In Vivo Electrochemical Measurements In

The Pennsylvania State University

The Graduate School

Department of Chemistry

IN VIVO ELECTROCHEMICAL MEASUREMENTS IN

DROSOPHILA MELANOGASTER

A Dissertation in

Chemistry

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

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.

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.

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 Methods...... 58 Results and Discussion...... 60 Conclusions...... 77 References...... 79

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Chapter 4: Oral Administration of Methylphenidate Blocks the Effect of Cocaine on Uptake at the Drosophila Dopamine Transporter...... 83 Introduction...... 84 Methods...... 87 Results and Discussion...... 89 Conclusions...... 97 References...... 98

Chapter 5: Methods for Stimulating Dopamine Release in the Drosophila CNS....103 Introduction...... 104 Methods...... 109 Results and Discussion...... 112 Conclusions...... 123 References...... 124

Chapter 6: Development and Characterization of a Voltammetric Carbon-Fiber Microelectrode pH Sensor...... 129 Introduction...... 130 Methods...... 132 Results and Discussion...... 135 Conclusions...... 149 References...... 152

Chapter 7: Future Directions for Quantifying Neurochemicals in Drosophila Using Electrochemical Detection...... 155 Investigating Alcohol Addiction with Drosophila...... 156 Quantifying the Kinetics of Dopamine Uptake in Drosophila...... 160 Improving the Detection of Stimulated Dopamine Release in Drosophila...... 163 References...... 166

Appendix...... 169

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List of Figures

Figure 1.1. Drosophila brain regions...... 3

Figure 1.2. MEKC-EC separations of homogenates from Drosophila...... 9

Figure 1.3. Mass spectrometric measurements of the Drosophila proteome...... 13

Figure 1.4. Microfluidic device for the analysis of Drosophila embryos...... 18

Figure 1.5. Measurements in Drosophila larvae following optogenetic stimulation...... 21

Figure 1.6. Investigating dopamine transporter function in adult Drosophila...... 24

Figure 2.1. Images of Drosophila taken during microsurgery...... 39

Figure 2.2. Confocal fluorescence micrographs of intact brains from adult transgenic TH-GAL4/UAS-GFP flies...... 41

Figure 2.3. Exogenously applied 1.0 mM dopamine detected in vivo in an adult wild- type fly...... 43

Figure 2.4. Voltammetric detection of exogenously applied dopamine solutions in the PAM area of an adult Drosophila brain...... 45

Figure 2.5. Effect of cocaine on dopamine uptake...... 47

Figure 2.6. Effect of TTX on dopamine uptake...... 50

Figure 3.1. In vivo detection of exogenously applied 1.0 mM dopamine in the adult Drosophila brain...... 62

Figure 3.2. Effect of 1.0 mM cocaine treatment on uptake of an exogenously applied 1.0 mM dopamine solution...... 64

Figure 3.3. Investigating dopamine transporter function...... 66

Figure 3.4. Comparison of wild-type and fmn mutant flies when 1.0 mM dopamine was exogenously applied before and after 1.0 mM cocaine treatment...... 67

Figure 3.5. Determining the physiological APAP concentration in the Drosophila CNS from a 1.0 mM APAP bath application...... 69

Figure 3.6. Comparison of wild-type and fmn mutant flies when 1.0 mM dopamine was exogenously applied before and after 10 min of various concentrations of cocaine treatments...... 71 ix

Figure 3.7. Comparison of wild-type and fmn mutant flies when 1.0 mM dopamine was exogenously applied before and after 1.0 stimulant treatment...... 74

Figure 4.1. Effect of orally consumed methylphenidate on cocaine inhibition of the dopamine transporter in the adult Drosophila brain...... 91

Figure 4.2. Effect of orally consumed methylphenidate on Drosophila dopamine transporter function...... 93

Figure 4.3. Comparison of dopamine concentration in the Drosophila CNS following drug treatments...... 96

