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Title	Genetic tools for imaging intracellular calcium dynamics in astrocytes
Publication Type	dissertation
School or College	College of Engineering
Department	Biomedical Engineering
Author	Gee, James Michael
Date	2016
Description	New evidence afforded by advanced live-tissue imaging techniques indicates that astrocytes, the predominant glial cell subtype, play a far more active role in synaptic physiology than was previously appreciated. Evolved iterations of genetically encoded calcium indicators, primarily the GCaMP variants, have enabled high spatiotemporal resolution detection of intracellular activity, but are limited by few options for gene transfection and expression. The goal of this dissertation work was to develop novel GCaMP-based tools for straightforward optical interrogation of astrocytic activity in rodent models of neuropathology. A Polr2a-targeted, Cre-dependent, CAG-driven, GCaMP5G-expressing reporter mouse line was constructed and designated "PC-G5-tdT". Detection of positive cells was facilitated by an IRES-tdTomato tag. PC-G5-tdT proved effective in diverse developmental contexts and reported intracellular calcium dynamics in somas and fine processes of astrocytes, microglia and neurons. Electrophysiological and behavioral analyses failed to detect a detrimental impact of GCaMP5G expression on nervous system performance. In acute brain slices prepared from a model of endotoxemia-induced neuroinflammation, a stereotyped sequence of astrocytic intrinsic activity was observed over the acute phase. At early time points, frequent somatic and distal process transients were observed but progressively declined with process event frequency lagging behind the soma. Several rat models of human neuropathology provide systems for researching basic mechanisms of disease. Unfortunately, transgenic rat technologies are immature and viral-based methods are hampered by side effects. In utero electroporation (IUE) is a proven method for transfecting astrocytes and neurons without major drawbacks. A toolset of IUE plasmids carrying CAG-driven, subcellular compartment-targeted GCaMP variants with optional cytosolic tdTomato co-expression was constructed. Stable expression was accomplished via random genomic integration of the reporter cassette through a binary plasmid system derived from the piggyBac transposon. Preparation- and age-specific patterns of activity were readily detected in astrocytes and neurons. In particular, organotypic slice culture astrocytes exhibited frequent global intrinsic transients whereas activity was restricted to distal astrocytic processes in acute brain slices prepared from older animals. This work has already stimulated progress in the field of glial cell physiology. Future application of these tools will advance our understanding of glial-neuronal interaction and possibly inform development of improved disease modification strategies.
Type	Text
Publisher	University of Utah
Subject	astrocyte; calcium imaging; GCaMP6; glia; in vivo; two-photon
Dissertation Name	Doctor of Philosophy
Language	eng
Rights Management	¬©James Michael Gee
Format	application/pdf
Format Medium	application/pdf
ARK	ark:/87278/s60g7sz2
DOI	https://doi.org/doi:10.26053/0H-385F-5P00
Setname	ir_etd
ID	1370644
Reference URL	https://collections.lib.utah.edu/ark:/87278/s60g7sz2

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Title	Page 113
Format	application/pdf
Setname	ir_etd
ID	1370757
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Reference URL	https://collections.lib.utah.edu/ark:/87278/s60g7sz2/1370757