The Pennsylvania State University The Graduate School Department of Chemistry IN VIVO ELECTROCHEMICAL MEASUREMENTS IN DROSOPHILA MELANOGASTER A Dissertation in Chemistry by Monique Adrianne Makos © 2010 Monique Adrianne Makos Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2010 The dissertation of Monique Adrianne Makos was reviewed and approved* by the following: Andrew G. Ewing Professor of Chemistry J. Lloyd Huck Chair in Natural Science Dissertation Advisor Chair of Committee Mary Elizabeth Williams Associate Professor of Chemistry Christine D. Keating Associate Professor of Chemistry Richard W. Ordway Associate Professor of Biology Michael L. Heien Assistant Professor of Chemistry Barbara J. Garrison Shapiro Professor of Chemistry Head of the Department of Chemistry *Signatures are on file in the Graduate School ii Thesis Abstract Carbon-fiber microelectrodes coupled with electrochemical detection have been extensively used for the analysis of biogenic amines. In order to determine the functional role of amines, in vivo studies have primarily used rats and mice as model organisms. This thesis concerns the development of an electrochemical detection method for in vivo measurements of dopamine in the nanoliter-sized adult Drosophila melanogaster central nervous system (CNS). A cylindrical carbon-fiber microelectrode was placed in a fly brain region containing a dense cluster of dopaminergic neurons while a micropipet injector was used to exogenously apply dopamine to the area. Changes in dopamine concentration in the fly were monitored in vivo with background-subtracted fast-scan cyclic voltammetry (FSCV). Distinct differences were found for the clearance of exogenously applied dopamine by the dopamine transporter in the brain of a wild-type fly vs. a mutant fly lacking dopamine transporter function. The measured current response due to oxidation of dopamine at the electrode surface increased significantly for wild- type flies following treatment with cocaine which is a known dopamine uptake blocker. The current remained unchanged for mutant flies under the same conditions. These results demonstrate the validity of using this novel analytical technique to monitor dopamine uptake in Drosophila. The in vivo method described in this thesis has been used to study mechanisms that underlie drug addiction from a physiological perspective. In addition to being a valuable tool for the analytical chemistry field, this work is of significant interest to the neuroscience community. Dopamine neurotransmission is believed to play a critical role in addiction reinforcing mechanisms of drugs of abuse. Little is known about the in vivo iii nature of drug interactions with invertebrate transporters, mainly because of the lack of techniques available for quantifying neurochemicals in such small native environments. Hence, the effects of several psychostimulants on dopamine clearance in the Drosophila melanogaster CNS have been investigated with in vivo electrochemical detection. FSCV was used to quantify changes in dopamine concentration in the fly brain when cells were exposed to cocaine, amphetamine, methamphetamine, or methylphenidate. Clearance of exogenously applied dopamine was significantly decreased in the wild-type fly following all drug treatments. In contrast, dopamine uptake remained unchanged when identical treatments were employed in mutant flies lacking functional dopamine transporters. Although the understanding of the complex actions of cocaine in the brain has improved, an effective drug treatment for cocaine addiction has yet to be found. During the last decade, methylphenidate has been investigated as a potential medication for cocaine addiction treatment. Methylphenidate binds the dopamine transporter and increases extracellular dopamine levels in the CNS similar to cocaine but is thought to elicit fewer addictive and reinforcing effects. Several studies that have investigated the effects of oral methylphenidate taken by cocaine users have reported mixed results. I utilized the Drosophila model system to investigate the mechanism behind treating cocaine addiction with methylphenidate. The results suggested oral consumption of methylphenidate sufficiently blocks the Drosophila dopamine transporter, and further inhibition of the transporter by cocaine applied directly to the brain was undetectable. These data highlight the possibility that methylphenidate could be used as a treatment for cocaine addiction and demonstrate the great potential of Drosophila as a model system for future drug abuse research. iv Chemical, electrical, and optogenetic methods to stimulate dopamine release in the adult Drosophila CNS with FSCV detection were investigated. The results suggested that the noninvasive optogenetic stimulation method is capable of initiating targeted neurochemical release in the Drosophila CNS. Dopamine release has been shown to cause pH fluctuations in the rat brain which can interfere with electrochemically measured signals; therefore, a pH sensor was developed for use in the fly. The fabrication and characterization of a novel voltammetric pH microelectrode sensor is described. This sensor has been used to detect pH changes in Drosophila associated with in vivo neurotransmitter release. Voltammetric pH sensors measure changes in the redox-potential of a surface-bound, electrochemically active species as a function of pH. While this approach to measuring pH has been demonstrated with a variety of quinone-modified electrodes, up until now, none have been developed with biocompatible materials that exhibit activity on a physiological time scale in a relevant pH range. Voltammetric reduction of the commercially available diazonium salt Fast Blue RR (FBRR) onto the carbon-fiber surface provided a one-step, reagentless procedure for surface modification of a carbon-fiber microelectrode. This produced a 5-µm diameter sensor with a pH-sensitive quinone molecule covalently bonded to the carbon surface. FSCV was used to probe the redox activity of the FBRR molecule as a function of pH. Calibration of the sensor in solutions ranging from pH 6.5 to 8.0 resulted in a linear pH- dependent anodic peak potential response. Flow-injection analysis was used to characterize the modified microelectrode which responded to acidic and basic changes as low as 0.005 pH units in < 2 s. The long-term stability of the FBRR microelectrode pH v sensor was tested by continuously applying potential to electrodes in pH 7.5 physiological saline solution for 2.5 h (corresponding to 45,000 voltammetric sweeps). This is an ample time window for in vivo electrochemical measurements in Drosophila melanogaster. Furthermore, the pH sensor was successfully used to measure dynamic pH fluctuations in vivo following dopamine release in the nanoliter-sized CNS of Drosophila. The results obtained from the analytical tools developed for in vivo detection of dopamine and pH changes in the fly suggest the validity of using Drosophila as a model system to study neurotransmission. vi Table of Contents List of Figures...................................................................................................................ix List of Tables...................................................................................................................xii List of Schemes...............................................................................................................xiii Abbreviations..................................................................................................................xiv Acknowledgements.........................................................................................................xvi Chapter 1: Chemical Measurements in Drosophila.......................................................1 Introduction.............................................................................................................2 Detection Methods for the Analysis of Drosophila Homogenates........................6 Analytical Techniques for Measuring the Physiology of Intact Flies..................16 Scope of the Thesis................................................................................................23 References..............................................................................................................26 Chapter 2: In Vivo Electrochemical Measurements of Exogenously Applied Dopamine in Drosophila melanogaster...........................................................................31 Introduction............................................................................................................32 Methods..................................................................................................................34 Results and Discussion...........................................................................................37 Conclusions.............................................................................................................51 References...............................................................................................................52 Chapter 3: Using In Vivo Electrochemistry to Study the Physiological Effects of Cocaine and Other Stimulants on the Drosophila melanogaster Dopamine Transporter......................................................................................................................55 Introduction...........................................................................................................56