Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Biomedical Engineering

First Advisor's Name

Dr. Jorge Riera Diaz

First Advisor's Committee Title

Committee Chair

Second Advisor's Name

Dr. Jeremy Chambers

Second Advisor's Committee Title

Committee Member

Third Advisor's Name

Dr. Timothy Allen

Third Advisor's Committee Title

Committee Member

Fourth Advisor's Name

Dr. Jacob McPherson

Fourth Advisor's Committee Title

Committee Member

Fifth Advisor's Name

Dr. James Schummers

Fifth Advisor's Committee Title

Committee Member

Sixth Advisor's Name

Dr. Wei-Chiang Lin

Sixth Advisor's Committee Title

Committee Member

Keywords

Astrocyte, Optogenetics, Channelrhodopsin-2, Calcium Imaging, Serotype

Date of Defense

4-23-2021

Abstract

Gliosis observed in several neurological disorders is associated with neuroinflammation and enhanced astrocytic Ca2+ levels. The inherent multicellular nature of this neuroinflammation poses challenges in deciphering the exact role of astrocytic Ca2+ signaling and whether it leads to the generation and/or exacerbation of neuroinflammation. These challenges are aggravated by the dearth of systematic characterization of a regulated method for eliciting astrocytic Ca2+ increases.

The primary goal of this dissertation is to address the lack of a characterized method by studying optogenetics for eliciting astrocytic Ca2+ increases. As part of this analysis, we aim to identify light stimulation paradigms resulting in consistent astrocytic Ca2+ increases and assess optogenetic construct serotypes yielding maximum target cell transduction. Firstly, a novel protocol was devised to perform simultaneous optogenetics and astrocytic Ca2+ imaging in adult murine brain slices. Neocortical astrocytes exhibited synchronous patterns of Ca2+ activity upon light stimulation, drastically different from resting spontaneous activity, and based on the effect of various light paradigms; we identified ix those conducive for robust astrocytic signaling. Secondly, a theoretical model was constructed to study the effect of short and long-term light stimulation of optogenetically-enabled (ChR2-expressing) astrocytes on their Ca2+ spiking activity and basal level. We further investigated how ChR2 gating dynamics, buffering, and coupling coefficient of Ca2+ influence astrocytic activity in a single cell and a network. The response of select variants of ChR2 to varying light stimulation paradigms and key parameters to design future constructs was explored. A preliminary evaluation revealed model similarities to our in situ experimental data. Finally, to facilitate future translational work and eventual comparison to current disease models, astrocytic transduction of various serotypes of an AAV optogenetic construct was assessed in vivo, and the serotype with maximal transduction efficiency was identified.

Overall, we identified light stimulation paradigms that lead to repeated robust activation of astrocytes and AAV serotypes with high astrocytic transduction efficiency, thereby verifying that via an analysis of light stimulation paradigms and serotype transduction patterns, optogenetics can be implemented for inducing astrocytic Ca2+ increases in a controlled and tunable manner.

Identifier

FIDC010179

Previously Published In

Balachandar, L., Montejo, K.A., Castano, E., Perez, M., Moncion, C., Chambers, J.W., Lujan, J.L. and Diaz, J.R., 2020. Simultaneous Ca2+ Imaging and Optogenetic Stimulation of Cortical Astrocytes in Adult Murine Brain Slices. Current Protocols in Neuroscience, 94(1), p.e110.

Moshkforoush, A., Balachandar, L., Moncion, C., Montejo, K.A. and Riera, J., 2021. Unraveling ChR2-driven stochastic Ca2+ dynamics in astrocytes: A call for new interventional paradigms. PLoS Computational Biology, 17(2), p.e1008648.

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