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Organotypic Entorhino-Hippocampal Slice Cultures—A Tool to Study the Molecular and Cellular Regulation of Axonal Regeneration and Collateral Sprouting In Vitro

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Book cover Neuroprotection Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 399))

Abstract

Organotypic slice cultures of the brain are widely used as a tool to study fundamental questions in neuroscience. In this chapter, we focus on a protocol based on organotypic slice cultures of mouse entorhinal cortex and hippocampus that can be employed to study axonal regeneration and collateral sprouting in the central nervous system in vitro. Using pharmacological as well as genetic approaches, axonal regeneration and sprouting can be influenced, and some of the molecular and cellular mechanisms involved in these processes can be identified. The protocol describes in detail (1) the generation of organotypic entorhino-hippocampal slice cultures, (2) the conditions needed for the analysis of axonal regeneration and collateral sprouting, respectively, (3) the lesioning technique, (4) tracing techniques to visualize regenerating entorhinal axons, and (5) an immunohistochemical technique to visualize sprouting fibers.

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References

  1. Frotscher, M., Zafirov, S., and Heimrich, B. (1995). Development of identified neuronal types and of specific synaptic connections in slice cultures of rat hippocampus. Prog. Neurobiol. 45, vii–xxviii.

    Article  Google Scholar 

  2. Del Rio, J. A., Heimrich, B., Borrell, V., Forster, E., Drakew, A., Alcantara, S., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., Derer, P., Frotscher, M., and Soriano, E. (1997). A role for Cajal-Retzius cells and reelin in the development of hippocampal connections. Nature 385, 70–74.

    Article  Google Scholar 

  3. Dusart, I., Airaksinen, M. S., and Sotelo, C. (1997). Purkinje cell survival and axonal regeneration are age dependent: an in vitro study. J. Neurosci. 17, 3710–3726.

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Frotscher, M., Drakew, A., and Heimrich, B. (2000). Role of afferent innervation and neuronal activity in dendritic development and spine maturation of fascia dentata granule cells. Cereb. Cortex 10, 946–951.

    Article  CAS  Google Scholar 

  6. Gimsa, U., Peter, S. V., Lehmann, K., Bechmann, I., and Nitsch, R. (2000). Axonal damage induced by invading T cells in organotypic central nervous system tissue in vitro: involvement of microglial cells. Brain Pathol. 10, 365–377.

    Article  CAS  Google Scholar 

  7. Kapfhammer, J. P. (2004). Cellular and molecular control of dendritic growth and development of cerebellar Purkinje cells. Prog. Histochem. Cytochem. 39, 131–182.

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Duport, S., Robert, F., Muller, D., Grau, G., Parisi, L., and Stoppini, L. (1998). An in vitro blood-brain barrier model: cocultures between endothelial cells and organotypic brain slice cultures. Proc. Natl. Acad. Sci. U. S. A. 95, 1840–1845.

    Article  CAS  Google Scholar 

  11. Dehghani, F., Conrad, A., Kohl, A., Korf, H. W., and Hailer, N. P. (2004). Clodronate inhibits the secretion of proinflammatory cytokines and NO by isolated microglial cells and reduces the number of proliferating glial cells in excitotoxically injured organotypic hippocampal slice cultures. Exp. Neurol. 189, 241–251.

    Article  CAS  Google Scholar 

  12. Radojevic, V. and Kapfhammer, J. P. (2004). Repair of the entorhino-hippocampal projection in vitro. Exp. Neurol. 188, 11–19.

    Article  Google Scholar 

  13. Huuskonen, J., Suuronen, T., Miettinen, R., van Groen, T., and Salminen, A. (2005). A refined in vitro model to study inflammatory responses in organotypic membrane culture of postnatal rat hippocampal slices. J. Neuroinflammation 2, 25.

    Article  Google Scholar 

  14. Noraberg, J., Poulsen, F. R., Blaabjerg, M., Kristensen, B. W., Bonde, C., Montero, M., Meyer, M., Gramsbergen, J. B., and Zimmer, J. (2005). Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair. Curr. Drug Targets. CNS. Neurol. Disord. 4, 435–452.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Bergold, P. J. and Casaccia-Bonnefil, P. (1997). Preparation of organotypic hippocampal slice cultures using the membrane filter method. Methods Mol. Biol. 72, 15–22.

    CAS  Google Scholar 

  17. Stoppini, L., Buchs, P. A., and Muller, D. (1993). Lesion-induced neurite sprouting and synapse formation in hippocampal organotypic cultures. Neuroscience 57, 985–994.

    Article  CAS  PubMed  Google Scholar 

  18. Diekmann, S., Nitsch, R., and Ohm, T. G. (1994). The organotypic entorhinalhippocampal complex slice culture of adolescent rats. A model to study transcellular changes in a circuit particularly vulnerable in neurodegenerative disorders. J. Neural Transm. Suppl. 44, 61–71.

    CAS  Google Scholar 

  19. Li, D., Field, P. M., and Raisman, G. (1996). Connectional specification of regenerating entorhinal projection neuron classes cannot be overridden by altered target availability in postnatal organotypic slice co-culture. Exp. Neurol. 142, 151–160.

