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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 217226 (2010) | Download Citation

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.

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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

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

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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

Image files

  1. 1.

    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.

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DOI

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

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