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:

Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration

Nature Protocols volume 5, pages 217–226 (2010)Cite this article

Subjects

Abstract

We present a method for using long-term organotypic slice co-cultures of the entorhino-hippocampal formation to analyze the axon-regenerative properties of a determined compound. The culture method is based on the membrane interphase method, which is easy to perform and is generally reproducible. The degree of axonal regeneration after treatment in lesioned cultures can be seen directly using green fluorescent protein (GFP) transgenic mice or by axon tracing and histological methods. Possible changes in cell morphology after pharmacological treatment can be determined easily by focal in vitro electroporation. The well-preserved cytoarchitectonics in the co-culture facilitate the analysis of identified cells or regenerating axons. The protocol takes up to a month.

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: GFAP-positive glial scar in long-term MICM EHC co-cultures.
Figure 2: MICM electroporation for morphological studies.
Figure 3: Illustration of the MICM entorhino-hippocampal co-culture protocol.
Figure 4: Axotomy of the EHC in MCIM platforms, drug treatment, quantification and corroboration of EHC regeneration.

Similar content being viewed by others

References

  1. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Sofroniew, M.V., Dreifuss, J.J. & Gahwiler, B.H. Slice cultures of rat hypothalamus examined by immunohistochemical staining for neurohypophyseal peptides and GFAP. Brain Res. Bull. 20, 669–674 (1988).

    Article  CAS  PubMed  Google Scholar 

  4. Zimmer, J. & Gahwiler, B.H. Cellular and connective organization of slice cultures of the rat hippocampus and fascia dentata. J. Comp. Neurol. 228, 432–446 (1984).

    Article  CAS  PubMed  Google Scholar 

  5. Gahwiler, B.H. Morphological differentiation of nerve cells in thin organotypic cultures derived from rat hippocampus and cerebellum. Proc. R. Soc. Lond., B, Biol. Sci. 211, 287–290 (1981).

    Article  CAS  PubMed  Google Scholar 

  6. Lonchamp, E., Dupont, J.L., Beekenkamp, H., Poulain, B. & Bossu, J.L. The mouse cerebellar cortex in organotypic slice cultures: an in vitro model to analyze the consequences of mutations and pathologies on neuronal survival, development, and function. Crit. Rev. Neurobiol. 18, 179–186 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Behan, M., Kroker, A. & Bolz, J. Cortical barrelfields in organotypic slice cultures from rat somatosensory cortex. Neurosci. Lett. 133, 191–194 (1991).

    Article  CAS  PubMed  Google Scholar 

  8. Li, D., Field, P.M., Starega, U., Li, Y. & Raisman, G. Entorhinal axons project to dentate gyrus in organotypic slice co-culture. Neuroscience 52, 799–813 (1993).

    Article  CAS  PubMed  Google Scholar 

  9. Ostergaard, K. Organotypic slice cultures of the rat striatum—I. A histochemical and immunocytochemical study of acetylcholinesterase, choline acetyltransferase, glutamate decarboxylase and GABA. Neuroscience 53, 679–693 (1993).

    Article  CAS  PubMed  Google Scholar 

  10. 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  PubMed  Google Scholar 

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

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  13. Gahwiler, B.H., Thompson, S.M. & Muller, D. Preparation and maintenance of organotypic slice cultures of CNS tissue. Curr. Protoc. Neurosci. Chapter 6, Unit 6 11 (2001).

  14. Cho, S., Wood, A. & Bowlby, M.R. Brain slices as models for neurodegenerative disease and screening platforms to identify novel therapeutics. Curr. Neuropharmacol. 5, 19–33 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bali, B., Nagy, Z. & Kovacs, K.J. Oxygen–glucose deprivation-induced changes in organotypic cultures of the rat hippocampus. Ideggyogy. Sz. 60, 140–143 (2007).

    PubMed  Google Scholar 

  16. Laake, J.H., Haug, F.M., Wieloch, T. & Ottersen, O.P. A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence. Brain Res. 4, 173–184 (1999).

    CAS  Google Scholar 

  17. Adamchik, Y., Frantseva, M.V., Weisspapir, M., Carlen, P.L. & Perez Velazquez, J.L. Methods to induce primary and secondary traumatic damage in organotypic hippocampal slice cultures. Brain Res. 5, 153–158 (2000).

    CAS  Google Scholar 

  18. Stoppini, L. et al. Infection of organotypic slice cultures from rat central nervous tissue with Trypanosoma brucei brucei . Int. J. Med. Microbiol. 290, 105–113 (2000).