    Article  CAS  PubMed  Google Scholar 

  20. Stoppini, L., Parisi, L., Oropesa, C., and Muller, D. (1997). Sprouting and functional recovery in co-cultures between old and young hippocampal organotypic slices. Neuroscience 80, 1127–1136.

    Article  CAS  PubMed  Google Scholar 

  21. Prang, P., Del Turco, D., and Deller, T. (2003). Associational sprouting in the mouse fascia dentata after entorhinal lesion in vitro. Brain Res. 978, 205–212.

    Article  CAS  PubMed  Google Scholar 

  22. van Groen, T., Miettinen, P., and Kadish, I. (2003). The entorhinal cortex of the mouse: organization of the projection to the hippocampal formation. Hippocampus 13, 133–149.

    Article  PubMed  Google Scholar 

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

    PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  25. Kluge, A., Hailer, N. P., Horvath, T. L., Bechmann, I. and Nitsch, R. (1998). Tracing of the entorhinal-hippocampal pathway in vitro. Hippocampus 8, 57–68.

    Article  CAS  PubMed  Google Scholar 

  26. Prang, P., Del Turco, D., and Kapfhammer, J. P. (2001). 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.

    Article  CAS  PubMed  Google Scholar 

  27. Muller, D., Djebbara-Hannas, Z., Jourdain, P., Vutskits, L., Durbec, P., Rougon, G., and Kiss, J. Z. (2000). Brain-derived neurotrophic factor restores long-term potentiation in polysialic acid-neural cell adhesion molecule-deficient hippocampus. Proc. Natl. Acad. Sci. U. S. A. 97, 4315–4320.

    Article  CAS  PubMed  Google Scholar 

  28. Schwab, M. H., Bartholomae, A., Heimrich, B., Feldmeyer, D., Druffel-Augustin, S., Goebbels, S., Naya, F. J., Zhao, S., Frotscher, M., Tsai, M. J., and Nave, K. A. (2000). Neuronal basic helix-loop-helix proteins (NEX and BETA2/Neuro D) regulate terminal granule cell differentiation in the hippocampus. J. Neurosci. 20, 3714–3724.

    CAS  PubMed  Google Scholar 

  29. Schrenk, K., Kapfhammer, J. P., and Metzger, F. (2002). Altered dendritic development of cerebellar Purkinje cells in slice cultures from protein kinase Cgammadeficient mice. Neuroscience 110, 675–689.

    Article  CAS  PubMed  Google Scholar 

  30. Teter, B., Xu, P. T., Gilbert, J. R., Roses, A. D., Galasko, D., and Cole, G. M. (2002). Defective neuronal sprouting by human apolipoprotein E4 is a gain-ofnegative function. J. Neurosci. Res. 68, 331–336.

    Article  CAS  PubMed  Google Scholar 

  31. Zhao, S., Forster, E., Chai, X., and Frotscher, M. (2003). Different signals control laminar specificity of commissural and entorhinal fibers to the dentate gyrus. J. Neurosci. 23, 7351–7357.

    CAS  PubMed  Google Scholar 

  32. Sole, M., Fontana, X., Gavin, R., Soriano, E., and Del Rio, J. A. (2004). Bcl-2 overexpression does not promote axonal regeneration of the entorhino-hippocampal connections in vitro after axotomy. Brain Res. 1020, 204–209.

    Article  CAS  PubMed  Google Scholar 

  33. Zhao, S., Chai, X., Forster, E., and Frotscher, M. (2004). Reelin is a positional signal for the lamination of dentate granule cells. Development 131, 5117–5125.

    Article  CAS  PubMed  Google Scholar 

  34. Zhao, S., Chai, X., Bock, H. H., Brunne, B., Forster, E., and Frotscher, M. (2006). Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for reelin and disabled 1. J. Comp Neurol. 495, 1–9.

    Article  CAS  PubMed  Google Scholar 

  35. Frotscher, M. and Heimrich, B. (1993). Formation of layer-specific fiber projections to the hippocampus in vitro. Proc. Natl. Acad. Sci. U. S. A. 90, 10400–10403.

    Article  CAS  PubMed  Google Scholar 

  36. Blasco-Ibanez, J. M. and Freund, T. F. (1997). Distribution, ultrastructure, and connectivity of calretinin-immunoreactive mossy cells of the mouse dentate gyrus. Hippocampus 7, 307–320.

    Article  CAS  PubMed  Google Scholar 

  37. Del Turco, D., Woods, A. G., Gebhardt, C., Phinney, A. L., Jucker, M., Frotscher, M., and Deller, T. (2003). Comparison of commissural sprouting in the mouse and rat fascia dentata after entorhinal cortex lesion. Hippocampus 13, 685–699.

    Article  PubMed  Google Scholar 

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Tiziana Borsello

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Del Turco, D., Deller, T. (2007). Organotypic Entorhino-Hippocampal Slice Cultures—A Tool to Study the Molecular and Cellular Regulation of Axonal Regeneration and Collateral Sprouting In Vitro. In: Borsello, T. (eds) Neuroprotection Methods and Protocols. Methods in Molecular Biology, vol 399. Humana Press. https://doi.org/10.1007/978-1-59745-504-6_5

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  • DOI: https://doi.org/10.1007/978-1-59745-504-6_5

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-666-5

  • Online ISBN: 978-1-59745-504-6

  • eBook Packages: Springer Protocols

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