Dec 12th, 2025 - Vincent Villette

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IBENS, ENS, PSL

Monitoring neuronal spiking and subthreshold activities in cerebral and cerebellar microcircuits using optical methods in head-fixed behaving mice

To understand how information is represented, processed, and propagated in the brain, technologies for recording transmembrane potential in vivo in neuronal populations with high ­fidelity will be essential. Direct cellular voltage imaging in vivo, was limited by the speed and sensitivity of both indicators and imaging modalities. We developed two microscopy techniques (ULoVE1 and 3D-CASH2) enabling the optical recordings of cortical population voltage signal at single cell and high temporal resolution (>2KHz) in awake behaving animal circumventing limitations of classical imaging approaches. Improved Genetically encoded voltage indicators enable to record up to 500µm of depth during tens of minutes (up to 1 hour). Also, red shifted versions3 enable to multiplexed with calcium indicators offering a large palette of tools to study cell and network mechanisms. In addition to present the journey we made with those optical developments and GEVI improvements, I will also shortly present the context of our physiology researches within the cerebellum that seeded and strengthened our interest to achieve fast populational GEVIs multiphoton optical recordings.

  1. Villette, V., Chavarha, M., Dimov, I.K., Bradley, J., Pradhan, L., Mathieu, B., Evans, S.W., Chamberland, S., Shi, D., Yang, R., et al. (2019). Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice. Cell 179, 1590-1608.e23. https://doi.org/10.1016/j.cell.2019.11.004.
  2. Akemann, W., Wolf, S., Villette, V., Mathieu, B., Tangara, A., Fodor, J., Ventalon, C., Léger, J.-F., Dieudonné, S., and Bourdieu, L. (2022). Fast optical recording of neuronal activity by three-dimensional custom-access serial holography. Nat Methods 19, 100–110. https://doi.org/10.1038/s41592-021-01329-7.
  3. Villette, V., Yang, S., Valenti, R., Macklin, J.J., Bradley, J., Mathieu, B., Lombardini, A., Podgorski, K., Dieudonné, S., Schreiter, E.R., et al. (2024). A novel rhodopsin-based voltage indicator for simultaneous two-photon optical recording with GCaMP in vivo. Preprint, https://doi.org/10.1101/2024.11.15.623698 https://doi.org/10.1101/2024.11.15.623698.