Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Spontaneous activity regulates Robo1 transcription to mediate a switch in thalamocortical axon growth

Nature Neuroscience volume 15, pages 1134–1143 (2012)Cite this article

Subjects

Abstract

Developing axons must control their growth rate to follow the appropriate pathways and establish specific connections. However, the regulatory mechanisms involved remain elusive. By combining live imaging with transplantation studies in mice, we found that spontaneous calcium activity in the thalamocortical system and the growth rate of thalamocortical axons were developmentally and intrinsically regulated. Indeed, the spontaneous activity of thalamic neurons governed axon growth and extension through the cortex in vivo. This activity-dependent modulation of growth was mediated by transcriptional regulation of Robo1 through an NF-κB binding site. Disruption of either the Robo1 or Slit1 genes accelerated the progression of thalamocortical axons in vivo, and interfering with Robo1 signaling restored normal axon growth in electrically silent neurons. Thus, modifications to spontaneous calcium activity encode a switch in the axon outgrowth program that allows the establishment of specific neuronal connections through the transcriptional regulation of Slit1 and Robo1 signaling.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The speed of growth of TCAs is developmentally regulated.
Figure 2: Spontaneous thalamic activity is developmentally regulated.
Figure 3: Manipulation of spontaneous thalamic activity alters the axon growth rate.
Figure 4: Silencing spontaneous thalamic activity attenuates TCA elongation in vivo.
Figure 5: Silencing thalamic activity upregulates Robo1 transcription.
Figure 6: Robo1 expression is regulated at the transcriptional level through a NF-κB binding site.
Figure 7: Loss of Robo1 increases the intrinsic capacity for thalamic outgrowth.
Figure 8: Robo1 controls axon extension in vivo downstream of spontaneous activity.

Similar content being viewed by others

References

  1. López-Bendito, G. et al. Tangential neuronal migration controls axon guidance: a role for neuregulin-1 in thalamocortical axon navigation. Cell 125, 127–142 (2006).

    Article  Google Scholar 

  2. Métin, C., Deleglise, D., Serafini, T., Kennedy, T.E. & Tessier-Lavigne, M. A role for netrin-1 in the guidance of cortical efferents. Development 124, 5063–5074 (1997).

    PubMed  Google Scholar 

  3. Garel, S. & Rubenstein, J.L.R. Intermediate targets in formation of topographic projections: inputs from the thalamocortical system. Trends Neurosci. 27, 533–539 (2004).

    Article  CAS  Google Scholar 

  4. Braisted, J.E. et al. Netrin-1 promotes thalamic axon growth and is required for proper development of the thalamocortical projection. J. Neurosci. 20, 5792–5801 (2000).

    Article  CAS  Google Scholar 

  5. Tucker, K.L., Meyer, M. & Barde, Y. Neurotrophins are required for nerve growth during development. Nat. Neurosci. 4, 29–37 (2001).

    Article  CAS  Google Scholar 

  6. Blackmore, M. & Letourneau, P.C. Changes within maturing neurons limit axonal regeneration in the developing spinal cord. J. Neurobiol. 66, 348–360 (2006).

    Article  CAS  Google Scholar 

  7. Filbin, M.T. Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS. Nat. Rev. Neurosci. 4, 703–713 (2003).

    Article  CAS  Google Scholar 

  8. Spitzer, N.C. Electrical activity in early neuronal development. Nature 444, 707–712 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Goldberg, J.L. et al. Retinal ganglion cells do not extend axons by default: promotion by neurotrophic signaling and electrical activity. Neuron 33, 689–702 (2002).

    Article  CAS  Google Scholar 

  11. Fields, R.D., Neale, E. & Nelson, P. Effects of patterned electrical activity on neurite outgrowth from mouse sensory neurons. J. Neurosci. 10, 2950–2964 (1990).

    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. Tang, F., Dent, E.W. & Kalil, K. Spontaneous calcium transients in developing cortical neurons regulate axon outgrowth. J. Neurosci. 23, 927–936 (2003).

    Article  CAS  Google Scholar 

  14. Cohan, C.S. & Kater, S. Suppression of neurite elongation and growth cone motility by electrical activity. Science 232, 1638–1640 (1986).