Figure 5.1. Cartoon depiction of the effects of blue light exposure on neurons expressing Channelrhodopsin-2...... 108

Figure 5.2. Schematic comparing three methods for stimulating neurotransmitter release in adult Drosophila...... 114

Figure 5.3. Effect of blue light stimulation on flies with genetically altered dopamine neurons...... 118

Figure 5.4. Voltammograms obtained during blue and red light stimulation of a TH- GAL4/UAS:ChR2 mutant fly...... 120

Figure 5.5. Spontaneous release of an electroactive species from a TH- GAL4/UAS:ChR2 mutant fly...... 122

Figure 6.1. Cyclic voltammograms of a carbon-fiber microelectrode before and after FBRR attachment...... 137

Figure 6.2. Electrochemical characterization of the FBRR microelectrode pH sensor in pH 7.5 AHL saline solution...... 143

Figure 6.3. Cyclic voltammograms of a microelectrode modified with FBRR in AHL saline solutions of different pH...... 145

Figure 6.4. The anodic peak potential as a function of AHL saline solution pH for FBRR-modified electrodes...... 146

Figure 6.5. Plot of anodic peak potential vs. time during flow injection changes of 0.2 pH units in AHL saline...... 148

Figure 6.6. Physiological pH measurements in an adult Drosophila CNS...... 150

Figure 7.1. The fly inebriometer...... 159

Figure 7.2. Modeling dopamine uptake...... 162

Figure 7.3. Voltammetric measurements of dopamine using an applied waveform of 1.0 V vs. a waveform extended to 1.4 V...... 165

List of Tables

Table 3.1. Change in [DA]max for four drugs of abuse...... 76

Table 5.1. Eliciting dopamine release via chemical stimulation...... 106

Table 6.1. Effect of varying voltammetric deposition parameters for FBRR reduction onto a carbon-fiber surface...... 139

Table 7.1. Drosophila mutants that display altered behavioral responses to ethanol....158

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List of Schemes

Scheme 6.1. Electrochemical deposition of FBRR salt onto the carbon-fiber microelectrode surface...... 136

Scheme 6.2. Proposed mechanism for the oxidation-reduction reaction of the surface- bound quinone derivative of FBRR...... 141

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Abbreviations

ACN: acetonitrile ADHD: attention deficit hyperactivity disorder AHL: adult-hemolymph like ANOVA: analysis of variance APAP: N-acetyl-p-aminophenol, acetaminophen CAT: catechol CE: capillary electrophoresis ChR2: Channelrhodopsin-2 CNS: central nervous system [DA]max: peak dopamine concentration DHBA: dihydroxylbenzylamine dsRNA: double-stranded RNA Epa: anodic peak potential Epc: cathodic peak potential FBRR: Fast Blue RR fmn: fumin (Drosophila mutant) FSCV: fast-scan cyclic voltammetry GABA: γ-aminobutyric acid GAL4/UAS: galactosidase-4-upstream activating sequence GFP: green fluorescent protein HPLC: high-performance liquid chromatography IC50: half maximal inhibitory concentration ISMs: ion-selective microelectrodes LC: liquid chromatography LC-IMS-MS: liquid chromatography-ion mobility spectrometry-mass spectrometry L-DOPA: L-3,4-dihydroxyphenylalanine LED: light-emitting diode LOD: limit of detection MALDI-TOF: matrix-assisted laser desorption ionization time-of-flight MB: mushroom body MEKC: micellar electrokinetic chromatography MEKC-EC: micellar electrokinetic chromatography with electrochemical detection mRNA: messenger RNA na5-HT: N-acetyl serotonin naDA: N-acetyl dopamine naOA: N-acetyl octopamine naTA: N-acetyl tyramine NMDA: N-methyl-D-aspartate QTOF: quadrupole time-of-flight PAM: protocerebral anterior medial PBS: phosphate-buffered saline PI: propidium iodide

xiv

RISC: RNA-induced silencing complex RNA: ribonucleic acid RNAi: RNA interference SDS: sodium dodecyl sulfate SEM: standard error of the mean siRNA: small interfering RNA S/N: signal-to-noise TEABF4: tetraethylammonium tetrafluoroborate TES: N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid TH: tyrosine hydroxylase TTX: tetrodotoxin UAS: upstream activating sequence VNC: ventral nerve cord

