Background & Aims

Optical imaging of spinal cord neural activity in the awake, behaving animal has clear and significant advantages (Cheng et al., 2019; Nelson et al., 2019; Iseppon et al., 2022). However, due to technical difficulties, few studies have recorded spinal cord neural activity, long-term, in awake animals (Cheng et al., 2016; Sekiguchi et al., 2016; Ju et al., 2022; Shekhtmeyster et al., 2023). We have recently solved the post-laminectomy fibrosis problem, which makes long-term imaging possible (Ahanonu et al, 2023). The problem of excessive spinal cord movement has also been addressed, allowing us to record long-term from the same population of spinal cord neurons and glia in behaving mice. With this new ability, we can now track cellular changes in the organization of dorsal horn circuits that process injury messages, before and after nerve injury, and with changes in behavior.

Methods

Our spinal optical imaging approach provides an expansive view of both sides of the spinal cord, within multiple segments of the lumbar enlargement. By incorporating fluoropolymer membranes, which provide long-term fibrosis regrowth inhibition, we reliably maintain clear optical access to the spinal cord, for many months to over a year. To handle the complex spinal cord motion that occurs during awake spinal cord imaging, we developed novel image registration pipelines, which are available within CIAtah, our existing Ca2+ imaging analysis pipeline (Corder et al., 2019). Using this method, we longitudinally recorded the activity of genetically-defined dorsal horn lamina I projection neurons (SCPNs; using Phox2a-Cre floxed-GCaMP6 mice) and tracked microglial expression (using CX3CR1 reporter mice) across the development of chronic pain phenotypes in the SNI (Spared Nerve Injury) model.

Results

Repeated imaging of single-cell neural activity of large numbers of SCPNs revealed that SCPNs predominantly display polymodal responses in awake animals. Individual response profiles were consistent over months before injury. Further, in contrast to anesthetized mice, in the awake state, lamina I projection neurons exhibit significant spontaneous and movement-evoked activity. We also demonstrate persistent microglial changes after SNI for months, consistent with previous immunohistochemical analyses using Iba1 antibodies, but we are now able to study the time course of the changes post-injury in individual animals.

Conclusions

Our ability to follow the same population of neurons over time will illuminate the tissue and nerve injury-induced changes during the transition from acute to chronic pain. In progress are studies that evaluate tissue and nerve injury-induced changes in spontaneous and evoked excitability of SCPNs. Future studies will determine the impact of existing and novel analgesics on the activity of these projection neurons, at different times during the development of chronic pain.

References

1. Ahanonu, B., Crowther, A., Kania, A., Casillas, M. R., & Basbaum, A. (2023). Long-term optical imaging of the spinal cord in awake, behaving animals. bioRxiv, 2023-05.
2. Cheng, Y. T., Lett, K. M., & Schaffer, C. B. (2019). Surgical preparations, labeling strategies, and optical techniques for cell-resolved, in vivo imaging in the mouse spinal cord. Experimental neurology, 318, 192-204.
3. Iseppon, F., Linley, J. E., & Wood, J. N. (2022). Calcium imaging for analgesic drug discovery. Neurobiology of Pain, 11, 100083.
4. Corder, G., Ahanonu, B., Grewe, B. F., Wang, D., Schnitzer, M. J., & Scherrer, G. (ensemble2019). An amygdalar neural that encodes the unpleasantness of pain. Science, 363(6424), 276-281.
5. Nelson, N. A., Wang, X., Cook, D., Carey, E. M., & Nimmerjahn, A. (2019). Imaging spinal cord activity in behaving animals. Experimental neurology, 320, 112974.
6. Sekiguchi, K. J., Shekhtmeyster, P., Merten, K., Arena, A., Cook, D., Hoffman, E., … & Nimmerjahn, A. (2016). Imaging large-scale cellular activity in spinal cord of freely behaving mice. Nature communications, 7(1), 11450.
7. Ju, F., Jian, W., Han, Y., Huang, T., Ke, J., Liu, Z., … & Wei, P. (2022). Long-term two-photon imaging of spinal cord in freely behaving mice. bioRxiv, 2022-01.
8. Cheng, Y. T., Ness, S. L., Hu, S. H., Raikin, J., Pan, L. D., Wang, T., … & Schaffer, C. B. (2016, October). In-vivo three-photon excited fluorescence imaging in the spinal cord of awake, locomoting mouse. In Laser Science (pp. JTh2A-183). Optica Publishing Group.
9. Shekhtmeyster, P., Duarte, D., Carey, E. M., Ngo, A., Gao, G., Olmstead, J. A., … & Nimmerjahn, A. (2023). Trans-segmental imaging in the spinal cord of behaving mice. Nature Biotechnology, 1-5.

Presenting Author

Andrew Crowther

Poster Authors

Andrew Crowther

PhD

University of California San Francisco

Lead Author

Biafra Ahanonu PHD

University of California, San Francisco

Lead Author

Mariela Rosa Casillas BS

University of California San Francisco

Lead Author

Artur Kania

IRCM (Instut de recherches cliniques de Montreal) / McGill University

Lead Author

Allan Basbaum

Univ of California - San Francisco

Lead Author

Topics

  • Pain Imaging