Designer indicators for two-photon recording of subthreshold voltage dynamics
Subthreshold voltage dynamics are critical for neuronal information integration, yet they remain understudied in vivo due to the limitations of current tools. While genetically encoded voltage indicators (GEVIs) offer a promising alternative, their application for deep-tissue recording using two-photon (2P) microscopy-a preferred method for deep-tissue recording-has been hindered by insufficient sensitivity for detecting millivolt-scale subthreshold signals. Here, we refined our multiparametric two-photon high-throughput screening platform to develop two novel GEVIs, JEDI3sub and JEDI3hyp, tailored explicitly for subthreshold voltage detection. Through fast 2P optical recording in awake, behaving mice, we demonstrated the superior sensitivity of JEDI3 indicators compared to JEDI-2P. We also showed that JEDI3sub can track population-level subthreshold optical tuning, while JEDI3hyp reliably captured subthreshold dynamics associated with sharp-wave ripple oscillations in hippocampal PV interneurons. Finally, JEDI3hyp facilitated extended imaging of brain-state-dependent, millivolt-scale subthreshold voltage changes across deep-layer somas, fine dendritic structures, and diverse cell types. By addressing the critical gap in 2P optical recording of subthreshold voltage dynamics, JEDI3 indicators open new avenues for studying neural information processing and its alterations in health and disease.
Authors
- Jonathan Bradley (ORCID: https://orcid.org/0000-0002-6933-1153)
- Xiaoyu Lu (ORCID: https://orcid.org/0000-0003-2426-1427)
- Cameron Smith (ORCID: https://orcid.org/0000-0002-2079-4667)
- Shanii Tabb
- Alex J. McDonald (ORCID: https://orcid.org/0000-0003-1214-3562)
- François St-Pierre (ORCID: https://orcid.org/0000-0001-8618-4135)
- Ryan G. Natan (ORCID: https://orcid.org/0000-0002-3123-4833)
- H. Liu
- Robert Law (ORCID: https://orcid.org/0000-0002-1220-3871)
- Mario Galdamez (ORCID: https://orcid.org/0000-0002-1626-4823)
- Matthew Shorey (ORCID: https://orcid.org/0000-0002-5105-4127)
- Barna Dudok (ORCID: https://orcid.org/0000-0001-5078-0090)
- Jacob Reimer (ORCID: https://orcid.org/0000-0003-4364-8846)
- Shuyuan Yang (ORCID: https://orcid.org/0000-0001-5977-9782)
- Vincent Villette
- Kevin L. Colbert
- Ryan Kroeger
- Zhuohe Liu (ORCID: https://orcid.org/0000-0002-1818-646X)
- Elsayed Zaabout (ORCID: https://orcid.org/0000-0002-5588-3635)
- Xiaoyu Dong (ORCID: https://orcid.org/0000-0003-0678-4056)
- S. Lai
- Laurent Bourdieu (ORCID: https://orcid.org/0000-0003-0195-8758)
- Michelle Ann Land
- Noura Hakam
- Ming Hu
- Annick Ayon
- Caroline Mailhes-Hamon
- Na Ji
- Jian Zhong
- Mate Marosi
Institutions
- Centre National de la Recherche Scientifique (FR)
- Inserm (FR)
- Baylor College of Medicine (US)
- Institut de Biologie de l'École Normale Supérieure (FR)
- Rice University (US)
- University of California, Berkeley (US)
Publication Details
- Journal
- bioRxiv (Cold Spring Harbor Laboratory)
- Published
- 2026-04-11
- DOI
- https://doi.org/10.64898/2026.04.08.717226
- Primary Topic
- Neural dynamics and brain function
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- National Science Foundation
- National Institutes of Health
- Intellectual and Developmental Disabilities Research Center