Acknowledgments

Graduate school has been a marathon, and I would like to acknowledge several people for their contributions to my run. While I have come into contact with many great scientists during the last five years, without two scientists in particular this thesis work would not have been accomplished. I would like to thank Andy Ewing for the standards he set for me. He expected no less than my personal best work, and I am a better scientist for it. In addition, he has been an example of how to work with people to attain a desired goal, which is knowledge I will carry with me long after I forget how to dissect a fruit fly.

I would like to thank Michael Heien for his significant contributions to my graduate school education. Without his technical skills in lab and his assistance with writing draft after draft (after draft) of papers, much of this thesis simply would not exist. My research was financially supported by National Institutes of Health Grant 5R01GM078385-02.

On a personal note, I would like to thank my Dad, my Sister, and my Grandma for their encouragement. My Mom especially has been a role model for my educational endeavors, as well as for all aspects of my life. If one day I can possess just half her ability to overcome life’s ups and downs, then I will consider myself successful indeed.

My education at PSU has given me the opportunity to meet two special people.

Donna Omiatek started as the labmate whose chair I was constantly backing up into with my own, but has since become my best friend. She has picked me up on many a rough day both inside and outside of the lab. Matthew Pond began as one of my many classmates, but will continue to be part of my life where ever I go. I appreciate his interest in my work here at PSU, as well as his support of my future goals.

Lastly, to Drosophilia...may you rest in peace.

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Chapter 1: Chemical Measurements in Drosophila*

*Reproduced with permission from Makos, M. A., Kuklinski, N. J., Berglund, E. C., Heien, M. L., and Ewing, A. G. (2009) Chemical Measurements in Drosophila, TrAC, Trends Anal. Chem. 28, 1223-1234. © 2009 Elsevier.

1 Introduction

Drosophila melanogaster has been extensively used as a model organism in genetics research and significantly contributed to the molecular, cellular, and evolutionary understandings of human behavior. Originally pioneered by Thomas H.

Morgan at the beginning of the last century, research utilizing the fruit fly has led to important insights into the mechanisms of human developmental and physiological processes. Recently, research has focused on developing analytical methods to obtain highly sensitive chemical quantification along with spatiotemporal information of

Drosophila (1). The fly matures relatively quickly, developing from an embryo, to larva

(divided into 1st, 2nd, and 3rd instar larva stages), to pupa, to a sexually mature adult in a span of ~12 days. An adult fly brain is approximately 5 nL in volume and comprised of several distinct structures which control specific tasks (Figure 1.1A) (2). The small dimensions are a challenge for researchers attempting chemical quantification in the fly and necessitate the use of techniques capable of handling mass-limited samples.

Although the adult fly has a more simple nervous system when compared to vertebrates, it is capable of higher-order brain functions including both aversive and appetitive learning and recalling learned information from prior experiences (3, 4). In addition,

Drosophila larvae can be used as a model for investigating basic neurotransmission and chemosensory pathways (5). Conservation between the Drosophila and mammalian proteomes is high with approximately half the protein sequences in the fly having similar counterparts in the human sequence (6). Many central nervous system (CNS) pathways are evolutionarily conserved between the two species because of the genetic similarity.

Figure 1.1. Drosophila brain regions. (A) A polygonal model of the Drosophila melanogaster brain. Major neuropil regions are highlighted in color (brown = mushroom body; beige = lateral horn; blue = antennal lobe; green = central complex; red = medulla; orange = lobula; yellow = lobula plate). (B) Tyrosine hydroxylase immunolabeling showing dopaminergic neuron patterns in multifocal confocal views of adult fly brain. (Reprinted from (7, 8), with permission from Elsevier and the Society for Neuroscience).

