643 (2002).

    Article  CAS  Google Scholar 

  23. Hamann, J., Rommelspacher, H., Storch, A., Reichmann, H. & Gille, G. Neurotoxic mechanisms of 2,9-dimethyl-beta-carbolinium ion in primary dopaminergic culture. J. Neurochem. 98, 1185–1199 (2006).

    Article  CAS  Google Scholar 

  24. Poulsen, K.T. et al. TGF beta 2 and TGF beta 3 are potent survival factors for midbrain dopaminergic neurons. Neuron 13, 1245–1252 (1994).

    Article  CAS  Google Scholar 

  25. Wurdak, H. et al. Inactivation of TGFbeta signaling in neural crest stem cells leads to multiple defects reminiscent of DiGeorge syndrome. Genes Dev. 19, 530–535 (2005).

    Article  CAS  Google Scholar 

  26. Ittner, L.M. et al. Compound developmental eye disorders following inactivation of TGFbeta signaling in neural-crest stem cells. J. Biol. 4, 11 (2005).

    Article  Google Scholar 

  27. Massague, J. How cells read TGF-beta signals. Nat. Rev. Mol. Cell Biol. 1, 169–178 (2000).

    Article  CAS  Google Scholar 

  28. Farkas, L.M., Dunker, N., Roussa, E., Unsicker, K. & Krieglstein, K. Transforming growth factor-beta(s) are essential for the development of midbrain dopaminergic neurons in vitro and in vivo . J. Neurosci. 23, 5178–5186 (2003).

    Article  CAS  Google Scholar 

  29. Ittner, L.M. & Gotz, J. Pronuclear injection for the production of transgenic mice. Nat. Protoc. 2, 1206–1215 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Mian Bi, Fabien Delerue and Denise Nergenau for technical assistance and Dr Victor Anggono for the dynamin antibody. L.M.I. has been supported by the DFG, the Australian Research Council (ARC) and the University of Sydney. J.G. is a Medical Foundation Fellow and has been supported by the University of Sydney, the National Health and Medical Research Council (NHMRC), the ARC, the New South Wales Government through the Ministry for Science and Medical Research (BioFirst Program), the Nerve Research Foundation, the Medical Foundation (University of Sydney) and the Judith Jane Mason and Harold Stannett Williams Memorial Foundation. P.G. is supported by a NSW cancer institute research infrastructure grant and is a Principal Research Fellow of the NHMRC.

Author information

Authors and Affiliations

  1. Oncology Research Unit, The Children's Hospital at Westmead, Sydney, Westmead, 2145, New South Wales, Australia

    Thomas Fath & Peter Gunning

  2. Alzheimer's and Parkinson's Disease Laboratory, The Brain and Mind Research Institute (BMRI), University of Sydney, Camperdown, Sydney, 2050, New South Wales, Australia

    Yazi D Ke, Jürgen Götz & Lars M Ittner

Authors
  1. Thomas Fath
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yazi D Ke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter Gunning
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jürgen Götz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lars M Ittner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Thomas Fath, Jürgen Götz or Lars M Ittner.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fath, T., Ke, Y., Gunning, P. et al. Primary support cultures of hippocampal and substantia nigra neurons. Nat Protoc 4, 78–85 (2009). https://doi.org/10.1038/nprot.2008.199

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2008.199

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing