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:

Preparation of organotypic hippocampal slice cultures: interface method

Nature Protocols volume 1, pages 1439–1445 (2006)Cite this article

Abstract

This protocol describes a method for making and culturing rat hippocampal organotypic slices on membrane inserts. Supplementary videos are included to demonstrate visually the different steps of the procedure. Cultured hippocampal slices has been increasingly used as a model for synaptic studies of the brain as they allow examination of mid to long term manipulations in a preparation where the gross cytoarchitecture of the hippocampus is preserved. Combining techniques such as molecular biology, electrophysiology and immunohistochemistry to study physiological or pathological processes can easily be applied to organotypic slices. The technique described here can be used to make organotypic slices from other parts of the brain, other rodent species and from a range of ages. This protocol can be completed in 3 h.

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: This photograph shows the 300 μm slice lying on the blade of the vibrotome.
Figure 2: Development in vitro results in a flattening of the hippocampal slice and in the appearance of flat and clean cell bodies.

Similar content being viewed by others

References

  1. Gahwiler, B.H. Organotypic cultures of neural tissue. Trends. Neurosci. 11, 484–489 (1988).

    Article  CAS  Google Scholar 

  2. Gahwiler, B.H. Nerve cells in culture: the extraordinary discovery by Ross Granville Harrison. Brain. Res. Bull. 50, 343–344 (1999).

    Article  CAS  Google Scholar 

  3. Harrison, R.G. Observations on the living developing nerve fiber. Proc. Soc. Exp. Biol. Med. 4, 140–143 (1907).

    Article  Google Scholar 

  4. Harrison, R.G. The outgrowth of the nerve fiber as a mode of protoplasmic movement. J. Exp. Zoo. 142, 5–73 (1959).

    Article  CAS  Google Scholar 

  5. Keshishian, H. Ross Harrison's “The outgrowth of the nerve fiber as a mode of protoplasmic movement”. J. Exp. Zool A Comp. Exp. Biol. 301, 201–203 (2004).

    Article  Google Scholar 

  6. Gahwiler, B.H. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Methods 4, 329–342 (1981).

    Article  CAS  Google Scholar 

  7. Hogue, M.J. Human fetal brain cells in tissue cultures: their identification and motility. J. Exp. Zool. 106, 85–107 (2006).

    Article  Google Scholar 

  8. Stoppini, L., Buchs, P.A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991).

    Article  CAS  Google Scholar 

  9. Gahwiler, B.H., Capogna, M., Debanne, D., McKinney, R.A. & Thompson, S.M. Organotypic slice cultures: a technique has come of age. Trends Neurosci. 20, 471–477 (1997).

    Article  CAS  Google Scholar 

  10. De Simoni, A., Griesinger, C.B. & Edwards, F.A. Development of rat CA1 neurones in acute versus organotypic slices: role of experience in synaptic morphology and activity. J. Physiol. 550, 135–147 (2003).

    Article  CAS  Google Scholar 

  11. Buchs, P.A. & Muller, D. Induction of long-term potentiation is associated with major ultrastructural changes of activated synapses. Proc. Natl. Acad. Sci. USA 93, 8040–8045 (1996).

    Article  CAS  Google Scholar 

  12. Debanne, D., Gahwiler, B.H. & Thompson, S.M. Cooperative interactions in the induction of long-term potentiation and depression of synaptic excitation between hippocampal CA3–CA1 cell pairs in vitro. Proc. Natl. Acad. Sci. USA 93, 11225–11230 (1996).

    Article  CAS  Google Scholar 

  13. Maletic-Savatic, M., Malinow, R. & Svoboda, K. Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283, 1923–1927 (1999).

    Article  CAS  Google Scholar 

  14. Collin, C., Miyaguchi, K. & Segal, M. Dendritic spine density and LTP induction in cultured hippocampal slices. J. Neurophysiol 77, 1614–1623 (1997).

    Article  CAS  Google Scholar 

  15. Li, Z. et al. Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin. Proc. Natl. Acad. Sci. USA 102, 6131–6136 (2005).

    Article  CAS  Google Scholar 

  16. Engert, F. & Bonhoeffer, T. Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 399, 66–70 (1999).

    Article  CAS  Google Scholar 

  17. Barria, A. & Malinow, R. NMDA receptor subunit composition controls synaptic plasticity by regulating binding to CaMKII. Neuron 48, 289–301 (2005).

    Article  CAS  Google Scholar 

  18. Leutgeb, J.K., Frey, J.U. & Behnisch, T. LTP in cultured hippocampal-entorhinal cortex slices from young adult (P25–30) rats. J. Neurosci. Methods 130, 19–32 (2003).

    Article  Google Scholar 

  19. Glover, C.P., Bienemann, A.S., Heywood, D.J., Cosgrave, A.S. & Uney, J.B. Adenoviral-mediated, high-level, cell-specific transgene expression: a SYN1-WPRE cassette mediates increased transgene expression with no loss of neuron specificity. Mol. Ther. 5, 509–516 (2002).

    Article  CAS  Google Scholar 

  20. Miyaguchi, K., Maeda, Y., Collin, C. & Sihag, R.K. Gene transfer into hippocampal slice cultures with an adenovirus vector driven by cytomegalovirus promoter: stable co-expression of green fluorescent protein and lacZ genes. Brain Res. Bull. 51, 195–202 (2000).

    Article  CAS  Google Scholar 

  21. De Paola, V., Arber, S. & Caroni, P. AMPA receptors regulate dynamic equilibrium of presynaptic terminals in mature hippocampal networks. Nat. Neurosci. 6, 491–500 (2003).

    Article  CAS  Google Scholar 

  22. Stricker, C. in Neuroscience Methods (ed. Martin, R.) (Harwood Academic Publishers, Amsterdam, 1997).

  23. Nagerl, U.V., Eberhorn, N., Cambridge, S.B. & Bonhoeffer, T. Bidirectional activity-dependent morphological plasticity in hippocampal neurons. Neuron 44, 759–767 (2004).

    Article  Google Scholar 

  24. Galimberti, I. et al. Long-term rearrangements of hippocampal mossy fiber terminal connectivity in the adult regulated by experience. Neuron 50, 749–763 (2006).

    Article  CAS  Google Scholar 

  25. 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 

  26. Nikonenko, I. et al. Integrins are involved in synaptogenesis, cell spreading, and adhesion in the postnatal brain. Brain Res. Dev. Brain Res. 140, 185–194 (2003).

    Article  CAS  Google Scholar 

  27. Linke, R., Heimrich, B. & Frotscher, M. Axonal regeneration of identified septohippocampal projection neurons in vitro. Neuroscience 68, 1–4 (1995).

    Article  CAS  Google Scholar 

  28. Lundstrom, K. et al. Semliki Forest virus vectors: efficient vehicles for in vitro and in vivo gene delivery. FEBS Lett. 504, 99–103 (2001).

    Article  CAS  Google Scholar 

  29. Lundstrom, K., Abenavoli, A., Malgaroli, A. & Ehrengruber, M.U. Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature sensitivity for long-term enhancement of transgene expression. Mol. Ther. 7, 202–209 (2003).

    Article  CAS  Google Scholar 

  30. Kakegawa, W., Tsuzuki, K., Yoshida, Y., Kameyama, K. & Ozawa, S. Input- and subunit-specific AMPA receptor trafficking underlying long-term potentiation at hippocampal CA3 synapses. Eur. J. Neurosci. 20, 101–110 (2004).

    Article  Google Scholar 

  31. Ehrengruber, M.U. et al. Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures. Proc. Natl. Acad. Sci. USA 96, 7041–7046 (1999).

    Article  CAS  Google Scholar 

  32. Perez Velazquez, J.L., Frantseva, M.V. & Carlen, P.L. In vitro ischemia promotes glutamate-mediated free radical generation and intracellular calcium accumulation in hippocampal pyramidal neurons. J. Neurosci. 17, 9085–9094 (1997).

    Article  CAS  Google Scholar 

  33. Newell, D.W., Barth, A., Papermaster, V. & Malouf, A.T. Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures. J. Neurosci. 15, 7702–7711 (1995).

    Article  CAS  Google Scholar 

  34. Schmidt, H. et al. Organotypic hippocampal cultures. A model of brain tissue damage in Streptococcus pneumoniae meningitis. J. Neuroimmunol. 113, 30–39 (2001).

    Article  CAS  Google Scholar 

  35. Gianinazzi, C. et al. Apoptosis of hippocampal neurons in organotypic slice culture models: direct effect of bacteria revisited. J. Neuropathol. Exp. Neurol. 63, 610–617 (2004).

    Article  Google Scholar 

  36. Naumann, T., Linke, R. & Frotscher, M. Fine structure of rat septohippocampal neurons: I. Identification of septohippocampal projection neurons by retrograde tracing combined with electron microscopic immunocytochemistry and intracellular staining. J. Comp. Neurol. 325, 207–218 (1992).

    Article  CAS  Google Scholar 

  37. Coltman, B.W., Earley, E.M., Shahar, A., Dudek, F.E. & Ide, C.F. Factors influencing mossy fiber collateral sprouting in organotypic slice cultures of neonatal mouse hippocampus. J. Comp. Neurol. 362, 209–222 (1995).

    Article  CAS  Google Scholar 

  38. Ghoumari, A.M. et al. Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum. Proc. Natl. Acad. Sci. USA 100, 7953–7958 (2003).

    Article  CAS  Google Scholar 

  39. Becq, H., Bosler, O., Geffard, M., Enjalbert, A. & Herman, J.P. Anatomical and functional reconstruction of the nigrostriatal system in vitro: selective innervation of the striatum by dopaminergic neurons. J. Neurosci. Res. 58, 553–566 (1999).

    Article  CAS  Google Scholar 

  40. Oishi, Y., Baratta, J., Robertson, R.T. & Steward, O. Assessment of factors regulating axon growth between the cortex and spinal cord in organotypic co-cultures: effects of age and neurotrophic factors. J. Neurotrauma 21, 339–356 (2004).

    Article  Google Scholar 

  41. Hilton, K.J., Bateson, A.N. & King, A.E. A model of organotypic rat spinal slice culture and biolistic transfection to elucidate factors that drive the preprotachykinin-A promoter. Brain Res. Brain. Res. Rev. 46, 191–203 (2004).

    Article  CAS  Google Scholar 

  42. Takuma, H., Sakurai, M. & Kanazawa, I. In vitro formation of corticospinal synapses in an organotypic slice co-culture. Neuroscience 109, 359–370 (2002).

    Article  CAS  Google Scholar 

  43. Gong, Q., Liu, W.L., Srodon, M., Foster, T.D. & Shipley, M.T. Olfactory epithelial organotypic slice cultures: a useful tool for investigating olfactory neural development. Int. J. Dev. Neurosci. 14, 841–852 (1996).

    Article  CAS  Google Scholar 

  44. Molnar, Z. & Blakemore, C. Development of signals influencing the growth and termination of thalamocortical axons in organotypic culture. Exp. Neurol. 156, 363–393 (1999).

    Article  CAS  Google Scholar 

  45. Letinic, K., Zoncu, R. & Rakic, P. Origin of GABAergic neurons in the human neocortex. Nature 417, 645–649 (2002).

    Article  CAS  Google Scholar 

  46. Dong, H.W. & Buonomano, D.V. A technique for repeated recordings in cortical organotypic slices. J. Neurosci. Methods 146, 69–75 (2005).

    Article  Google Scholar 

  47. Krassioukov, A.V. et al. An in vitro model of neurotrauma in organotypic spinal cord cultures from adult mice. Brain Res. Brain Res. Protoc. 10, 60–68 (2002).

    Article  Google Scholar 

  48. Tom, V.J., Doller, C.M., Malouf, A.T. & Silver, J. Astrocyte-associated fibronectin is critical for axonal regeneration in adult white matter. J. Neurosci. 24, 9282–9290 (2004).

    Article  CAS  Google Scholar 

  49. Mtchedlishvili, Z. & Kapur, J. High-affinity, slowly desensitizing GABAA receptors mediate tonic inhibition in hippocampal dentate granule cells. Mol. Pharmacol. 69, 564–575 (2006).

