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Phenotyping sensory nerve endings in vitro in the mouse

Nature Protocols volume 4, pages 174–196 (2009)Cite this article

Abstract

This protocol details methods to identify and record from cutaneous primary afferent axons in an isolated mammalian skin–saphenous nerve preparation. The method is based on extracellular recordings of propagated action potentials from single-fiber receptive fields. Cutaneous nerve endings show graded sensitivities to various stimulus modalities that are quantified by adequate and controlled stimulation of the superfused skin with heat, cold, touch, constant punctate pressure or chemicals. Responses recorded from single-fibers are comparable with those obtained in previous in vivo experiments on the same species. We describe the components and the setting-up of the basic equipment of a skin–nerve recording station (few days), the preparation of the skin and the adherent saphenous nerve in the mouse (15–45 min) and the isolation and recording of neurons (approximately 1–3 h per recording). In addition, stimulation techniques, protocols to achieve single-fiber recordings, issues of data acquisition and action potential discrimination are discussed in detail.

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Figure 1: The experimental setup: schematic representation of data capture.
Figure 2: Typical view of a recording in Dapsys.
Figure 3: Typical view of an offline analysis in Spike2.
Figure 4: Custom-made gold wire electrodes and glassrods.
Figure 5: The experimental set-up: schematic representation of organ bath and fluid circulation.
Figure 6: Anatomical context of the saphenous nerve (lower leg, adapted from ref. 82).
Figure 7: The surgical procedure in a left leg saphenous nerve preparation.
Figure 8: Views of organ bath and recording chamber.
Figure 9: Sweep showing several C-fiber action potential waveforms.
Figure 10: The marking test.
Figure 11: The parts of an application system for thermostatic control.
Figure 12: Views of organ bath and isolation of a receptive field with a hollow steel cylinder.
Figure 13: Factors that influence the shape of the action potential waveform.
Figure 14: Electrostimulation, marking test and latency analysis.
Figure 15: Troubleshooting a single-fiber recording.
Figure 16: Thermosensitive C-fibers with ongoing activity at 32 °C.
Figure 17: Heat responses of polymodal nociceptors (CMH).
Figure 18: Cold responses of cold nociceptors (CMC).

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Acknowledgements

We thank Richard Lewis (Department of Molecular Pharmacology, Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia) and Richard Koerber (Department of Neurobiology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA) for critical comments on the manuscript. This work was supported by the European Union (EU) and K.Z. was supported by the Deutsche Forschungsgemeinschaft (DFG).

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Authors and Affiliations

  1. Department of Physiology and Pathophysiology, Friedrich-Alexander University of Erlangen-Nuremberg, Universitätsstraβe 17, Erlangen, 91054, Germany

    Katharina Zimmermann, Alexander Hein, Ulrich Hager & Peter W Reeh

  2. Department of Cardiology, Department of Neurobiology, Howard Hughes Medical Institute, Children's Hospital Boston, Harvard Medical School, 320 Longwood Ave., Boston, 02115, Massachusetts, USA

    Katharina Zimmermann, Jan Stefan Kaczmarek & David E Clapham

  3. Department of Mathematics and Computer Science, Bethel University, 3900 Bethel Drive, St. Paul, 55112, Minnesota, USA

    Brian P Turnquist

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Correspondence to Katharina Zimmermann or Peter W Reeh.

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Zimmermann, K., Hein, A., Hager, U. et al. Phenotyping sensory nerve endings in vitro in the mouse. Nat Protoc 4, 174–196 (2009). https://doi.org/10.1038/nprot.2008.223

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