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Spectrally distinct channelrhodopsins for two-colour optogenetic peripheral nerve stimulation

Nature Biomedical Engineering volume 2, pages 485–496 (2018)Cite this article

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Abstract

Technologies for peripheral nerve stimulation have conventionally relied on the anatomic placement of electrodes adjacent to subsets of sensory fibres or motor fibres that selectively target an end effector. Here, we demonstrate the use of optogenetics to directly target the innervating fibres of an end effector by relying on retrograde transfection of adeno-associated virus serotype 6 to restrict axonal opsin expression to the desired fibre targets. By using an in vivo screen in rats, we identify the first channelrhodopsins as well as a halorhodopsin that respond to red light in the peripheral nerve. Combining two channelrhodopsins with spectrally distinct activation profiles allowed us to drive opposing muscle activity via two-colour illumination of the same mixed nerve. We also show halorhodopsin-mediated reductions in electrically evoked muscle tremor spectrally optimized for deep peripheral nerves. Our non-invasive peripheral neurostimulator with targeted multi-fascicle resolution enables scientific and clinical exploration, such as motor control in paralysis, biomimetic sensation feedback for amputees and targeted inhibition of muscle tremor.

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Fig. 1: Comparison of channelrhodopsin activity in the peripheral nerve.
Fig. 2: Comparison of channelrhodopsin expression in the peripheral nerve.
Fig. 3: Optogenetic activation with two unique wavelengths drives opposing ankle movements.
Fig. 4: Fluence rate modelling reveals moderately increased levels of red light within deeper tissues.
Fig. 5: Loss of expression is opsin dependent with highly expressing nerves requiring low fluence rates for activation.
Fig. 6: Jaws inhibits motor activity.

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References

  1. Navarro, X. et al. A critical review of interfaces with the peripheral nervous system for the control of neuroprostheses and hybrid bionic systems. J. Peripher. Nerv. Syst. 10, 229–258 (2005).

    Article  PubMed  Google Scholar 

  2. Raspopovic, S. et al. Restoring natural sensory feedback in real-time bidirectional hand prostheses. Sci. Transl. Med. 6, 222ra19 (2014).

    Article  PubMed  Google Scholar 

  3. Boretius, T. et al. A transverse intrafascicular multichannel electrode (TIME) to interface with the peripheral nerve. Biosens. Bioelectron. 26, 62–69 (2010).

    Article  PubMed  CAS  Google Scholar 

  4. Musick, K. M., Chew, D. J., Fawcett, J. W. & Lacour, S. P. PDMS microchannel regenerative peripheral nerve interface. In 2013 6th International IEEE/EMBS Conference on Neural Engineering (NER) 649–652 (2013).

  5. Maimon, B. et al. Assessment of nerve regeneration through a novel microchannel array. Int. J. Phys. Med. Rehabil. 4, 332 (2016).

    Article  Google Scholar 

  6. Dyer, K. R. & Duncan, I. D. The intraneural distribution of myelinated fibres in the equine recurrent laryngeal nerve. Brain 110, 1531–1543 (1987).

    Article  PubMed  Google Scholar 

  7. Stewart, J. D. Peripheral nerve fascicles: anatomy and clinical relevance. Muscle Nerve 28, 525–541 (2003).

    Article  PubMed  Google Scholar 

  8. Thompson, A. C., Stoddart, P. R. & Jansen, E. D. Optical stimulation of neurons. Curr. Mol. Imaging 3, 162–177 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Peterson, E. J. & Tyler, D. J. Motor neuron activation in peripheral nerves using infrared neural stimulation. J. Neural Eng. 11, 16001 (2014).

    Article  CAS  Google Scholar 

  10. Iyer, S. M. et al. Virally mediated optogenetic excitation and inhibition of pain in freely moving nontransgenic mice. Nat. Biotechnol. 32, 274–278 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Llewellyn, M. E., Thompson, K. R., Deisseroth, K. & Delp, S. L. Orderly recruitment of motor units under optical control in vivo. Nat. Med. 16, 1161–1165 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Towne, C., Montgomery, K. L., Iyer, S. M., Deisseroth, K. & Delp, S. L. Optogenetic control of targeted peripheral axons in freely moving animals. PLoS ONE 8, e72691 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Lin, J. Y. et al. A user’s guide to channelrhodopsin variants: features, limitations and future developments. Exp. Physiol. 96, 19–25 (2011).

    Article  PubMed  Google Scholar 

  14. Stujenske, J. M., Spellman, T. & Gordon, J. A. Modeling the spatiotemporal dynamics of light and heat propagation for in vivo optogenetics. Cell. Rep. 12, 525–534 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Maimon, B. et al. Transdermal optogenetic peripheral nerve stimulation. J. Neural Eng. 14, 034002 (2017).

