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Self-assembled ultraflexible probes for long-term neural recordings and neuromodulation

Nature Protocols volume 18, pages 1712–1744 (2023)Cite this article

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Abstract

Ultraflexible microelectrode arrays (MEAs) that can stably record from a large number of neurons after their chronic implantation offer opportunities for understanding neural circuit mechanisms and developing next-generation brain–computer interfaces. The implementation of ultraflexible MEAs requires their reliable implantation into deep brain tissues in a minimally invasive manner, as well as their precise integration with optogenetic tools to enable the simultaneous recording of neural activity and neuromodulation. Here, we describe the process for the preparation of elastocapillary self-assembled ultraflexible MEAs, their use in combination with adeno-associated virus vectors carrying opsin genes and promoters to form an optrode probe and their in vivo experimental use in the brains of rodents, enabling electrophysiological recordings and optical modulation of neuronal activity over long periods of time (on the order of weeks to months). The procedures, including device fabrication, probe assembly and implantation, can be completed within 3 weeks. The protocol is intended to facilitate the applications of ultraflexible MEAs for long-term neuronal activity recording and combined electrophysiology and optogenetics. The protocol requires users with expertise in clean room facilities for the fabrication of ultraflexible MEAs.

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Fig. 1: Structure design of ultraflexible MEAs.
Fig. 2: Fabrication process of ultraflexible MEAs.
Fig. 3: Structure of ultraflexible MEAs.
Fig. 4: Flip-chip bonding process of ultraflexible MEAs to FPCs.
Fig. 5: Release and electroplating processes of ultraflexible MEAs.
Fig. 6: Elastocapillary self-assembly process of ultraflexible MEAs.
Fig. 7: Elastocapillary self-assembly process of optrodes.
Fig. 8: Preparation process of AAV-delivery optrodes.
Fig. 9: Implantation process of self-assembled probes.
Fig. 10: Experiment setup for simultaneous electrophysiological recording and optogenetic stimulation.
Fig. 11: Preparation process of brain slices with implanted probes.
Fig. 12: Example interfacing results between chronically implanted ultraflexible MEA and neurons.
Fig. 13: Example neuronal recording results by chronically implanted probes.

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Data availability

The raw recording data used to generate Fig. 13b–d have been deposited on Figshare (https://doi.org/10.6084/m9.figshare.c.6393534.v1). Source data are provided with this paper.

References

  1. Buzsaki, G. Large-scale recording of neuronal ensembles. Nat. Neurosci. 7, 446–451 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Jun, J. J. et al. Fully integrated silicon probes for high-density recording of neural activity. Nature 551, 232–236 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kollo, M. et al. CHIME: CMOS-hosted in vivo microelectrodes for massively scalable neuronal recordings. Front. Neurosci. 14, 834 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Obaid, A. et al. Massively parallel microwire arrays integrated with CMOS chips for neural recording. Sci. Adv. 6, eaay2789 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gilletti, A. & Muthuswamy, J. Brain micromotion around implants in the rodent somatosensory cortex. J. Neural Eng. 3, 189–195 (2006).

    Article  PubMed  Google Scholar 

  6. Biran, R., Martin, D. C. & Tresco, P. A. Neuronal cell loss accompanies the brain tissue response to chronically implanted silicon microelectrode arrays. Exp. Neurol. 195, 115–126 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Polikov, V. S., Tresco, P. A. & Reichert, W. M. Response of brain tissue to chronically implanted neural electrodes. J. Neurosci. Methods 148, 1–18 (2005).

    Article  PubMed  Google Scholar 

  8. Williams, J. C., Rennaker, R. L. & Kipke, D. R. Long-term neural recording characteristics of wire microelectrode arrays implanted in cerebral cortex. Brain Res. Protoc. 4, 303–313 (1999).

    Article  CAS  Google Scholar 

  9. Kozai, T. D. Y. et al. Chronic tissue response to carboxymethyl cellulose based dissolvable insertion needle for ultra-small neural probes. Biomaterials 35, 9255–9268 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. Khodagholy, D. et al. Highly conformable conducting polymer electrodes for in vivo recordings. Adv. Mater. 23, H268–H272 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Tian, B. et al. Macroporous nanowire nanoelectronic scaffolds for synthetic tissues. Nat. Mater. 11, 986–994 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kim, T.-i et al. Injectable, cellular-scale optoelectronics with applications for wireless optogenetics. Science 340, 211–216 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Liu, J. et al. Syringe-injectable electronics. Nat. Nanotechnol. 10, 629–636 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Someya, T., Bao, Z. & Malliaras, G. G. The rise of plastic bioelectronics. Nature 540, 379–385 (2016).

    Article  CAS  PubMed  Google Scholar 

  15. Luan, L. et al. Ultraflexible nanoelectronic probes form reliable, glial scar-free neural integration. Sci. Adv. 3, e1601966 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Park, S. et al. One-step optogenetics with multifunctional flexible polymer fibers. Nat. Neurosci. 20, 612–619 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Chung, J. E. et al. High-density, long-lasting, and multi-region electrophysiological recordings using polymer electrode arrays. Neuron 101, 21–31 (2019).

    Article  CAS  PubMed  Google Scholar 

  18. Guan, S. et al. Elastocapillary self-assembled neurotassels for stable neural activity recordings. Sci. Adv. 5, eaav2842 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Musk, E. An integrated brain-machine interface platform with thousands of channels. J. Med. Internet Res. 21, e16194 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Yang, X. et al. Bioinspired neuron-like electronics. Nat. Mater. 18, 510–517 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fu, T.-M. et al. Stable long-term chronic brain mapping at the single-neuron level. Nat. Methods 13, 875–882 (2016).

