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Membrane potential shifts caused by diffusible guidance signals direct growth-cone turning

Nature Neuroscience volume 11, pages 762–771 (2008)Cite this article

Abstract

Plasma membrane potentials gate the ion channel conductance that controls external signal–induced neuronal functions. We found that diffusible guidance molecules caused membrane potential shifts that resulted in repulsion or attraction of Xenopus laevis spinal neuron growth cones. The repellents Sema3A and Slit2 caused hyperpolarization, and the attractants netrin-1 and BDNF caused depolarization. Clamping the growth-cone potential at the resting state prevented Sema3A-induced repulsion; depolarizing potentials converted the repulsion to attraction, whereas hyperpolarizing potentials had no effect. Sema3A increased the intracellular concentration of guanosine 3′,5′-cyclic monophosphate ([cGMP]i) by soluble guanylyl cyclase, resulting in fast onset and long-lasting hyperpolarization. Pharmacological increase of [cGMP]i caused protein kinase G (PKG)-mediated depolarization, switching Sema3A-induced repulsion to attraction. This bimodal switch required activation of either Cl− or Na+ channels, which, in turn, regulated the differential intracellular Ca2+ concentration increase across the growth cone. Thus, the polarity of growth-cone potential shifts imposes either attraction or repulsion, and Sema3A achieves this through cGMP signaling.

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Figure 1: Repellent and attractant guidance molecules cause growth-cone membrane hyperpolarization and depolarization, respectively, in cultured Xenopus spinal neurons.
Figure 2: Sema3A-induced fast-onset and long-lasting growth-cone membrane hyperpolarization.
Figure 3: The growth-cone membrane potential indicates the direction of growth-cone turning.
Figure 4: Repulsive Sema3A-induced growth-cone membrane hyperpolarization is mediated by soluble guanylyl cyclase–produced cGMP.
Figure 5: Sema3A-induced bidirectional growth-cone turning is mediated by cGMP-dependent hyperpolarization, which causes repulsion, and PKG-dependent depolarization, which causes attraction.
Figure 6: Sema3A-induced membrane potential shifts determine the differential magnitude of [Ca2+]i increase across the growth cone.

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References

  1. Hille, B. Ion Channels of Excitable Membranes (Sinauer, Sunderland, Massachusetts, 2001).

    Google Scholar 

  2. Gomez, T.M. & Spitzer, N.C. In vivo regulation of axon extension and pathfinding by growth-cone calcium transients. Nature 397, 350–355 (1999).

    Article  CAS  Google Scholar 

  3. Goodman, C.S. Mechanisms and molecules that control growth cone guidance. Annu. Rev. Neurosci. 19, 341–377 (1996).

    Article  CAS  Google Scholar 

  4. Yu, T.W. & Bargmann, C.I. Dynamic regulation of axon guidance. Nat. Neurosci. 4, 1169–1176 (2001).

    Article  CAS  Google Scholar 

  5. Mueller, B.K. Growth cone guidance: steps towards a deeper understanding. Annu. Rev. Neurosci. 22, 351–358 (1999).

    Article  CAS  Google Scholar 

  6. Song, H.J. & Poo, M.M. Signal transduction underlying growth cone guidance by diffusible factors. Curr. Opin. Neurobiol. 9, 355–363 (1999).

    Article  CAS  Google Scholar 

  7. Tessier-Lavigne, M. & Goodman, C.S. The molecular biology of axon guidance. Science 274, 1123–1133 (1996).

    Article  CAS  Google Scholar 

  8. Höpker, V.H., Shewan, D., Tessier-Lavigne, M., Poo, M.M. & Holt, C. Growth cone attraction to netrin-1 is converted to repulsion by laminin-1. Nature 401, 69–73 (1999).

    Article  Google Scholar 

  9. He, Z., Wang, K.C., Koprivica, V., Ming, G.L. & Song, H.J. Knowing how to navigate: mechanisms of semaphorin signaling in the nervous system. Sci. STKE 119, RE1 (2002).

    Google Scholar 

  10. Pasterkamp, R.J. & Kolodkin, A.L. Semaphorin junction: making tracks toward neural connectivity. Curr. Opin. Neurobiol. 13, 79–89 (2003).

    Article  CAS  Google Scholar 

  11. Henley, J.R. & Poo, M. Guiding neuronal growth cones using Ca2+ signals. Trends Cell Biol. 14, 320–330 (2004).

    Article  CAS  Google Scholar 

  12. Gomez, T.M. & Zheng, J.Q. The molecular basis for calcium-dependent axon pathfinding. Nat. Rev. Neurosci. 7, 115–125 (2006).

