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Date of Award
Doctor of Philosophy (PhD)
Calcium (Ca2+) signals control key functions in almost all type of cells, including neurons. Imaging calcium in a large scale and in high resolution is particularly important. The information in spatiotemporal calcium dynamics allows us to further explore the cellular mechanisms associated with behavior and physiology. In this thesis, I have utilized an integrated microscope in freely moving mice expressing GCaMP6f to investigate the calcium dynamics of dorsal-CA1 hippocampal neurons in nicotine-reward contextual associations. Also by implementing similar approaches, we imaged early embryonic calcium activity in GCaMP6s transgenic zebrafish and developed an approach to automatically track calcium events that dramatically improves the efficiency and accuracy of the research. Furthermore, we collaborated with Rogers’ lab in University of Illinois Urbana-Champaign (UIUC) to develop and optimize a wireless photometry device system. The wireless system was applied to record calcium dynamics during different behavioral experiments to investigate the deep brain region basolateral amygdala (BLA), and comparted to traditional fiber photometry system to prove the efficiency of the new system.
We showed the development of specific neuronal ensembles which both encode nicotine-reward contextual memories and are necessary for the expression of place preference. We demonstrated that dorsal CA1 neuronal activity is necessary for both acquisition and expression of a nicotine conditional place preference (CPP), but is not necessarily required for learning simple operant-based reward tasks (i.e. sucrose self-administration). We reported that dorsal-CA1 Ca2+ transient activity is tightly locked to subsequent nicotine-reward seeking behavior and this activity is increased when animal encounters related contextual cues. This may be the result of recalling the reward-context association or being in a context that has been previously associated with reward. These results suggest that dorsal-CA1 ensembles specifically encode nicotine-associated spatial cues related to contextual information. We conclude that specific neuronal ensembles within the CA1 are recruited by nicotine-reward contextual pairings, and that their activity is necessary for the expression of this behavior, suggesting that nicotine engages and potentiates CA1 activity to inform subsequent cued contextual behaviors. We also explored the possibility of modeling CA1 neural activity to predict speed during locomotion.
Using the Tg[βactin2:GCaMP6s]stl351 mouse line featuring strong GCaMP6s expression from cleavage through gastrula stages, we detected higher frequency of Ca2+ transients in the superficial blastomeres during the blastula stages preceding the midblastula transition. Additionally, changes in GCaMP6s fluorescence also revealed that dorsal-biased Ca2+ signaling that follows the midblastula transition persisted longer during gastrulation, compared with earlier studies. We observed that dorsal-biased Ca2+ signaling is diminished in ventralized ichabod/ß-catenin2 mutant embryos and ectopically induced in dorsalized embryos by excess ß-catenin. During gastrulation, we directly visualized Ca2+ signaling in the dorsal forerunner cells, which form in a Nodal signaling dependent manner and later give rise to the laterality organ. We found that excess Nodal increases the number and the duration of Ca2+ transients specifically in the dorsal forerunner cells.
Furthermore, we explored the capabilities for wireless recording of neural activity in behaving mice using GCaMP6f in deep brain regions. We introduced a wireless injectable fluorescence photometer that integrates miniaturized light sources and photodetectors on flexible, needle-shaped polymer supports, suitable for injection into the deep brain at sites of interest. In vivo studies in freely moving animals demonstrated that this technology allows high fidelity recording of calcium fluorescence in deep brain structures, with measurement characteristics that match or exceed those associated with fiber photometry systems. We applied in vivo recording with both the wireless device and fiber photometry system in the foot-shock test and pharmacological manipulations. Results suggest that the wireless photometry system is highly biocompatible and efficiently collects fluorescence dynamics in freely moving mice during behavior. The resulting capabilities in optical recordings of neuronal dynamics in untethered, freely moving animals have potential for widespread applications in neuroscience research.
Michael R. Bruchas
Dennis Barbour, Robert Gereau, Timothy Holy, Baranidharan Raman,
Available for download on Wednesday, December 15, 2117