    Article  PubMed  Google Scholar 

  16. Lin, J. Y., Knutsen, P. M., Muller, A., Kleinfeld, D. & Tsien, R. Y. ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nat. Neurosci. 16, 1499–1508 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Klapoetke, N. C. et al. Independent optical excitation of distinct neural populations. Nat. Methods 11, 338–346 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Fenno, L., Yizhar, O., & Deisseroth, K. The development and application of optogenetics. Annu. Rev. Neurosci. 34, 389–412 (2011).

    Article  PubMed  CAS  Google Scholar 

  19. Prigge, M. et al. Color-tuned channelrhodopsins for multiwavelength optogenetics. J. Biol. Chem. 287, 31804–31812 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Lin, J. Y., Lin, M. Z., Steinbach, P. & Tsien, R. Y. Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys. J. 96, 1803–1814 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Chuong, A. S. et al. Noninvasive optical inhibition with a red-shifted microbial rhodopsin. Nat. Neurosci. 17, 1123–1129 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Mason, M. R. et al. Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons. Mol. Ther. 18, 715–724 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Hareendran, S. et al. Adeno-associated virus (AAV) vectors in gene therapy: immune challenges and strategies to circumvent them. Rev. Med. Virol. 23, 399–413 (2013).

    Article  PubMed  CAS  Google Scholar 

  24. Mingozzi, F. et al. Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood 122, 23–36 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Wang, H. et al. High-speed mapping of synaptic connectivity using photostimulation in channelrhodopsin-2 transgenic mice. Proc. Natl Acad. Sci. USA 104, 8143–8148 (2007).

    Article  PubMed  CAS  Google Scholar 

  26. Zepetnek, J. E. T., de Gordon, T., Stein, R. B. & Zung, H. V. Comparison of force and EMG measures in normal and reinnervated tibialis anterior muscles of the rat. Can. J. Physiol. Pharmacol. 69, 1774–1783 (1991).

    Article  Google Scholar 

  27. Schmalbruch, H. Fiber composition of the rat sciatic nerve. Anat. Rec. 215, 71–81 (1986).

    Article  PubMed  CAS  Google Scholar 

  28. Mense, S. Muscle pain: mechanisms and clinical significance. Dtsch Arztebl. Int. 105, 214–219 (2008).

    PubMed  PubMed Central  Google Scholar 

  29. Mohan, R., Tosolini, A. P. & Morris, R. Targeting the motor end plates in the mouse hindlimb gives access to a greater number of spinal cord motor neurons: an approach to maximize retrograde transport. Neuroscience 274, 318–330 (2014).

    Article  PubMed  CAS  Google Scholar 

  30. Tozburun, S., Cilip, C. M., Lagoda, G. A., Burnett, A. L. & Fried, N. M. Continuous-wave infrared optical nerve stimulation for potential diagnostic applications. J. Biomed. Opt. 15, 55012 (2010).

    Article  Google Scholar 

  31. Gray, S. J. et al. Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Hum. Gene Ther. 22, 1143–1153 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Hofherr, A., Fakler, B. & Klöcker, N. Selective Golgi export of Kir2.1 controls the stoichiometry of functional Kir2.x channel heteromers. J. Cell. Sci. 118, 1935–1943 (2005).

    Article  PubMed  CAS  Google Scholar 

  33. Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Alusi, S. H., Glickman, S., Aziz, T. Z. & Bain, P. G. Tremor in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 66, 131–134 (1999).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. O'Suilleabhain, P. E. & Matsumoto, J. Y. Time-frequency analysis of tremors. Brain 121, 2127–2134 (1998).

    Article  PubMed  Google Scholar 

  36. Harmaline induced tremors. PsychoGenics http://www.psychogenics.com/harmaline.html (accessed 2 June 2017).

  37. McGie, S. C., Zariffa, J., Popovic, M. R. & Nagai, M. K. Short-term neuroplastic effects of brain-controlled and muscle-controlled electrical stimulation. Neuromodulation Technol. Neural Interface 18, 233–240 (2015).

    Article  Google Scholar 

  38. Arcourt, A. et al. Touch receptor-derived sensory information alleviates acute pain signaling and fine-tunes nociceptive reflex coordination. Neuron 93, 179–193 (2017).

    Article  PubMed  CAS  Google Scholar 

  39. Williams, E. K. et al. Sensory neurons that detect stretch and nutrients in the digestive system. Cell 166, 209–221 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Chang, R. B., Strochlic, D. E., Williams, E. K., Umans, B. D. & Liberles, S. D. Vagal sensory neuron subtypes that differentially control breathing. Cell 161, 622–633 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Bashkatov, A. N. et al. In vivo and in vitro study of control of rat skin optical properties by acting of osmotical liquid. Biomed. Photon. Optoelectronic Imaging 4224, 300–311 (2000).

