Date of Award
Doctor of Philosophy (PhD)
Epilepsy affects almost 1% of the global population, and further study is necessary to reveal diagnostic biomarkers and therapeutic targets . Second-by-second quantification of neurotransmitter molecules like L-glutamate is expected to elucidate the underlying mechanisms of epilepsy [2-4], and enzyme-based microelectrode probes offer improved temporal and spatial resolution over existing tools due to their smaller profiles and higher sampling frequencies [5, 6]. To that end, we tested a ceramic enzyme-based microelectrode array (MEA) probe in the hippocampi of Male Sprague-Dawley rats in vivo for the detection of L-glutamate during status epilepticus. Current induced by hydrogen peroxide, the reporter molecule resulting from enzyme-mediated breakdown of glutamate, was quantified using fixed potential amperometry. Our analysis used moving averages to investigate the glutamate-induced changes in mean current and peak-to-peak current amplitude. Fourier analysis provided insight into changes in frequency content. Our results indicated an increasing trend in glutamate current over the duration of status epilepticus, but this was not statisticallyhigher than the values observed pre-treatment. These experiments provided valuable information for the next phases of our tool development process.
The in vivo experiments highlight the need for a probe that can be removed and replaced in long-term experiments, which led us to pursue wire-based probes. Initial experiments with the new probes used brain slices extracted from male Sprague-Dawley rats and measured glutamate concentration changes due to electrical stimuli. In early prototyping, manual operation of the electronic stimulus isolator revealed a need for more precise stimulation timing. The Arduino Uno microcontroller provided a platform for developing a pulse generator capable of timing additional experiments.
Finally, we tested the novel enzyme-coated, wire-based glutamate probes in hippocampal slices using the custom pulse generator to drive electrical stimuli at various frequencies. Our investigation plotted maximum concentration of glutamate, maximum current, rise time, response pulse width, and fall time following stimuli. Our results indicated a significantly higher concentration following high-frequency stimulation and little difference in rise time across stimuli, which revealed a potential direction for exploring sources of excitation and inhibition. Significant differences in pulse width and rise time were likely coupled to concentration maxima.
Doughty, Phillip Timothy, "" (2019). Dissertation. 834.