    Article  CAS  Google Scholar 

  15. Borodinsky, L.N. et al. Activity-dependent homeostatic specification of transmitter expression in embryonic neurons. Nature 429, 523–530 (2004).

    Article  CAS  Google Scholar 

  16. 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 

  17. Marek, K.W., Kurtz, L. & Spitzer, N. cJun integrates calcium activity and tlx3 expression to regulate neurotransmitter specification. Nat. Neurosci. 13, 944–950 (2010).

    Article  CAS  Google Scholar 

  18. López-Bendito, G. & Molnár, Z. Thalamocortical development: how are we going to get there? Nat. Rev. Neurosci. 4, 276–289 (2003).

    Article  Google Scholar 

  19. Skaliora, I., Adams, R. & Blakemore, C. Morphology and growth patterns of developing thalamocortical axons. J. Neurosci. 20, 3650–3662 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Burrone, J., O'Byrne, M. & Murthy, V.N. Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons. Nature 420, 414–418 (2002).

    Article  CAS  Google Scholar 

  22. Demarque, M. & Spitzer, N.C. Activity-dependent expression of Lmx1b regulates specification of serotonergic neurons modulating swimming behavior. Neuron 67, 321–334 (2010).

    Article  CAS  Google Scholar 

  23. Mizuno, H., Hirano, T. & Tagawa, Y. Evidence for activity-dependent cortical wiring: formation of interhemispheric connections in neonatal mouse visual cortex requires projection neuron activity. J. Neurosci. 27, 6760–6770 (2007).

    Article  CAS  Google Scholar 

  24. Itoh, K., Ozaki, M., Stevens, B. & Fields, R. Activity-dependent regulation of N-cadherin in DRG neurons: differential regulation of N-cadherin, NCAM, and L1 by distinct patterns of action potentials. J. Neurobiol. 33, 735–748 (1997).

    Article  CAS  Google Scholar 

  25. Dolmetsch, R.E., Lewis, R.S., Goodnow, C.C. & Healy, J.I. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 386, 855–858 (1997).

    Article  CAS  Google Scholar 

  26. Fajardo, O., Meseguer, V., Belmonte, C. & Viana, F. TRPA1 channels: novel targets of 1,4-dihydropyridines. Channels (Austin) 2, 429–438 (2008).

    Article  Google Scholar 

  27. Sugiyama, C. et al. Activator protein-1 responsive to the group II metabotropic glutamate receptor subtype in association with intracellular calcium in cultured rat cortical neurons. Neurochem. Int. 51, 467–475 (2007).

    Article  CAS  Google Scholar 

  28. Xiang, G. et al. Identification of activity-dependent gene expression profiles reveals specific subsets of genes induced by different routes of Ca2+ entry in cultured rat cortical neurons. J. Cell. Physiol. 212, 126–136 (2007).

    Article  CAS  Google Scholar 

  29. Bagri, A. et al. Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain. Neuron 33, 233–248 (2002).

    Article  CAS  Google Scholar 

  30. 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 

  31. Sann, S.B., Xu, L., Nishimune, H., Sanes, J.R. & Spitzer, N.C. Neurite outgrowth and in vivo sensory innervation mediated by a Ca(V)2.2-laminin beta 2 stop signal. J. Neurosci. 28, 2366–2374 (2008).

    Article  CAS  Google Scholar 

  32. Porter, B.E., Weis, J. & Sanes, J.R. A motoneuron-selective stop signal in the synaptic protein S-laminin. Neuron 14, 549–559 (1995).

    Article  CAS  Google Scholar 

  33. Butler, A.K., Dantzker, J., Shah, R. & Callaway, E. Development of visual cortical axons: layer-specific effects of extrinsic influences and activity blockade. J. Comp. Neurol. 430, 321–331 (2001).

    Article  CAS  Google Scholar 

  34. Uesaka, N., Hayano, Y., Yamada, A. & Yamamoto, N. Interplay between laminar specificity and activity-dependent mechanisms of thalamocortical axon branching. J. Neurosci. 27, 5215–5223 (2007).

    Article  CAS  Google Scholar 

  35. Nishimaru, H., Iizuka, M., Ozaki, S. & Kudo, N. Spontaneous motoneuronal activity mediated by glycine and GABA in the spinal cord of rat fetuses in vitro. J. Physiol. (Lond.) 497, 131–143 (1996).

