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.
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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.).
The authors declare no competing financial interests.
Supplementary Figures 1–9 (PDF 16642 kb)
Speed of growth of TCA travelling at the vTel. (AVI 4100 kb)
Speed of growth of TCA extending at the neocortex. (AVI 6660 kb)
Speed of growth of TCA travelling at the angle at the PSPB. (AVI 3588 kb)
Speed of growth of TCA travelling at the entrance of the neocortex. (AVI 1077 kb)
Spontaneous activity in the thalamus of E12.5 embryo. (AVI 5969 kb)
Spontaneous activity in the thalamus of E16.5 embryo. (AVI 7184 kb)
Spontaneous activity in early thalamocortical growth cones. (AVI 4007 kb)
Spontaneous activity in late thalamocortical growth cones. (AVI 3428 kb)
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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
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