    Article  CAS  PubMed  Google Scholar 

  19. Mayer, D., Fischer, H., Schneider, U., Heimrich, B. & Schwemmle, M. Borna disease virus replication in organotypic hippocampal slice cultures from rats results in selective damage of dentate granule cells. J. Virol. 79, 11716–11723 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Falsig, J. et al. A versatile prion replication assay in organotypic brain slices. Nat. Neurosci. 11, 109–117 (2008).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  22. del Rio, J.A., Heimrich, B., Soriano, E., Schwegler, H. & Frotscher, M. Proliferation and differentiation of glial fibrillary acidic protein-immunoreactive glial cells in organotypic slice cultures of rat hippocampus. Neuroscience 43, 335–347 (1991).

    Article  CAS  PubMed  Google Scholar 

  23. De Simoni, A. & Yu, L.M. Preparation of organotypic hippocampal slice cultures: interface method. Nat. Protoc. 1, 1439–1445 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Gogolla, N., Galimberti, I., DePaola, V. & Caroni, P. Preparation of organotypic hippocampal slice cultures for long-term live imaging. Nat. Protoc. 1, 1165–1171 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Caeser, M. & Aertsen, A. Morphological organization of rat hippocampal slice cultures. J. Comp. Neurol 307, 87–106 (1991).

    Article  CAS  PubMed  Google Scholar 

  26. Xie, F. & Zheng, B. White matter inhibitors in CNS axon regeneration failure. Exp. Neurol. 209, 302–312 (2008).

    Article  CAS  PubMed  Google Scholar 

  27. Fitch, M.T. & Silver, J. CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure. Exp. Neurol. 209, 294–301 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Paxinos, G. & Franklin, K.B.J. The Mouse Brain in Stereotaxic Coordinates (Academic Press, San Diego, 2001).

  29. Li, D., Field, P.M., Yoshioka, N. & Raisman, G. Axons regenerate with correct specificity in horizontal slice culture of the postnatal rat entorhino-hippocampal system. Eur. J. Neurosci. 6, 1026–1037 (1994).

    Article  CAS  PubMed  Google Scholar 

  30. Woodhams, P.L. & Atkinson, D.J. Regeneration of entorhino-dentate projections in organotypic slice cultures: mode of axonal regrowth and effects of growth factors. Exp. Neurol. 140, 68–78 (1996).

    Article  CAS  PubMed  Google Scholar 

  31. Woodhams, P.L., Atkinson, D.J. & Raisman, G. Rapid decline in the ability of entorhinal axons to innervate the dentate gyrus with increasing time in organotypic co-culture. Eur. J. Neurosci. 5, 1596–1609 (1993).

    Article  CAS  PubMed  Google Scholar 

  32. Heimrich, B. & Frotscher, M. Slice cultures as a model to study entorhinal-hippocampal interaction. Hippocampus 3 Spec No, 11–17 (1993).

    CAS  PubMed  Google Scholar 

  33. Frotscher, M. & Heimrich, B. Formation of layer-specific fiber projections to the hippocampus in vitro . Proc. Natl. Acad. Sci. USA 90, 10400–10403 (1993).

    Article  CAS  PubMed  Google Scholar 

  34. Del Rio, J.A. et al. A role for Cajal–Retzius cells and reelin in the development of hippocampal connections. Nature 385, 70–74 (1997).

    Article  CAS  PubMed  Google Scholar 

  35. Prang, P., Del Turco, D. & Kapfhammer, J.P. Regeneration of entorhinal fibers in mouse slice cultures is age dependent and can be stimulated by NT-4, GDNF, and modulators of G-proteins and protein kinase C. Exp. Neurol. 169, 135–147 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Mingorance, A. et al. Regeneration of lesioned entorhino-hippocampal axons in vitro by combined degradation of inhibitory proteoglycans and blockade of Nogo-66/NgR signaling. FASEB J. 20, 491–493 (2006).

    Article  CAS  PubMed  Google Scholar 

  37. del Rio, J.A., Sole, M., Borrell, V., Martinez, A. & Soriano, E. Involvement of Cajal–Retzius cells in robust and layer-specific regeneration of the entorhino-hippocampal pathways. Eur. J. Neurosci. 15, 1881–1890 (2002).

    Article  PubMed  Google Scholar 

  38. Murphy, R.C. & Messer, A. Gene transfer methods for CNS organotypic cultures: a comparison of three nonviral methods. Mol. Ther. 3, 113–121 (2001).

    Article  CAS  PubMed  Google Scholar 

  39. Flames, N. et al. Short- and long-range attraction of cortical GABAergic interneurons by neuregulin-1. Neuron 44, 251–261 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Barth, A., Barth, L. & Newell, D.W. Combination therapy with MK-801 and alpha-phenyl-tert-butyl-nitrone enhances protection against ischemic neuronal damage in organotypic hippocampal slice cultures. Exp. Neurol. 141, 330–336 (1996).

