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 language | English |
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Pages (from-to) | 1028-1037 |
Number of pages | 10 |
Journal | Nature Protocols |
Volume | 9 |
Issue number | 5 |
DOIs | |
Publication status | Published - 2014 |
Keywords
- Animals
- Axotomy
- Fluorescence
- Hippocampus
- Mice
- Nerve Regeneration
- Neural Pathways
- Single-Cell Analysis