The goals of this research proposal are to develop and use electrochemistry-based probes to monitor neurotransmitters in intact brain tissue. This work is motivated by the belief that monitoring the concentration changes of neurotransmitters in real time provides the most direct way to understand the processes that regulate and control neuronal communication. The neurotransmitter target molecules are dopamine, 5-hydroxytryptamine, and norepinephrine. These molecules are electroactive and are thus a natural target for electrochemical methods. In addition, the three molecules are vital to normal brain function. The information gained by observing these molecules undergoing their job as neurotransmitters will enable their role and their regulation in the brain to be more clearly understood. Furthermore, since real time information is not available for any other neurotransmitters in intact tissue, the information gained will help establish the ways in which neurotransmitters interact with neurons. In addition, we propose to measure local oxygen concentrations and pH. These are both indices of metabolic activity and are closely coupled to neurotransmitter activity. The specific plans of the proposed research are: 1. To improve the response time of carbon-fiber electrodes to neurotransmitter changes. Adsorption of neurotransmitters on carbon-fiber microelectrodes contributes to the sensitivity of these probes in vivo, but it also decreases response time. We propose to characterize the kinetics and mechanism of the adsorption and to minimize the temporal distortion. 2. Design of carbon-fiber surfaces for catecholamine detection. Two surface modification schemes are proposed to improve sensitivity and response time. Carbon surfaces will be exposed to activated carbon to remove impurities and will be covered with quinones. Both methods have been successfully used at large glassy carbon electrodes to improve responses to these neurotransmitters. 3. Probing the regulation of extracellular dopamine. The function of dopamine autoreceptors will be probed to distinguish between autoreceptors regulation of synthesis and release. In addition, we will probe the mechanism of the dopamine transporter in intact tissue. 4. Probing extracellular regulation of norepinephrine. Voltammetric techniques will be used to measure extracellular norepinephrine, an important neurotransmitter in the CNS. The rates of release, uptake, and extrasynaptic diffusion will be studied in several regions of the mouse brain. 5. In vivo electrochemical signals and local blood flow. The carbon fiber electrode can be used to detect extracellular 02 and pH in the brain. 02 levels are related to local blood flow. In this work, we plan to probe the relation between local pH and these other two indices. Our hypothesis is that these measures provide a general view of neuronal activation.
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