    Article  CAS  PubMed  Google Scholar 

  41. Eyupoglu, I.Y., Savaskan, N.E., Brauer, A.U., Nitsch, R. & Heimrich, B. Identification of neuronal cell death in a model of degeneration in the hippocampus. Brain Res. 11, 1–8 (2003).

    Google Scholar 

  42. Frotscher, M. & Heimrich, B. Lamina-specific synaptic connections of hippocampal neurons in vitro . J. Neurobiol. 26, 350–359 (1995).

    Article  CAS  PubMed  Google Scholar 

  43. Del Rio, J.A. et al. Differential survival of Cajal–Retzius cells in organotypic cultures of hippocampus and neocortex. J. Neurosci. 16, 6896–6907 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Del Turco, D. & Deller, T. Organotypic entorhino-hippocampal slice cultures—a tool to study the molecular and cellular regulation of axonal regeneration and collateral sprouting in vitro . Methods Mol. Biol. 399, 55–66 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Hechler, D., Nitsch, R. & Hendrix, S. Green-fluorescent-protein-expressing mice as models for the study of axonal growth and regeneration in vitro . Brain Res. Rev. 52, 160–169 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Koprivica, V. et al. EGFR activation mediates inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans. Science 310, 106–110 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Montolio, M. et al. A semaphorin 3A inhibitor blocks axonal chemorepulsion and enhances axon regeneration. Chem. Biol. 16, 691–701 (2009).

    Article  CAS  PubMed  Google Scholar 

  48. Gogolla, N., Galimberti, I., DePaola, V. & Caroni, P. Long-term live imaging of neuronal circuits in organotypic hippocampal slice cultures. Nat. Protoc. 1, 1223–1226 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. 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  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  51. GrandPre, T., Li, S. & Strittmatter, S.M. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature 417, 547–551 (2002).

    Article  CAS  PubMed  Google Scholar 

  52. Pratt, T., Sharp, L., Nichols, J., Price, D.J. & Mason, J.O. Embryonic stem cells and transgenic mice ubiquitously expressing a tau-tagged green fluorescent protein. Dev. Biol. 228, 19–28 (2000).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by grants from the MICINN, the Generalitat de Catalunya, the Instituto Carlos III, the FP7-EU program (PRIORITY) and Fundació la Caixa. The authors thank all the members of our labs for their contributions during these years toward improving the techniques explained in this paper. We also thank Isabel Jimenez for her technical assistance and the Language Advisory Service at the University of Barcelona for their editorial help.

Author information

Authors and Affiliations

  1. Molecular and Cellular Neurobiotechnology, Catalonian Institute of Bioengineering (IBEC), Science Park of Barcelona, Barcelona, Spain

    José Antonio del Río

  2. Department of Cell Biology, Faculty of Biology, University of Barcelona, Barcelona, Spain

    José Antonio del Río & Eduardo Soriano

  3. Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Sevilla, Spain,

    José Antonio del Río & Eduardo Soriano

  4. Developmental Neurobiology and Neuronal Regeneration, Institute for Research in Biomedicine (IRB), Science Park of Barcelona, Barcelona, Spain

    Eduardo Soriano

Authors
  1. José Antonio del Río
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eduardo Soriano
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.A.d.R. performed the experiments illustrated in the paper and developed the electroporation device showed in Figure 2; and J.A.d.R. and E.S. wrote the paper.

Corresponding author

Correspondence to José Antonio del Río.

Supplementary information

Supplementary Fig. 1 | EH co-cultures using the MCIM method and EHC labeling, intact vs complex co-cultures.

A) Example of EHC at 3 DIV (A-B) and after 15 DIV (C-D). A is a phase contrast image of the culture and B is the corresponding fluorescence photomicrograph. Complex co-cultures in A-C were prepared using a Tau-eGFP expressing mice kindly provided by Prof. Manson (Edinburgh, UK)52. In these co-cultures the entorhinal cortex is taken from an eGFP +/- mouse and the hippocampus from an eGFP -/- mouse. Growing axons (arrows in C) in the hippocampal fields can be seen directly under the fluorescence microscope. D) Example of a Biocytin-labeled EHC in an intact co-culture after the above-mentioned protocol. The location of the Biocytin crystal in the entorhinal cortex is labeled by an asterisk. Arrows point to anterogradely labeled axons in the hippocampus. Abbreviations as in Figure 1. Scale bar: A = 250 µm, pertains to B-D. (JPG 1021 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

del Río, J., Soriano, E. Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration. Nat Protoc 5, 217–226 (2010). https://doi.org/10.1038/nprot.2009.202

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.202

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: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research