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Regulation of axon growth in vivo by activity-based competition

Abstract

The formation of functional neural networks requires precise regulation of the growth and branching of the terminal arbors of axons, processes known to be influenced by early network electrical activity1,2,3. Here we show that a rule of activity-based competition between neighbouring axons appears to govern the growth and branching of retinal ganglion cell (RGC) axon arbors in the developing optic tectum of zebrafish. Mosaic expression of an exogenous potassium channel or a dominant-negative SNARE protein was used to suppress electrical or neurosecretory activity in subsets of RGC axons. Imaging in vivo showed that these forms of activity suppression strongly inhibit both net growth and the formation of new branches by individually transfected RGC axon arbors. The inhibition is relieved when the activity of nearby ‘competing’ RGC axons is also suppressed. These results therefore identify a new form of activity-based competition rule that might be a key regulator of axon growth and branch initiation.

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Figure 1: Kir2.1 overexpression inhibits zebrafish neuronal calcium spiking; expression in single RGCs inhibits axon arbor growth.
Figure 2: VAMPm overexpression suppresses presynaptic vesicle function in zebrafish neurons; expression in single RGCs inhibits axon arbor growth.
Figure 3: Transfection of multiple neighbouring RGC axons with Kir2.1 or VAMPm mitigates their growth-suppressing effects compared with transfection of a single axon.
Figure 4: Selective activity suppression inhibits axon motility and branch formation rate with little effect on branch stability.

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Acknowledgements

We thank T. R. Clandinin, R. W. Tsien, R. W. Aldrich, L. Luo and the Smith laboratory for comments on the manuscript, and C. M. Niell for developing the Matlab routines used in image analysis. We thank T. Roeser for isolating the brn3c promoter. The US National Institutes of Health and the Vincent Coates Foundation provided financial support. J.Y.H. was supported by a Stanford Graduate Fellowship and a Coates Foundation Fellowship. M.C.S. was supported by a predoctoral fellowship from the American Heart Association.

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Correspondence to Jackie Yuanyuan Hua.

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Supplementary information

Supplementary Figure S1

This document contains Supplementary Figure S1 and accompanying legend. The figure shows that Kir2.1 and VAMP-GFP coexpress with high efficiency. (DOC 406 kb)

Supplementary Movie S1

This movie shows the growth of a VAMP-GFP expressing RGC axon arbor. Images were collected at two-minute intervals for 48 minutes. Scale bars represent 5µm in all movies. Auto-fluorescence of the skin is seen in the upper left corner of this movie and in some of the other movies presented (QuickTime; 5MB). (MOV 517 kb)

Supplementary Movie S2

This movie shows the growth of a Kir2.1 and VAMP-GFP expressing RGC axon arbor. Images were collected at two-minute intervals for 48 minutes. The axon shows less exploratory behaviour compared with control axons expressing VAMP-GFP (Supplementary movie 1). (MOV 514 kb)

Supplementary Movie S3

This movie shows the growth of a VAMPm expressing RGC axon arbor. Images were collected at two-minute intervals for 48 minutes. The axon shows less exploratory behaviour compared with control axons expressing VAMP-GFP (Supplementary movie 1). (MOV 707 kb)

Supplementary Movie S4

This movie shows the growth of a VAMP-GFP expressing RGC axon arbor. Images were collected at 20-minute intervals for 8 hours. In each of this movie and Supplementary movie S5-6, a new branch formed during the imaging period and lasted beyond 4 hours is pointed out by arrowhead in the last time point of imaging. (MOV 700 kb)

Supplementary Movie 5

This movie shows the growth of a Kir2.1 and VAMP-GFP expressing RGC axon arbor. Images were collected at 20-minute intervals for 8 hours. The axon is less exploratory compared with control (Supplementary movie S5), but new branch stability is not significantly affected. A new branch that lasted for more than 4 hours, and was likely stabilized, is pointed out by arrowhead in the last time point of this movie. (MOV 1534 kb)

Supplementary Movie S6

This movie shows the growth of a VAMPm expressing RGC axon arbor. Images were collected at 20-minute intervals for 8 hours. The axon is less exploratory compared to control (Supplementary movie S5), but new branch stability is not significantly affected. (MOV 591 kb)

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Hua, J., Smear, M., Baier, H. et al. Regulation of axon growth in vivo by activity-based competition. Nature 434, 1022–1026 (2005). https://doi.org/10.1038/nature03409

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