    Article  CAS  Google Scholar 

  36. Scain, A.L. et al. Glycine release from radial cells modulates the spontaneous activity and its propagation during early spinal cord development. J. Neurosci. 30, 390–403 (2010).

    Article  CAS  Google Scholar 

  37. Picken Bahrey, H.L. & Moody, W.J. Early development of voltage-gated ion currents and firing properties in neurons of the mouse cerebral cortex. J. Neurophysiol. 89, 1761–1773 (2003).

    Article  Google Scholar 

  38. Xiao, Q., Xu, L. & Spitzer, N.C. Target-dependent regulation of neurotransmitter specification and embryonic neuronal calcium spike activity. J. Neurosci. 30, 5792–5801 (2010).

    Article  CAS  Google Scholar 

  39. Ibarretxe, G., Perrais, D., Jaskolski, F., Vimeney, A. & Mulle, C. Fast regulation of axonal growth cone motility by electrical activity. J. Neurosci. 27, 7684–7695 (2007).

    Article  CAS  Google Scholar 

  40. Gutierrez, H. & Davies, A.M. Regulation of neural process growth, elaboration and structural plasticity by NF-kappaB. Trends Neurosci. 34, 316–325 (2011).

    Article  CAS  Google Scholar 

  41. Gutierrez, H., O'Keeffe, G.W., Gavalda, N., Gallagher, D. & Davies, A.M. Nuclear factor kappa B signaling either stimulates or inhibits neurite growth depending on the phosphorylation status of p65/RelA. J. Neurosci. 28, 8246–8256 (2008).

    Article  CAS  Google Scholar 

  42. Riquelme, D. et al. High-frequency field stimulation of primary neurons enhances ryanodine receptor–mediated Ca2+ release and generates hydrogen peroxide, which jointly stimulate NF-kappaB activity. Antioxid. Redox Signal. 14, 1245–1259 (2011).

    Article  CAS  Google Scholar 

  43. Tcherkezian, J., Brittis, P.A., Thomas, F., Roux, P.P. & Flanagan, J.G. Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation. Cell 141, 632–644 (2010).

    Article  CAS  Google Scholar 

  44. Jung, H., Yoon, B.C. & Holt, C.E. Axonal mRNA localization and local protein synthesis in nervous system assembly, maintenance and repair. Nat. Rev. Neurosci. 13, 308–324 (2012).

    Article  CAS  Google Scholar 

  45. Andrews, W. et al. Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain. Development 133, 2243–2252 (2006).

    Article  CAS  Google Scholar 

  46. López-Bendito, G. et al. Robo1 and Robo2 cooperate to control the guidance of major axonal tracts in the mammalian forebrain. J. Neurosci. 27, 3395–3407 (2007).

    Article  Google Scholar 

  47. Zallen, J.A., Yi, B.A. & Bargmann, C.I. The conserved immunoglobulin superfamily member SAX-3/Robo directs multiple aspects of axon guidance in C. elegans. Cell 92, 217–227 (1998).

    Article  CAS  Google Scholar 

  48. Hadjantonakis, A.K., Gertsenstein, M., Ikawa, M., Okabe, M. & Nagy, A. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 76, 79–90 (1998).

    Article  CAS  Google Scholar 

  49. Andrews, W. et al. The role of Slit-Robo signaling in the generation, migration and morphological differentiation of cortical interneurons. Dev. Biol. 313, 648–658 (2008).

    Article  CAS  Google Scholar 

  50. Long, H. et al. Conserved roles for Slit and Robo proteins in midline commissural axon guidance. Neuron 42, 213–223 (2004).