    Article  CAS  Google Scholar 

  13. Hong, K., Nishiyama, M., Henley, J., Tessier-Lavigne, M. & Poo, M. Calcium signalling in the guidance of nerve growth by netrin-1. Nature 403, 93–98 (2000).

    Article  CAS  Google Scholar 

  14. Wang, G.X. & Poo, M. Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones. Nature 434, 898–904 (2005).

    Article  CAS  Google Scholar 

  15. Ming, G., Henley, J., Tessier-Lavigne, M., Song, H.-J. & Poo, M. Electrical activity modulates growth cone guidance by diffusible factors. Neuron 29, 441–452 (2001).

    Article  CAS  Google Scholar 

  16. Henley, J.R., Huang, K.H., Wang, D. & Poo, M.M. Calcium mediates bidirectional growth cone turning induced by myelin-associated glycoprotein. Neuron 44, 909–916 (2004).

    Article  CAS  Google Scholar 

  17. Nishiyama, M. et al. Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning. Nature 423, 990–995 (2003).

    Article  CAS  Google Scholar 

  18. Shim, S. et al. XTRPC1-dependent chemotropic guidance of neuronal growth cones. Nat. Neurosci. 8, 730–735 (2005).

    Article  CAS  Google Scholar 

  19. Song, H. et al. Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science 281, 1515–1518 (1998).

    Article  CAS  Google Scholar 

  20. Ayoob, J.C., Yu, H., Terman, J.R. & Kolodkin, A.L. The Drosophila receptor guanylyl cyclase Gyc76C is required for semaphorin-1a-Plexin A-mediated axonal repulsion. J. Neurosci. 24, 6639–6649 (2004).

    Article  CAS  Google Scholar 

  21. Song, H.-J., Ming, G.-L. & Poo, M.M. cAMP-induced switching in turning direction of nerve growth cones. Nature 388, 275–279 (1997).

    Article  CAS  Google Scholar 

  22. Ming, G.-L., Song, H.-J., Berninger, B., Holt, C.E., Tessier-Lavigne, M. & Poo, M.M. cAMP-dependent growth cone guidance by netrin-1. Neuron 19, 1225–1235 (1997).

    Article  CAS  Google Scholar 

  23. Polleux, F., Morrow, T. & Ghosh, A. Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404, 567–573 (2000).

    Article  CAS  Google Scholar 

  24. Hong, K. et al. A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion. Cell 97, 927–941 (1999).

    Article  CAS  Google Scholar 

  25. Stein, E. & Tessier-Lavigne, M. Hierarchical organization of guidance receptors: silencing of netrin attraction by slit through a Robo/DCC receptor complex. Science 291, 1928–1938 (2001).

    Article  CAS  Google Scholar 

  26. Ben-Ari, Y. Excitatory actions of GABA during development: the nature of the nurture. Nat. Rev. Neurosci. 3, 728–739 (2002).

    Article  CAS  Google Scholar 

  27. O'Dowd, D.K., Ribera, A.B. & Spitzer, N.C. Development of voltage-dependent calcium, sodium, and potassium currents in Xenopus spinal neurons. J. Neurosci. 8, 792–805 (1988).

    Article  CAS  Google Scholar 

  28. Nakamura, F., Tanaka, M., Takahashi, T., Falb, R.G. & Strittmatter, S.M. Neuropilin-1 extracellular domains mediate semaphoring D/III-induced growth cone collapse. Neuron 21, 1093–1100 (1998).

    Article  CAS  Google Scholar 

  29. Goshima, Y. et al. A novel action of collapsin: collapsin-1 increases antero- and retrograde axoplasmic transport independently of growth cone collapse. J. Neurobiol. 33, 316–328 (1997).

    Article  CAS  Google Scholar 

  30. de la Torre, J.R. et al. Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC. Neuron 19, 1211–1224 (1997).

    Article  CAS  Google Scholar 

  31. Zecevic, D. Multiple spike-initiation zones in single neurons revealed by voltage-sensitive dyes. Nature 381, 322–325 (1996).

    Article  CAS  Google Scholar 

  32. Zheng, J.Q., Felder, M., Connor, J.A. & Poo, M.M. Turning of nerve growth cones induced by neurotransmitters. Nature 368, 140–144 (1994).

    Article  CAS  Google Scholar 

  33. Chen, C.K. The vertebrate phototransduction cascade: amplification and termination mechanisms. Rev. Physiol. Biochem. Pharmacol. 154, 101–121 (2005).

