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Ryk controls remapping of motor cortex during functional recovery after spinal cord injury

Nature Neuroscience volume 19, pages 697–705 (2016)Cite this article

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Abstract

Limited functional recovery can be achieved through rehabilitation after incomplete spinal cord injury. Eliminating the function of a repulsive Wnt receptor, Ryk, in mice and rats by either conditional knockout in the motor cortex or monoclonal antibody infusion resulted in increased corticospinal axon collateral branches with presynaptic puncta in the spinal cord and enhanced recovery of forelimb reaching and grasping function following a cervical dorsal column lesion. Using optical stimulation, we observed that motor cortical output maps underwent massive changes after injury and that hindlimb cortical areas were recruited to control the forelimb over time. Furthermore, a greater cortical area was dedicated to controlling the forelimb in Ryk conditional knockout mice than in controls (wild-type or heterozygotes). In the absence of weekly task-specific training, recruitment of ectopic cortical areas was greatly reduced and there was no significant functional recovery even in Ryk conditional knockout mice. Our study provides evidence that maximal circuit reorganization and functional recovery can be achieved by combining molecular manipulation and targeted rehabilitation.

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Figure 1: Ryk conditional deletion enhances motor function recovery from spinal cord injury.
Figure 2: Ryk conditional deletion enhances corticospinal axon sprouting after spinal cord injury.
Figure 3: Changes in corticospinal connectivity after C5 dorsal column lesion.
Figure 4: A second injury at C3 eliminates enhanced recovery.
Figure 5: Monoclonal Ryk antibody infusion promotes functional recovery from spinal cord injury.
Figure 6: Cortical map reorganization during recovery from spinal cord injury.
Figure 7: Forelimb motor map representations move into quiescent former hindlimb cortical areas.
Figure 8: Cortical map reorganization and functional recovery from spinal cord injury are dependent upon rehabilitative training.

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Acknowledgements

We would like to thank Z. He, B. Zheng, F. Wang, L. Wang and the members of the Zou lab for critical reading of the manuscript, as well as comments and suggestions. This work was supported by grants to Y.Z. (RO1 NS047484, R21 NS081738, Wings for Life Foundation, and International Foundation for Research in Paraplegia).

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Author notes

  1. Edmund R Hollis II & Shih-Hsiu Wang

    Present address: Present addresses: Burke Medical Research Institute, White Plains, New York, USA, and Brain and Mind Research Institute at Weill Cornell Medicine, New York, New York, USA (E.R.H.); Department of Pathology and Cell Biology, Columbia University School of Medicine, New York, New York, USA (S.-H.W.).,

Authors and Affiliations

  1. Biological Sciences Division, Neurobiology Section, University of California, San Diego, La Jolla, California, USA

    Edmund R Hollis II, Nao Ishiko, Ting Yu, Chin-Chun Lu, Ariela Haimovich, Kristine Tolentino, Alisha Richman, Anna Tury, Maysam Pessian, Euna Jo & Yimin Zou

  2. Solomon H. Snyder Department of Neuroscience, Howard Hughes Medical Institute, The Johns Hopkins School of Medicine, Baltimore, Maryland, USA

    Shih-Hsiu Wang & Alex Kolodkin

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Contributions

Y.Z. and E.R.H. designed the experiments. E.R.H., N.I., T.Y., C.-C.L., A.H., K.T., A.R., A.T., M.P. and E.J. performed all the experiments under the supervision of Y.Z. Y.Z. designed the antigen for the Ryk monoclonal antibody. C.-C.L. and A.R. prepared the antigen. S.-H.W. generated the hybridomas using the antigen under the supervision of A.K. A.T. and E.R.H. screened for the hybridoma and tested the function of the Ryk monoclonal antibody in vitro and in vivo.

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Correspondence to Yimin Zou.

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Integrated supplementary information

Supplementary Figure 1 Full Western blots from Figure 1c and Supplementary Figure 3a.

(a) Specificity of Ryk monoclonal antibody from Supplementary Fig. 3a. (b) Western blot of postnatal day 7 motor cortex extract from mice infected at postnatal day 0 with AAV2/1 synapsin Cre from Figure 1c. E18.5 cortex from two separate Ryk KO embryonic mouse cortices as control in right two lanes. GAPDH loading control from same blot.

Supplementary Figure 2 Sample frames from forelimb reach video.

Example at 13 weeks after C5 dorsal column lesion (1 week after C3 sham operation) demonstrates the recovery of skilled forelimb grasp. Successful reach requires the use of a grasping motion as a sweeping motion would result in the pellet being dropped in either the gap between the food pellet platform (black) and the main enclosure, or through the wire-frame floor of the enclosure.

Supplementary Figure 3 Ryk monoclonal antibody infusion in rats.

(a) Specificity of Ryk monoclonal antibody. Full-length blot presented in Supplementary Fig. 1. (b) Timeline outlining experimental details of Ryk monoclonal antibody infusion after bilateral C5 dorsal column lesion in rats.

Supplementary Figure 4 Axon collateralization increased after Ryk monoclonal antibody infusion caudal to the injury.

Rats infused for 28 days with Ryk monoclonal antibody had greater levels of collateralization caudal to the lesion than control IgG infused rats (n = 6 (IgG control) 5 (Ryk monoclonal) rats, one-tailed t-test P = 0.0196 t(6) = 2.594, data presented as mean ± s.e.m.). Injury site is at 0 μm, caudal is represented with positive numbers. Axon index is thresholded pixels in sagittal spinal cord divided by thresholded pixels in transverse pyramids.

Supplementary Figure 5 Optogenetic mapping example.

Sedated mice with unilateral cranial windows were stimulated with 470nm LED by fiber optic cable to evoke muscle movements. Two examples of motor maps from one animal, pre- and 3 days post-C5 dorsal column lesion are shown.

Supplementary Figure 6 Optogenetic mapping after spinal cord injury in mice that received weekly training.

(a-b) Maps of evoked motor output in control (a) and Ryk conditional deleted mice (b) shift after spinal cord injury. Red corresponds to a larger number of mice responsive at a given location, blue to a smaller number (n = number of mice per condition/time point). (c) Proportion of motor cortex occupied by characterized motor output changes over time in response to weekly training and Ryk conditional deletion.

Supplementary Figure 7 Recovery of skilled forelimb reach for mice used in cortical mapping experiments.

Behavioral performance on skilled forelimb reach task shows enhanced recovery after Ryk conditional deletion in contralateral motor cortex (weeks 1-8 after C5 lesion, n = 10 (control) 11 (Ryk cKO) mice, repeated measures ANOVA P=0.0304 F(1,19)=5.472). Secondary C3 eliminates enhanced recovery after Ryk conditional deletion (n = 9 (control) 8 (Ryk cKO)), while unilateral pyramidotomy (n = 8 (control) 7 (Ryk cKO)) completely ablates the ability of mice to perform the task. Data presented as mean±s.e.m.

Supplementary Figure 8 Optogenetic mapping after spinal cord injury without weekly training.

(a-b) Maps of evoked motor output in control (a) and Ryk conditional deleted mice (b) shift after spinal cord injury. Red corresponds to a larger number of mice responsive at a given location, blue to a smaller number. (c) Proportions of motor cortex occupied by characterized motor output are relatively stable in the absence of training after injury.

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Hollis, E., Ishiko, N., Yu, T. et al. Ryk controls remapping of motor cortex during functional recovery after spinal cord injury. Nat Neurosci 19, 697–705 (2016). https://doi.org/10.1038/nn.4282

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