    Article  CAS  Google Scholar 

  51. Agmon, A. & Connors, B.W. Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro. Neuroscience 41, 365–379 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank N. García and E. San Martin for outstanding technical assistance. We are also grateful to A. Nagy (Samuel Lunenfeld Research Institute) for the EGFP mice, M. Valdeolmillos (Instituto de Neurociencias de Alicante) for providing the calcium imaging set-up and helping with quantification of calcium activity in growth cones, F. Martini for advice on calcium imaging, G. Expósito for advice on two-photon imaging, O. Marín (Instituto de Neurociencias de Alicante) for reagents, M. Maravall for advice on data analysis, F. Viana (Instituto de Neurociencias de Alicante) for providing the inducible CHO-A1 cells, and M. Dominguez and A. Gontijo for access and advice on qPCR equipment. We are grateful to A. Nieto, P. Arlotta, E. Herrera and M. Valdeolmillos for advice and critical reading of the manuscript. We are also grateful to members of the López-Bendito laboratory and Herrera laboratory for stimulating discussions and comments. C.M. is recipient of a JAE-Predoctoral Fellowship from the CSIC. This work was supported by grants from the Spanish Ministerio de Ciencia e Innovacion (BFU2006-07138 to J.L. and BFU2009-08261 to G.L.-B.), an Human Frontier Science Program Organization grant (RGP29/2008), the Consolider programme (CSD2007-00023) and an European Research Council grant (ERC-2009-StG_20081210 to G.L.-B.).

Author information

Author notes

  1. Erik Mire and Cecilia Mezzera: These authors contributed equally to this work.

Authors and Affiliations

  1. Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas (UMH-CSIC), San't Joan d'Alacant, Spain

    Erik Mire, Cecilia Mezzera, Eduardo Leyva-Díaz, Ana V Paternain, Lisa Bluy, Mar Castillo-Paterna, María José López, Sandra Peregrín, Joan Galcerán, Juan Lerma & Guillermina López-Bendito

  2. Ecole Normale Supérieure, Institut de Biologie de l'ENS, Paris, France

    Paola Squarzoni & Sonia Garel

  3. INSERM, U1024, Avenir Team, Paris, France

    Paola Squarzoni & Sonia Garel

  4. CNRS, UMR 8197, Paris, France

    Paola Squarzoni & Sonia Garel

  5. Genentech, South San Francisco, California, USA

    Marc Tessier-Lavigne

  6. The Rockefeller University, New York, New York, USA

    Marc Tessier-Lavigne

Authors
  1. Erik Mire
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cecilia Mezzera
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eduardo Leyva-Díaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ana V Paternain
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paola Squarzoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lisa Bluy
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mar Castillo-Paterna
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. María José López
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sandra Peregrín
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Marc Tessier-Lavigne
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Sonia Garel
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Joan Galcerán
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Juan Lerma
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Guillermina López-Bendito
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.L.-B. conceived the idea. E.M. and G.L.-B. designed the study. E.M., C.M., E.L.-D. and G.L.-B. performed the in vitro, ex vivo and in utero electroporation experiments. E.L.-D. and L.B. performed the luciferase assays. A.V.P. performed the electrophysiological recordings. P.S. and S.G. performed the experiments on Slit1 mutant mice. M.C.-P. performed the semiquantitative PCR and the collapse assay. M.J.L. performed the calcium recordings in CHO-A1 cells. S.P. subcloned the Kir2.1 plasmid. M.T.-L. produced the Robo1, Robo2 and Robo1; Robo2 mutant mice. J.G. supervised the luciferase assays. J.L. supervised the electrophysiological experiments. E.M., C.M. and G.L.-B. conducted the data analysis and wrote the paper.

Corresponding author

Correspondence to Guillermina López-Bendito.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 (PDF 16642 kb)

Supplementary Video 1

Speed of growth of TCA travelling at the vTel. (AVI 4100 kb)

Supplementary Video 2

Speed of growth of TCA extending at the neocortex. (AVI 6660 kb)

Supplementary Video 3

Speed of growth of TCA travelling at the angle at the PSPB. (AVI 3588 kb)

Supplementary Video 4

Speed of growth of TCA travelling at the entrance of the neocortex. (AVI 1077 kb)

Supplementary Video 5

Spontaneous activity in the thalamus of E12.5 embryo. (AVI 5969 kb)

Supplementary Video 6

Spontaneous activity in the thalamus of E16.5 embryo. (AVI 7184 kb)

Supplementary Video 7

Spontaneous activity in early thalamocortical growth cones. (AVI 4007 kb)

Supplementary Video 8

Spontaneous activity in late thalamocortical growth cones. (AVI 3428 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mire, E., Mezzera, C., Leyva-Díaz, E. et al. Spontaneous activity regulates Robo1 transcription to mediate a switch in thalamocortical axon growth. Nat Neurosci 15, 1134–1143 (2012). https://doi.org/10.1038/nn.3160

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.3160

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing