Description
- Abstract:
- Engineered three-dimensional primary cortical microtissues provide a physiologically relevant in vitro model of cortical network organization, but their electrophysiological characterization has been limited to calcium imaging and extracellular multielectrode array recordings, which do not capture membrane potential dynamics or subthreshold voltage activity at single-cell resolution. This thesis investigates the integration of Voltron2, a chemigenetic genetically encoded voltage indicator, into an established three-dimensional cortical microtissue platform to enable optical electrophysiology at the level of individual neurons. Four primary objectives were addressed: validation of Voltron2 expression and dye labeling under Borton Lab culture conditions, optimization of adeno-associated viral titer for transduction, identification of acquisition parameters required for voltage signal detection, and development of an electrical stimulation platform compatible with simultaneous confocal imaging. Dual-vector AAV transduction produced sparse, neuron-specific Voltron2 expression, confirmed by immunohistochemistry, while viral titer was found to be a key determinant of optical separability, with higher titers producing overlapping fluorescence that limited single-cell resolution. Stimulus-evoked voltage responses were detected in individual neurons at later culture timepoints. In parallel, impedance characterization of a custom stimulation plate cover identified a frequency range that enabled reproducible and consistent field stimulation across electrode sites. Together, these results establish a framework for applying genetically encoded voltage indicators to three-dimensional neural microtissues and demonstrate the feasibility of resolving single-cell voltage dynamics in a physiologically relevant in vitro system.
- Notes:
- Thesis (Sc. M.)--Brown University, 2026
Citation
Ash, William James,
"Optical Electrophysiology of Engineered Three-Dimensional Cortical Microtissues Using Genetically Encoded Voltage Indicators"
(2026).
Biomedical Engineering Theses and Dissertations.
Brown Digital Repository. Brown University Library.
https://repository.library.brown.edu/studio/item/bdr:vfxprsv9/
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Collection:
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Biomedical Engineering Theses and Dissertations
Theses and Dissertations for the Biomedical Engineering department....