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Targeted axon-attached recording with fluorescent patch-clamp pipettes in brain slices

Nature Protocols volume 7, pages 1228–1234 (2012)Cite this article

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Abstract

Understanding the physiology of axons in the central nervous system requires experimental access to intact axons. This protocol describes how to perform cell-attached recordings from narrow axon fibers (ϕ <1 μm) in acute and cultured brain slice preparations (with a success rate of ∼50%). By using fluorophore-coated glass pipettes and Nipkow disk confocal microscopy, fluorescently labeled axons can be visually targeted under online optical control. In the cell-attached configuration, axonal action potentials are extracellularly recorded as unit-like, sharp negative currents. The axon morphology labeling and cell-attached recordings of axons can be completed within 1–2 h. The recordings are stable for at least 30 min.

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Figure 1: Confocal images of axonal fibers in a hippocampal CA3 pyramidal cell.
Figure 2: Online targeting of an axon fiber under confocal monitoring.
Figure 3: Dual patch-clamp recordings from the soma and the axon of a hippocampal CA3 pyramidal cell.
Figure 4: Latency of axonal APs.

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References

  1. Debanne, D. Information processing in the axon. Nat. Rev. Neurosci. 5, 304–316 (2004).

    Article  CAS  Google Scholar 

  2. Astman, N., Gutnick, M.J. & Fleidervish, I.A. Persistent sodium current in layer 5 neocortical neurons is primarily generated in the proximal axon. J. Neurosci. 26, 3465–3473 (2006).

    Article  CAS  Google Scholar 

  3. de Polavieja, G.G., Harsch, A., Kleppe, I., Robinson, H.P. & Juusola, M. Stimulus history reliably shapes action potential waveforms of cortical neurons. J. Neurosci. 25, 5657–5665 (2005).

    Article  CAS  Google Scholar 

  4. Shu, Y., Hasenstaub, A., Duque, A., Yu, Y. & McCormick, D.A. Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential. Nature 441, 761–765 (2006).

    Article  CAS  Google Scholar 

  5. Kole, M.H., Letzkus, J.J. & Stuart, G.J. Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy. Neuron 55, 633–647 (2007).

    Article  CAS  Google Scholar 

  6. Shu, Y., Yu, Y., Yang, J. & McCormick, D.A. Selective control of cortical axonal spikes by a slowly inactivating K+ current. Proc. Natl. Acad. Sci. USA 104, 11453–11458 (2007).

    Article  CAS  Google Scholar 

  7. Alle, H. & Geiger, J.R. Combined analog and action potential coding in hippocampal mossy fibers. Science 311, 1290–1293 (2006).

    Article  CAS  Google Scholar 

  8. Scott, R., Ruiz, A., Henneberger, C., Kullmann, D.M. & Rusakov, D.A. Analog modulation of mossy fiber transmission is uncoupled from changes in presynaptic Ca2+. J. Neurosci. 28, 7765–7773 (2008).

    Article  CAS  Google Scholar 

  9. Christie, J.M., Chiu, D.N. & Jahr, C.E. Ca2+-dependent enhancement of release by subthreshold somatic depolarization. Nat. Neurosci. 14, 62–68 (2011).

    Article  CAS  Google Scholar 

  10. Sasaki, T., Matsuki, N. & Ikegaya, Y. Effects of axonal topology on the somatic modulation of synaptic outputs. J. Neurosci. 32, 2868–2876 (2012).

    Article  CAS  Google Scholar 

  11. Sasaki, T., Matsuki, N. & Ikegaya, Y. Action-potential modulation during axonal conduction. Science 331, 599–601 (2011).

    Article  CAS  Google Scholar 

  12. Awatramani, G.B., Price, G.D. & Trussell, L.O. Modulation of transmitter release by presynaptic resting potential and background calcium levels. Neuron 48, 109–121 (2005).

    Article  CAS  Google Scholar 

  13. Hori, T. & Takahashi, T. Mechanisms underlying short-term modulation of transmitter release by presynaptic depolarization. J. Physiol. 587, 2987–3000 (2009).

    Article  CAS  Google Scholar 

  14. Alle, H. & Geiger, J.R. Analog signalling in mammalian cortical axons. Curr. Opin. Neurobiol. 18, 314–320 (2008).

    Article  CAS  Google Scholar 

  15. Geiger, J.R. & Jonas, P. Dynamic control of presynaptic Ca2+ inflow by fast-inactivating K+ channels in hippocampal mossy fiber boutons. Neuron 28, 927–939 (2000).