3 Neurochemical basis for observed behaviors. Studies of neurotransmitters in the CNS are underway in Drosophila to elucidate the roles of neurochemicals in human behavior.

Biogenic amines, namely dopamine, serotonin, and tyramine, are known to be involved in physiological processes found in both mammalian and Drosophila systems (9-11). For example, dopamine has been implicated in human and fly behaviors such as reward and motivation, sleep cycles, alcohol tolerance, and sensitivity to addictive drugs (12-14). In addition, the neurotransmitter octopamine is thought to control many behaviors in the fly that norepinephrine regulates in mammals (15). This evidence suggests many of the neurotransmitter systems that regulate behavior are comparable between mammals and

Drosophila.

Genetic manipulation for chemical analysis and behavioral studies. The Drosophila proteome was one of the first species with a fully sequenced genome (16). The process of producing mutants to display a desired behavior via genetic manipulation is a relatively straightforward task with the fruit fly. The Drosophila genome contains little genetic redundancy, or multiple genes performing the same biochemical function, which facilitates identification of individual genes and molecules that influence a particular behavior (2, 17). Many complex behavioral patterns found in mammalian systems with regards to learning and memory, courtship, alcohol tolerance, and circadian rhythms have been studied in the fruit fly through the use of genetic mutants (17-20).

Controlling genetic mutations in Drosophila is possible with the galactosidase-4- upstream activating sequence (GAL4/UAS) system. The landmark development of the

GAL4/UAS system by Brand and Perrimon in 1993 allows for the rapid generation of flies containing targeted gene expression (21). Briefly, GAL4 is a gene that encodes for

4 the yeast transcription activator protein Gal4 and can be expressed in various subsets of fly tissue. Thousands of GAL4 driver or enhancer lines have been created that direct transcription to different regions and/or types of cells in the fly (22). For instance, the

TH-GAL4 driver line produces flies with GAL4 only in neurons where tyrosine hydroxylase (TH) is present (23). GAL4 remains inactive in the fly until it binds an

UAS. Many UAS responder lines have been created to contain the UAS region by a desired protein like the green fluorescent protein (GFP). For example, the UAS-GFP responder line can be crossed with the TH-GAL4 driver line to produce flies with GFP transcription in their TH-containing neurons (24). Because TH is the enzyme involved in the rate limiting step of dopamine synthesis, TH-GAL4 targets GFP expression in dopamine neurons. This allows for the visualization of dopaminergic neurons in

Drosophila. Tools for fluorescent immunodetection of specific neuron clusters in the fly brain are available as well. Figure 1.1B is an example of a fly brain image utilizing TH immunolabeling.

Drosophila mutants have been successfully used to model several human neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and

Huntington’s disease. These diseases are characterized by the late onset of progressive neurodegeneration and/or formation of abnormal neuronal inclusions or protein aggregates (25, 26). While genetic mutants have helped in linking particular genes to a specific disease, little is known about the mechanisms leading up to these pathologies.

The ability to quantify all neuropeptides, amino acids, and neurotransmitters in

Drosophila is a goal researchers are moving towards. Obtaining spatiotemporal information along with chemical quantification will provide a more analytical view of

5 Drosophila and could lead to a better understanding of the physiological mechanisms that underlie human behaviors, addictions, and neurodegenerative diseases.

Detection Methods for the Analysis of Drosophila Homogenates

Techniques that are used to separate and quantify mass-limited samples include capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), and mass spectrometry. Indeed, these methods are sensitive and selective making them capable of measuring and identifying multiple compounds in a complex biological sample. This ability allows the determination of different neurochemicals that are within the brain which is crucial to understanding changes throughout disease states.

