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Direct live monitoring of heterotypic axon-axon interactions in vitro

Abstract

This protocol describes an optimized method for direct in vitro monitoring of homo- and heterotypic axon-axon interactions involved in the developmental assembly of neural circuits. The assay exploits a classical example of heterotypic axonal interactions by modeling the sequential extension of spinal motor and somatosensory neuron axons, but the procedure should be readily adaptable to other neuron types. The protocol is based on the rapid isolation and primary culture of genetically identified motor neurons combined with straightforward vital dye labeling and culture of dorsal root ganglion sensory neurons. Subsequently, axonal interactions are directly monitored via live fluorescence microscopy, whereas axon type identities can be unambiguously delineated throughout the experiments. Through chemical compound application or by using neurons derived from genetically engineered mice, the protocol facilitates the dissection of molecular pathways driving the axonal interactions that are crucial for neural pathway and circuit assembly. The whole procedure can be completed in 3 d.

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Figure 1
Figure 2: Procedure for performing MN explant culture from E12.5 Hb9eGFP transgenic embryo.
Figure 3: Procedure for DRG culture from E12.5 embryo.
Figure 4: Examples of flash-fixed MN-DRG explant cocultures.
Figure 5: Imaging setup based on the Olympus cell^M IX81/Yokogawa DSU-based spinning-disk system used in our laboratory.
Figure 6: Homotypic or heterotypic growth cones interactions in MN-DRG explant cocultures traced by live axon imaging.

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Acknowledgements

We thank A. Klusowski and E. Ling for technical assistance and Olympus-Germany for providing the DSU. This work was supported by the Emmy Noether Program of the Deutsche Forschungsgemeinschaft (DFG), as well as the DFG Research Center for Molecular Physiology of the brain. The ENI-G is a cooperation of the University of Göttingen Medical School and the Max Planck Gesellschaft.

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Authors and Affiliations

Authors

Contributions

L.W. designed and executed experiments and analyzed the data. T.M. devised the protocol and supervised the experiments. L.W. and T.M. co-wrote the manuscript.

Corresponding author

Correspondence to Till Marquardt.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Movie 1

Example of a sensory growth cone crossing an interjecting sensory axon. Total duration of movie sequence: 56 min (see Fig. 6a). (AVI 3714 kb)

Supplementary Movie 2

Sensory growth cone (DiI: red) encountering an interjecting motor axon (GFP: green). This encounter leads to reorientation of the sensory axon trajectory, and tracking of the sensory growth cone along the length of the motor axon shaft (see Fig. 6b). Note: tracking is typically accompanied by numerous transient sensory growth cone filopodial-motor axon membrane contacts. Total duration of movie sequence: 189 min. (AVI 12636 kb)

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Wang, L., Marquardt, T. Direct live monitoring of heterotypic axon-axon interactions in vitro. Nat Protoc 7, 351–363 (2012). https://doi.org/10.1038/nprot.2011.442

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