    CAS  PubMed  Google Scholar 

  34. Toyofuku, T. et al. FARP2 triggers signals for Sema3A-mediated axonal repulsion. Nat. Neurosci. 8, 1712–1719 (2005).

    Article  CAS  Google Scholar 

  35. Terman, J.R. & Kolodkin, A.L. Nervy links protein kinase A to Plexin-mediated semaphorin repulsion. Science 303, 1204–1207 (2004).

    Article  CAS  Google Scholar 

  36. Behar, O., Mizuno, K., Badminton, M. & Woolf, C.J. Semaphorin 3A growth cone collapse requires a sequence homologous to tarantula hanatoxin. Proc. Natl. Acad. Sci. USA 96, 13501–13505 (1999).

    Article  CAS  Google Scholar 

  37. Shewan, D., Dwivedy, A., Anderson, R. & Holt, C.E. Age-related changes underlie switch in netrin-1 responsiveness as growth cones advance along visual pathway. Nat. Neurosci. 5, 955–962 (2002).

    Article  CAS  Google Scholar 

  38. Campbell, D.S. et al. Semaphorin 3A elicits stage-dependent collapse, turning, and branching in Xenopus retinal growth cones. J. Neurosci. 21, 8538–8547 (2001).

    Article  CAS  Google Scholar 

  39. Huber, A.B., Kolodkin, A.L., Ginty, D.D. & Cloutier, J.F. Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Annu. Rev. Neurosci. 26, 509–563 (2003).

    Article  CAS  Google Scholar 

  40. Guirland, C., Suzuki, S., Kojima, M., Lu, B. & Zheng, J.Q. Lipid rafts mediate chemotropic guidance of nerve growth cones. Neuron 42, 51–62 (2004).

    Article  CAS  Google Scholar 

  41. Togashi, K. et al. Cyclic GMP-gated CNG channels function in Sema3A-induced growth cone repulsion. Neuron (in the press).

  42. Patel, N.B. & Poo, M.M. Perturbation of the direction of neurite growth by pulsed and focal electric fields. J. Neurosci. 4, 2939–2947 (1984).

    Article  CAS  Google Scholar 

  43. Zheng, J.Q. Turning of nerve growth cones induced by direct focal elevation of intracellular Ca2+ concentration. Nature 403, 89–93 (2000).

    Article  CAS  Google Scholar 

  44. Spitzer, N.C., Kingston, P.A., Manning, T.J. Jr & Conklin, M.W. Outside and in: development of neuronal excitability. Curr. Opin. Neurobiol. 12, 315–323 (2002).

    Article  CAS  Google Scholar 

  45. Crair, M.C. Neuronal activity during development: permissive or instructive? Curr. Opin. Neurobiol. 9, 88–93 (1999).

    Article  CAS  Google Scholar 

  46. Hanson, M.G. & Landmesser, L.T. Normal patterns of spontaneous activity are required for correct motor axon guidance and the expression of specific guidance molecules. Neuron 43, 687–701 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank C.-S. Lim for NP-1 (0111) immunocytochemistry, Y. Goshima for the Sema3A and SNP-1 proteins, S.M. Strittmatter for the NP1-0111 construct, E. Stein for the Slit2 proteins, M. Ichikawa, K. Tsubokura and B. Okura for technical assistance with the optical imaging system, and W. Jelinek and N. Cowan for critical comments on the manuscript. This work was supported by grants from the US National Institutes of Health National Institute of Neurological Disorders and Stroke, Pew Scholars Program in the Biomedical Sciences and the New York State Spinal Cord Injury Research Program (K.H.).

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  1. Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, 10016-6402, New York, USA

    Makoto Nishiyama, Melanie J von Schimmelmann, Kazunobu Togashi, William M Findley & Kyonsoo Hong

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  1. Makoto Nishiyama
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Contributions

M.N. designed and performed growth-cone electrophysiology, optical and Ca2+ imaging, and contributed to the preparation of the manuscript. M.J.v.S. performed cGMP immunostaining and single-neuron injection of the Ca2+ indicator. K.T. developed the image analysis software and analyzed imaging data. W.M.F. performed growth-cone membrane potential measurements. K.H. supervised the project, performed growth-cone turning assays and prepared the manuscript.

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Correspondence to Kyonsoo Hong.

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Nishiyama, M., von Schimmelmann, M., Togashi, K. et al. Membrane potential shifts caused by diffusible guidance signals direct growth-cone turning. Nat Neurosci 11, 762–771 (2008). https://doi.org/10.1038/nn.2130

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