Historically, analytical techniques have used homogenate methods to contend with the hard, exterior cuticle of the fly. Whole fly heads are easily pulverized using small tissue grinders; however, significant matrix effects from whole fly head samples can interfere with quantification. Another approach is to dissect the brains from the head by hand prior to homogenization. Unwanted signals from the fly head matrix are reduced, but preparation is more time consuming and requires knowledge of dissection techniques.

Capillary electrophoresis. CE separates ionic species according to their electrophoretic mobilities by applying a voltage over a narrow capillary filled with electrolytic solution.

The small injection volumes associated with CE (nanoliters to femtoliters) make it an excellent method to study volume-limited samples such as Drosophila (27, 28).

Moreover, CE has high resolving power due to its plug-like flow and minimal diffusion.

Neutral molecules can be separated with CE by utilizing a surfactant to carry out micellar

6 electrokinetic chromatography (MEKC). In MEKC, adding the surfactant sodium dodecyl sulfate (SDS) to the running buffer at levels above the critical micelle concentration results in the formation of micelles. The interaction of neutral molecules with the charged micelles causes retention in the capillary which can lead to the separation of neutral molecules.

The Ewing laboratory has developed a procedure using MEKC to measure and quantify biogenic amines, their metabolites, and their precursors in Drosophila. End- column amperometry is used to selectively detect electroactive species providing a simple and sensitive detection method without the need for derivatization. Using MEKC coupled to electrochemical detection (MEKC-EC), different anatomical regions of

Drosophila have been investigated including whole body homogenates (29), whole head homogenates (29), single head homogenates (27), and more recently dissected brains.

Pioneering work by Ream et al. attempted to identify neurotransmitters in

Drosophila using MEKC which resulted in the identification of four species: dopamine, tyramine, serotonin, and the dopamine precursor L-3,4-dihydroxyphenylalanine (L-

DOPA) (29). Migration times from standards obtained both before and after the fly sample were used for peak identification as well as normalization to the migration time of an internal standard, dihydroxylbenzylamine (DHBA), to compensate for possible peak drifting. A collection of either heads or bodies (thoraces and abdomens) were homogenized and separated using TES (N-tris(hydroxymethyl)methyl-2- aminoethanesulfonic acid)/SDS buffer. A higher abundance of dopamine in samples from the body was observed when compared to the head only. This may be a consequence of dopamine being a main component in sclerotization (hardening) of the

7 cuticle of the fly body. The levels of L-DOPA remained unaltered between the two fly preparations. Furthermore, the levels of serotonin and tyramine were found to be higher in the head with tyramine levels close to the limit of detection in the body.

The type of buffer used affects the resolution of separations of the biogenic amines. By using MEKC and a 25 mM borate/SDS buffer instead of TES/SDS, additional monoamines and metabolites were separated and identified (30). Borate (at basic pH) forms a complex with analytes possessing vicinal hydroxyl groups, imparting negative charge to the complex, which made it possible to separate a standard of 14 neurochemicals. Here, catechol (CAT) was used for the internal standard. In addition to previously identified molecules, dopamine, tyramine, L-DOPA, and octopamine were identified in homogenized samples from Drosophila heads. The N-acetylated metabolites N-acetyl dopamine (naDA), N-acetyl octopamine (naOA), and N-acetyl serotonin (na5-HT) were identified as well. The excellent separation ability of the borate/SDS buffer with MEKC-EC was demonstrated by comparing electropherograms from wild-type Drosophila to a mutant form, inactive, which expresses lower levels of octopamine and tyramine. As expected, the amounts of naOA, tyramine, and octopamine were reduced in the mutant vs. the wild-type fly, with tyramine being present at levels below the limit of detection in the mutant (Figure 1.2A).

The small sample volumes that can be analyzed using CE allow the study of the variability within a population of flies that arises from individual fly-to-fly differences.

