Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits.
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.
1] Axonal Growth and Regeneration, Max Planck Institute of Neurobiology, Martinsried, Germany. [2].
Susana
S
Gomis-Rüth
1] Axonal Growth and Regeneration, Max Planck Institute of Neurobiology, Martinsried, Germany. [2] Axon Growth and Regeneration, Deutsches Zentrum für Neurodegenerative Erkrankungen, Bonn, Germany. [3] [4].
Michael
0000000153630389
M
Stiess
Division of Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
Corette J
0000000190734099
CJ
Wierenga
1] Axonal Growth and Regeneration, Max Planck Institute of Neurobiology, Martinsried, Germany. [2] Axon Growth and Regeneration, Deutsches Zentrum für Neurodegenerative Erkrankungen, Bonn, Germany.
Liane
L
Meyn
1] Axonal Growth and Regeneration, Max Planck Institute of Neurobiology, Martinsried, Germany. [2] Axon Growth and Regeneration, Deutsches Zentrum für Neurodegenerative Erkrankungen, Bonn, Germany.
Frank
F
Bradke
D000818:Animals; D019771:Axotomy / Q000379:methods*; D005453:Fluorescence; D006624:Hippocampus / Q000166:cytology*; D051379:Mice; D009416:Nerve Regeneration / Q000502:physiology*; D009434:Neural Pathways / Q000166:cytology*; D059010:Single-Cell Analysis / Q000379:methods*