    Article  CAS  Google Scholar 

  50. Lo, D.C., McAllister, A.K. & Katz, L.C. Neuronal transfection in brain slices using particle-mediated gene transfer. Neuron 13, 1263–1268 (1994).

    Article  CAS  Google Scholar 

  51. McAllister, A.K., Lo, D.C. & Katz, L.C. Neurotrophins regulate dendritic growth in developing visual cortex. Neuron 15, 791–803 (1995).

    Article  CAS  Google Scholar 

  52. McAllister, A.K. Biolistic transfection of cultured organotypic brain slices. Methods Mol. Biol. 245, 197–206 (2004).

    CAS  PubMed  Google Scholar 

  53. O'Brien, J.A., Holt, M., Whiteside, G., Lummis, S.C. & Hastings, M.H. Modifications to the hand-held gene gun: improvements for in vitro biolistic transfection of organotypic neuronal tissue. J. Neurosci. Methods 112, 57–64 (2001).

    Article  CAS  Google Scholar 

  54. Rathenberg, J., Nevian, T. & Witzemann, V. High-efficiency transfection of individual neurons using modified electrophysiology techniques. J. Neurosci. Methods 126, 91–98 (2003).

    Article  Google Scholar 

  55. Beach, R.L., Bathgate, S.L. & Cotman, C.W. Identification of cell types in rat hippocampal slices maintained in organotypic cultures. Brain Res. 255, 3–20 (1982).

    Article  CAS  Google Scholar 

  56. Benediktsson, A.M., Schachtele, S.J., Green, S.H. & Dailey, M.E. Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures. J. Neurosci. Methods 141, 41–53 (2005).

    Article  Google Scholar 

  57. London, J.A., Biegel, D. & Pachter, J.S. Neurocytopathic effects of beta-amyloid-stimulated monocytes: a potential mechanism for central nervous system damage in Alzheimer disease. Proc. Natl. Acad. Sci. USA 93, 4147–4152 (1996).

    Article  CAS  Google Scholar 

  58. Noraberg, J. et al. Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair. Curr. Drug Targets CNS Neurol. Disord. 4, 435–452 (2005).

    Article  CAS  Google Scholar 

  59. Noraberg, J., Kristensen, B.W. & Zimmer, J. Markers for neuronal degeneration in organotypic slice cultures. Brain Res. Brain Res. Protoc. 3, 278–290 (1999).

    Article  CAS  Google Scholar 

  60. Kim, J.A. et al. Cytoskeleton disruption causes apoptotic degeneration of dentate granule cells in hippocampal slice cultures. Neuropharmacology 42, 1109–1118 (2002).

    Article  CAS  Google Scholar 

  61. Moroni, F. et al. Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia. Cell Death Differ. 8, 921–932 (2001).

    Article  CAS  Google Scholar 

  62. Heinemann, U. et al. Cell death and metabolic activity during epileptiform discharges and status epilepticus in the hippocampus. Prog. Brain Res. 135, 197–210 (2002).

    Article  CAS  Google Scholar 

  63. Heinemann, U. et al. Coupling of electrical and metabolic activity during epileptiform discharges. Epilepsia 43 (Suppl. 5): 168–173 (2002).

    Article  Google Scholar 

  64. Kovacs, R. et al. Free radical-mediated cell damage after experimental status epilepticus in hippocampal slice cultures. J. Neurophysiol 88, 2909–2918 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Dr. Frances A. Edwards, Dr. Yukiko Goda and Professor David Attwell for use of laboratory equipment and facilities. We also thank Professor David Attwell for valuable comments on the manuscript.

Author information

Authors and Affiliations

  1. Department of Physiology, University College London, Gower Street, London, WCIE 6BT, UK

    Anna De Simoni

  2. Medical Research Council Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, Gower Street, London, WC1E 6BT, UK

    Lily MY Yu

Authors
  1. Anna De Simoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lily MY Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anna De Simoni.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

De Simoni, A., MY Yu, L. Preparation of organotypic hippocampal slice cultures: interface method. Nat Protoc 1, 1439–1445 (2006). https://doi.org/10.1038/nprot.2006.228

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.228

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