    Article  CAS  Google Scholar 

  42. Bashkatov, A. N., Genina, E. A. & Tuchin, V. V. Optical properties of skin, subcutaneous, and muscle tissues: a review. J. Innov. Opt. Health Sci. 4, 9–38 (2011).

    Article  Google Scholar 

  43. Islam, M. S. et al. Extracting structural features of rat sciatic nerve using polarization-sensitive spectral domain optical coherence tomography. J. Biomed. Opt. 17, 56012 (2012).

    Article  Google Scholar 

  44. Hendriks, B. H. W. et al. Nerve detection with optical spectroscopy for regional anesthesia procedures. J. Transl. Med. 13, 380 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Yaroslavsky, A. N. et al. Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range. Phys. Med. Biol. 47, 2059 (2002).

    Article  PubMed  CAS  Google Scholar 

  46. Pennes, H. H. Analysis of tissue and arterial blood temperatures in the resting human forearm. J. Appl. Physiol. 1, 93–122 (1948).

    Article  PubMed  CAS  Google Scholar 

  47. Ward, S. R. & Lieber, R. L. Density and hydration of fresh and fixed human skeletal muscle. J. Biomech. 38, 2317–2320 (2005).

    Article  PubMed  Google Scholar 

  48. Hasgall, P. et al. IT’IS Database for Thermal and Electromagnetic Parameters of Biological Tissues Version 3.0 (IT’IS Foundation, 2015); https://doi.org/10.13099/VIP21000-03-0

  49. Fagher, B. & Monti, M. Thermogenic effect of two β-adrenoceptor blocking drugs, propranolol and carvedilol, on skeletal muscle in rats. A microcalorimetric study. Thermochim. Acta 251, 183–189 (1995).

    Article  CAS  Google Scholar 

  50. Allen, J. T., Allen, J. T. & Bowman, H. F. The simultaneous measurement of thermal conductivity, thermal diffusivity, and perfusion in small volumes of tissue. J. Biomech. Eng. 106, 192 (1984).

    Article  PubMed  Google Scholar 

  51. Rins, M., Diez, I., Calpena, A. C., & Obach, R. Skin density in the hairless rat. Evidence of regional differences. Eur. J. Drug. Metab. Pharmacokinet. Spec. No. 3, 456–457 (1991).

    PubMed  Google Scholar 

  52. Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank C. Towne for his assistance in crafting the motivation for this project. We thank E. Boyden for his assistance and consultation on this project. Furthermore, we thank K. Cormier, C. Condon, M. Brown, as well as the entire Koch Histology Core for their countless hours of painstaking histology efforts. In addition, we thank N. Klapoetke, O. Shemesh, K. Piatkevich, E. Revol, P. Calvaresi, C. Varela, A. Harding, M. Fahmi, C. Lee, H.-J. Suk, D. Park, M. Diaz, A. Schneider and S. Osseiran for their advice, guidance and assistance. This work was funded by the MIT Media Lab Consortium.

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Authors and Affiliations

  1. MIT Media Lab, Center for Extreme Bionics, Massachusetts Institute of Technology, Cambridge, MA, USA

    Benjamin E. Maimon, Shriya Srinivasan, Anthony N. Zorzos & Hugh M. Herr

  2. Harvard-MIT Program in Health Sciences and Technology (HST), Massachusetts Institute of Technology, Cambridge, MA, USA

    Benjamin E. Maimon & Shriya Srinivasan

  3. Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Kaitlyn Sparks

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Contributions

B.E.M. and H.M.H. contributed to idea conception, study design and data analysis. B.E.M. oversaw experiment conduction and data analysis for in vivo experiments including histology and fluence modelling. K.S. contributed to experiment conduction, data collection and histology processing. S.S. performed in vitro screen experiments along with corresponding data processing. A.N.Z. contributed to idea conception and experiment planning as well as assisted with Monte Carlo modelling. B.E.M. wrote the manuscript with all authors contributing to editing the text.

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Correspondence to Hugh M. Herr.

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Supplementary Video 1

Two-colour independent peripheral nerve stimulation (transdermal and exposed nerves), as well as high-frequency CsChrimson activation and JAWS tremor inhibition.

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Maimon, B.E., Sparks, K., Srinivasan, S. et al. Spectrally distinct channelrhodopsins for two-colour optogenetic peripheral nerve stimulation. Nat Biomed Eng 2, 485–496 (2018). https://doi.org/10.1038/s41551-018-0255-5

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