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. 2010 Feb;5(2):217-26. doi: 10.1038/nprot.2009.202. Epub 2010 Jan 21.

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.

MeSH terms

  • Animals
  • Animals, Newborn
  • Axons / drug effects
  • Axons / physiology*
  • Biolistics / methods
  • Brain / surgery
  • Cell Death
  • Coculture Techniques
  • Dissection / methods
  • Electroporation / methods
  • Entorhinal Cortex / cytology
  • Entorhinal Cortex / drug effects
  • Entorhinal Cortex / physiology*
  • Glial Fibrillary Acidic Protein
  • Green Fluorescent Proteins / genetics
  • Hippocampus / cytology
  • Hippocampus / drug effects
  • Hippocampus / physiology*
  • Mice
  • Mice, Transgenic
  • Nerve Regeneration / drug effects*
  • Nerve Tissue Proteins / analysis

Substances

  • Glial Fibrillary Acidic Protein
  • Nerve Tissue Proteins
  • glial fibrillary astrocytic protein, mouse
  • Green Fluorescent Proteins