Using the Biophotonics Toolbox to Study Neurodegeneration In Vitro

In neurons, intracellular calcium has the dual role of charge carrier and intracellular messenger. The signaling function of calcium has aspects that are particularly important to neurons. Synaptic transmission, the form of secretion that leads to the release of neurotransmitters, the process of learning and the formation and consolidation of memory, long term potentiation or depression of synaptic transmission, the direct coupling between depolarization of the plasma membrane and the increase of intracellular calcium are all under the control of calcium signals. Neuronal viability depends on proper functioning of all these processes and to a greater degree depend on the precise temporal and spatial regulation of calcium signals than most other eukaryotic cells. The primary focus of this work is the development of experimental and analytical tools for studies of chronic neurodegenerative disorders. This work investigates the spontaneous calcium activity in primary, dissociated cortical neurons as compared to Huntington’s disease (HD) model cells. The relative fluorescence variation of calcium-sensitive Fluo 4 is measured and analyzed using a custom, GUI-based software tool: Fluorescence Emissions Signal Analyzer (FESA). Characterization of intracellular calcium activity with well-defined parameters within emissions signal activity across several timepoints throughout incubation highlight calcium dysregulation among HD model cells. In addition, methods for optimizing the viability of long-term neuronal cultures are investigated and discussed. Furthermore, a fluorescence microscopy system capable of measuring and generating optically-coupled shear stresses via laser-induced shockwaves is described. Shear stress induced by laser-induced shockwaves is a contactless model for traumatic brain injury (TBI) in vitro that offers high levels of reproducibility and spatiotemporal precision. Results from this work have potential applications towards high-throughput methods for the discovery of novel, therapeutic avenues for chronic neurodegenerative disorders such as HD and TBI