Electrochemical Studies of Neuroactive Molecules with Fast-Scan Cyclic Voltammetry at Carbon-Fiber Microelectrodes.

HUGHES, STEPHANIE CRISTINA. Electrochemical Studies of Neuroactive Molecules with Fast-Scan Cyclic Voltammetry at Carbon-Fiber Microelectrodes. (Under the direction of Dr. Leslie A. Sombers.) Understanding the chemical dynamics of neuroactive molecules is critical to understanding the mechanisms underlying neurodegenerative disease states such as Parkinson’s Disease (PD). Electrochemical techniques are well-suited to monitor the chemical dynamics of neuroactive molecules. In particular, fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes is a prominent neuroanalytical tool that combines unparalleled micron spatial resolution and sub-second temporal resolution to monitor dynamic changes in neuroactive molecules in real-time to elucidate the chemical dynamics that underlie brain function and dysfunction in neurodegenerative disease states such as PD. This research aims to provide the framework for developing and modifying analytical tools to monitor neuroactive molecules such as nitric oxide (NO), and builds upon current techniques to monitor dopamine (DA) dynamics following pharmacological treatment with L-3,4-dihydroxyphenylalanine (L-DOPA) in living brain tissue in real-time. NO is a highly reactive free radical reactive nitrogen species (RNS) that has been implicated in the pathogenesis and progression of PD. NO quickly reacts to produce more aggressive RNS such at peroxynitrite that interrupt normal cell signaling and ultimately lead to cell death. In this study, FSCV at carbon-fiber microelectrodes was used to detect NO in vitro with a well-established waveform. The use of Nafion-coated carbon-fiber microelectrodes enhanced the detection of NO. Future studies examining NO with FSCV should focus on optimizing FSCV parameters to better-resolve the voltammetric signature of NO in addition to establishing selectivity against common interferents both in vitro and in brain slice tissue. In the future, this approach will enable the detection of NO in living tissue capable of addressing the role of RNS such as NO, in oxidative stress in PD. The effects of L-DOPA on DA dynamics from single pulse electrical stimulations, as well as artificial phasic stimulation paradigms in striatal brain slices were assessed. In the presence of 50 μM L-DOPA the ability to detect DA at Nafion-coated carbon-fiber microelectrodes in vitro was hindered and caused irreversible fouling of the electrode. A more clinically relevant dose of L-DOPA (1 μM) significantly increased electrically-evoked DA release at Nafion-coated carbon-fiber microelectrodes in a phasic firing paradigm. Release and reuptake parameters of DA by way of the DA transporter (DAT) were assessed and DA reuptake was decreased following a more clinically relevant dose of L-DOPA. This work builds on the understanding DA release and reuptake dynamics following L-DOPA treatment and will provide the foundation to assess the effects of L-DOPA on DA release and reuptake parameters in Parkinsonian animals to build a more complete picture of the chemical dynamics underlying neurodegenerative disease states such as PD. © Copyright 2013 by Stephanie Cristina Hughes All Rights Reserved Electrochemical Studies of Neuroactive Molecules with Fast-Scan Cyclic Voltammetry at Carbon-Fiber Microelectrodes by Stephanie Cristina Hughes A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science