Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits

Susana Gomis-Rüth, Michael Stiess, Corette J Wierenga, Liane Meyn, Frank Bradke

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

An understanding of the molecular mechanisms of axon regeneration after injury is key for the development of potential therapies. Single-cell axotomy of dissociated neurons enables the study of the intrinsic regenerative capacities of injured axons. This protocol describes how to perform single-cell axotomy on dissociated hippocampal neurons containing synapses. Furthermore, to axotomize hippocampal neurons integrated in neuronal circuits, we describe how to set up coculture with a few fluorescently labeled neurons. This approach allows axotomy of single cells in a complex neuronal network and the observation of morphological and molecular changes during axon regeneration. Thus, single-cell axotomy of mature neurons is a valuable tool for gaining insights into cell intrinsic axon regeneration and the plasticity of neuronal polarity of mature neurons. Dissociation of the hippocampus and plating of hippocampal neurons takes ∼2 h. Neurons are then left to grow for 2 weeks, during which time they integrate into neuronal circuits. Subsequent axotomy takes 10 min per neuron and further imaging takes 10 min per neuron.

Original languageEnglish
Pages (from-to)1028-1037
Number of pages10
JournalNature Protocols
Volume9
Issue number5
DOIs
Publication statusPublished - 2014

Keywords

  • Animals
  • Axotomy
  • Fluorescence
  • Hippocampus
  • Mice
  • Nerve Regeneration
  • Neural Pathways
  • Single-Cell Analysis

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