    Article  CAS  PubMed  Google Scholar 

  22. Zhang, F. et al. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nat. Protoc. 5, 439–456 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Anikeeva, P. et al. Optetrode: a multichannel readout for optogenetic control in freely moving mice. Nat. Neurosci. 15, 163–170 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Buzsáki, G. et al. Tools for probing local circuits: high-density silicon probes combined with optogenetics. Neuron 86, 92–105 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Zou, L. et al. Self-assembled multifunctional neural probes for precise integration of optogenetics and electrophysiology. Nat. Commun. 12, 5871 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. McCall, J. G. et al. Fabrication and application of flexible, multimodal light-emitting devices for wireless optogenetics. Nat. Protoc. 8, 2413–2428 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. McCall, J. G. et al. Preparation and implementation of optofluidic neural probes for in vivo wireless pharmacology and optogenetics. Nat. Protoc. 12, 219–237 (2017).

    Article  CAS  PubMed  Google Scholar 

  28. Gao, L. et al. Free-standing nanofilm electrode arrays for long-term stable neural interfacings. Adv. Mater. 34, e2107343 (2022).

    Article  PubMed  Google Scholar 

  29. Kozai, T. D. Y. & Kipke, D. R. Insertion shuttle with carboxyl terminated self-assembled monolayer coatings for implanting flexible polymer neural probes in the brain. J. Neurosci. Methods 184, 199–205 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Zhao, Z. et al. Nanoelectronic coating enabled versatile multifunctional neural probes. Nano Lett. 17, 4588–4595 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wu, F. et al. Monolithically integrated μLEDs on silicon neural probes for high-resolution optogenetic studies in behaving animals. Neuron 88, 1136–1148 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Watakabe, A. et al. Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex. Neurosci. Res. 93, 144–157 (2015).

    Article  PubMed  Google Scholar 

  33. Bennett, A. et al. Thermal stability as a determinant of AAV serotype identity. Mol. Ther. Methods Clin. Dev. 6, 171–182 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Deisseroth, K. Optogenetics: 10 years of microbial opsins in neuroscience. Nat. Neurosci. 18, 1213–1225 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Owen, S. F., Max, H. L. & Anatol, C. K. Thermal constraints on in vivo optogenetic manipulations. Nat. Neurosci. 22, 1061–1065 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yang, Y. et al. Preparation and use of wireless reprogrammable multilateral optogenetic devices for behavioral neuroscience. Nat. Protoc. 17, 1073–1096 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. van Asperen, J. V. et al. Determining glioma cell invasion and proliferation in ex vivo organotypic mouse brain slices using whole-mount immunostaining and tissue clearing. STAR Protoc. 3, 101703 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the Fabrication Lab at NCNST for the microfabrication facilities and support and the Animal Resource Center at NCNST for animal housing and care. This work is supported by the National Natural Science Foundation of China (Grant Nos. 21790393, 61971150 and 32061143013) and the National Key Research and Development Program of China (Grant No. 2021ZD0202200).

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Author notes

  1. These authors contributed equally: Shouliang Guan, Huihui Tian, Yinan Yang.

Authors and Affiliations

  1. CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, China

    Shouliang Guan, Huihui Tian, Yinan Yang, Mengcheng Liu, Jianfei Ding, Jinfen Wang & Ying Fang

  2. CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China

    Shouliang Guan, Yinan Yang & Ying Fang

  3. University of Chinese Academy of Sciences, Beijing, China

    Yinan Yang & Ying Fang

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Contributions

Y.F., H.T. and S.G. conceived the project and designed the detailed experimental protocol. S.G., J.D. and J.W. fabricated and characterized the devices. S.G., Y.Y. and M.L. performed animal surgery, electrophysiological recordings and corresponding analysis. Y.F., H.T. and S.G. wrote the paper. Y.F. acquired funding and supervised the project.

Corresponding author

Correspondence to Ying Fang.

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The authors declare no competing interests.

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Peer review information

Nature Protocols thanks Shadi A. Dayeh and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Guan, S. et al. Sci. Adv. 5, eaav2842 (2019): https://doi.org/10.1126/sciadv.aav2842

Zou, L. et al. Nat. Commun. 12, 5871 (2021): https://doi.org/10.1038/s41467-021-26168-0

Gao, L. et al. Adv. Mater. 34, e2107343 (2022): https://doi.org/10.1002/adma.202107343

Supplementary information

Reporting Summary

Supplementary Data 1

Eight-filament ultraflexible microelectrode arrays

Supplementary Data 2

16-filament ultraflexible microelectrode arrays

Supplementary Data 3

120-pin FPC

Supplementary Data 4

120-pin PCB

Supplementary Data 5

3D-printed MEA holder

Supplementary Data 6

3D-printed optical fiber holder

Supplementary Data 7

3D-printed optrode cover

Supplementary Data 8

Stainless steel head plate

Source data

Source Data Fig. 13

Stastical data for Fig. 13, e–g

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Guan, S., Tian, H., Yang, Y. et al. Self-assembled ultraflexible probes for long-term neural recordings and neuromodulation. Nat Protoc 18, 1712–1744 (2023). https://doi.org/10.1038/s41596-023-00824-9

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