    Article  CAS  Google Scholar 

  16. Bischofberger, J., Engel, D., Li, L., Geiger, J.R. & Jonas, P. Patch-clamp recording from mossy fiber terminals in hippocampal slices. Nat. Protoc. 1, 2075–2081 (2006).

    Article  CAS  Google Scholar 

  17. Forsythe, I.D. Direct patch recording from identified presynaptic terminals mediating glutamatergic EPSCs in the rat CNS, in vitro. J. Physiol. 479 (Part 3), 381–387 (1994).

    Article  Google Scholar 

  18. Palmer, L.M. et al. Initiation of simple and complex spikes in cerebellar Purkinje cells. J. Physiol. 588, 1709–1717 (2010).

    Article  CAS  Google Scholar 

  19. Rudolph, S., Overstreet-Wadiche, L. & Wadiche, J.I. Desynchronization of multivesicular release enhances Purkinje cell output. Neuron 70, 991–1004 (2011).

    Article  CAS  Google Scholar 

  20. Atherton, J.F., Wokosin, D.L., Ramanathan, S. & Bevan, M.D. Autonomous initiation and propagation of action potentials in neurons of the subthalamic nucleus. J. Physiol. 586, 5679–5700 (2008).

    Article  CAS  Google Scholar 

  21. Monsivais, P., Clark, B.A., Roth, A. & Hausser, M. Determinants of action potential propagation in cerebellar Purkinje cell axons. J. Neurosci. 25, 464–472 (2005).

    Article  CAS  Google Scholar 

  22. Ishikawa, D. et al. Fluorescent pipettes for optically targeted patch-clamp recordings. Neural Netw. 23, 669–672 (2010).

    Article  Google Scholar 

  23. Sasaki, T., Takahashi, N., Matsuki, N. & Ikegaya, Y. Fast and accurate detection of action potentials from somatic calcium fluctuations. J. Neurophysiol. 100, 1668–1676 (2008).

    Article  CAS  Google Scholar 

  24. Takahashi, N., Sasaki, T., Matsumoto, W., Matsuki, N. & Ikegaya, Y. Circuit topology for synchronizing neurons in spontaneously active networks. Proc. Natl. Acad. Sci. USA 107, 10244–10249 (2010).

    Article  CAS  Google Scholar 

  25. Koyama, R. et al. A low-cost method for brain slice cultures. J. Pharmacol. Sci. 104, 191–194 (2007).

    Article  CAS  Google Scholar 

  26. De Simoni, A. & Yu, L.M. Preparation of organotypic hippocampal slice cultures: interface method. Nat. Protoc. 1, 1439–1445 (2006).

    Article  CAS  Google Scholar 

  27. Debanne, D. et al. Paired-recordings from synaptically coupled cortical and hippocampal neurons in acute and cultured brain slices. Nat. Protoc. 3, 1559–1568 (2008).

    Article  CAS  Google Scholar 

  28. Takahashi, N. et al. High-speed multineuron calcium imaging using Nipkow-type confocal microscopy. Curr. Protoc. Neurosci. 57, 2.14.1–2.14.10 (2011).

    Article  Google Scholar 

  29. Perkins, K.L. Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices. J. Neurosci. Methods 154, 1–18 (2006).

    Article  CAS  Google Scholar 

  30. Takahashi, N. et al. Locally synchronized synaptic inputs. Science 335, 353–356 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by the Funding Program for Next Generation World-Leading Researchers (LS023).

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Authors and Affiliations

  1. Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan

    Takuya Sasaki, Norio Matsuki & Yuji Ikegaya

Authors
  1. Takuya Sasaki
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  2. Norio Matsuki
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  3. Yuji Ikegaya
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Contributions

T.S. collected experimental data and carried out the data analysis. T.S. and Y.I. wrote the manuscript. N.M. supervised the project and provided feedback on the manuscript.

Corresponding author

Correspondence to Takuya Sasaki.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Targeted axon-attached recording from an axon of a hippocampal pyramidal cell. (AVI 8228 kb)

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Sasaki, T., Matsuki, N. & Ikegaya, Y. Targeted axon-attached recording with fluorescent patch-clamp pipettes in brain slices. Nat Protoc 7, 1228–1234 (2012). https://doi.org/10.1038/nprot.2012.061

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