A novel high-throughput screening system for directed evolution of robust optical reporters of electrical activity in the brain
Abstract/Contents
- Abstract
- Dynamic patterns of neuronal activity within intact neuronal circuits underly the astounding complexity of the brain. To decipher this code, we need tools that can monitor neuronal electrical activity with sufficient resolution in space and time, in awake behaving animals. Genetically encoded voltage indicators (GEVIs) offer great potential in achieving this goal but require significant improvements before they can be routinely deployed in vivo. A major limitation to developing better voltage sensors has been a lack of high-throughput screening methods that can reproducibly induce a fast change in transmembrane potential of a cell. In my thesis work, I present the development of a new electroporation-based screening approach, and mutant library construction and expression methods that enabled functional testing of GEVI variants at unprecedented throughput. I further describe using the screening system and a mechanism-guided approach to improve ASAP-family voltage indicators by directed evolution. One of engineered variants, ASAP3, exhibits up to 51% fluorescence change in the physiological range, millisecond response kinetics, efficient membrane localization and full activity under two-photon illumination. Finally, I show imaging ASAP3 signals in the awake mammalian brain using volumetric kHz-rate random-access multiphoton microscopy. The improved responsivity of ASAP3 to voltage allowed imaging of oscillations and spikes in single cells in single trials, revealing correlations between neuronal activity and animal behavior.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2018; ©2018 |
| Publication date | 2018; 2018 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Chavarha, Mariya |
|---|---|
| Degree supervisor | Lin, Michael |
| Thesis advisor | Lin, Michael |
| Thesis advisor | Baer, Thomas, 1945- |
| Thesis advisor | Bryant, Zev David |
| Degree committee member | Baer, Thomas, 1945- |
| Degree committee member | Bryant, Zev David |
| Associated with | Stanford University, Department of Bioengineering. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Mariya Chavarha. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2018. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2018 by Mariya Chavarha
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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