Doctor of Philosophy (PhD)
First Advisor's Name
Dr. Jorge Riera Diaz
First Advisor's Committee Title
Second Advisor's Name
Dr. Jeremy Chambers
Second Advisor's Committee Title
Third Advisor's Name
Dr. Timothy Allen
Third Advisor's Committee Title
Fourth Advisor's Name
Dr. Jacob McPherson
Fourth Advisor's Committee Title
Fifth Advisor's Name
Dr. James Schummers
Fifth Advisor's Committee Title
Sixth Advisor's Name
Dr. Wei-Chiang Lin
Sixth Advisor's Committee Title
Astrocyte, Optogenetics, Channelrhodopsin-2, Calcium Imaging, Serotype
Date of Defense
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.
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.
Balachandar, Lakshmini, "Stimulation Paradigms and Transduction Patterns for Optogenetic Intervention of Astrocytes" (2021). FIU Electronic Theses and Dissertations. 4821.
Available for download on Thursday, September 14, 2023
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