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.
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Acknowledgements
We thank B. Garvalov and K. Flynn for their suggestions and revisions to this manuscript. F.B. was supported by Wings for Life (WfL), the International Foundation for Research in Paraplegia (IRP) and the Deutsche Forschungsgemeinschaft (DFG). S.G.-R. was supported by a grant from Boehringer Ingelheim Fonds. M.S. was supported by a European Molecular Biology Organization (EMBO) long-term fellowship and is supported by the Human Frontier Science Program.
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S.G.-R., L.M. and F.B. conceived and designed the protocol and experimental procedures. S.G.-R., M.S., L.M. and C.J.W. performed cultures and experiments. S.G.-R., M.S. and F.B. wrote the manuscript. F.B. supervised the project.
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Gomis-Rüth, S., Stiess, M., Wierenga, C. et al. Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits. Nat Protoc 9, 1028–1037 (2014). https://doi.org/10.1038/nprot.2014.069
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DOI: https://doi.org/10.1038/nprot.2014.069
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