This is accomplished by analyzing one fly head at a time (27). Following homogenization of a single fly head in 250 nL of perchloric acid, three individual fly

Figure 1.2. MEKC-EC separations of homogenates from Drosophila. (A) Enlarged portion of an electropherogram (left) includes peaks naOA (2), naDA (3), na5-HT (5), octopamine (6), dopamine (8). Electropherogram (right) compares wild-type (WT, black trace) and mutant (inactive, blue trace) head homogenates emphasizing the internal standard CAT (11) and tyramine (9). Separation was run with borate buffer. There is not a detectable level of tyramine in the mutant form. (B) Electropherogram of a single head with TES running buffer highlighting L-DOPA (1), naOA (2), naDA (3), naTA (4), na5- HT (5), octopamine (6), DHBA (7), dopamine (8), serotonin (10). Tyramine (9) is not visible on this scale. (C) Electropherogram of hand dissected brain where naTA (4) and CAT (11) are visible. The working electrode was held at +750 mV vs. a Ag/AgCl reference electrode for all separations. (Reprinted from (27, 30), with permission from the American Chemical Society).

9 heads were analyzed and compared. This procedure resulted in reproducible identification of nine neurochemicals (Figure 1.2B), including N-acetyl tyramine (naTA).

Despite the resolving power of MEKC, unidentified electroactive species coelute with some neurochemicals. To reduce this problem, dissected Drosophila brains can be separated using borate/SDS buffer (Figure 1.2C). By removal of fly components thought to contain electroactive molecules (e.g., the cuticle, antennae, and eyes) the electropherogram becomes easier to interpret. This is observed when comparing Figure

1.2A, B with Figure 1.2C, where the number of large, overloading, unidentified peaks is reduced in the single brain electropherogram. In addition, the dopamine from the cuticles is not measured which allows the amount of dopamine in the CNS of the fly to be determined.

High-performance liquid chromatography. HPLC has been used to quantify the amount of biogenic amines, their metabolites, and their precursors in the Drosophila

CNS. The aim of these studies was to determine the function of molecules and their localization within the fly head. HPLC is an improved form of column chromatography where solvent is pushed though the column under high pressures (up to 40 MPa). The high pressure allows for faster separation times and smaller column particles, yielding improved resolution. Typically, a C-18 column with an acidic mobile phase and electrochemical detector has been used to separate and detect compounds (31-35).

Early reports using HPLC demonstrated the separation and quantification of dopamine, L-DOPA, and α-methyldopa in 1-4 week old brains and retinas of wild-type flies and ebony mutant flies, which have a darker pigment and impaired vision (31).

Although the levels of all three analytes were variable over time, the authors did report

10 that these analytes were more abundant in the retina than in the brain and more abundant in the heads of mutant ebony flies than the heads of wild-type flies.

Hardie and Hirsh expanded the number of neurotransmitters analyzed with HPLC by quantifying dopamine, octopamine, tyramine, and serotonin in the brains and whole heads of Drosophila white-eyed (white) mutants (32). They noted that nearly 75% of the total dopamine within the white mutant head is located outside of the brain. In contrast, the percentages of octopamine, tyramine, and serotonin present outside of the brain range from only 1 to 37% when compared to the amount in the brain. The quantitative nature of HPLC has been utilized to examine the role of tyramine in cocaine sensitization studies of the inactive and the TβHM18 Drosophila mutants (10). The inactive mutant, named for the low activity level of the mutant flies, was found to have approximately

60% less tyramine than wild-type flies, despite similar levels of dopamine. While these mutants displayed expected behavioral responses to cocaine upon their first exposure, with repeated cocaine exposure minimal behavioral sensitization to cocaine was observed. The TβHM18 line has a null mutation in the gene that codes for tyramine β- hydroxylase, the enzyme used to convert tyramine into octopamine. TβHM18 mutant flies were found to have almost an order of magnitude greater amount of tyramine and near- normal cocaine sensitization when compared to the wild-type fly, ruling out octopamine as the biogenic amine contributing to this cocaine sensitization. These two comparisons showed that tyramine plays a critical role in cocaine sensitization and later helped to confirm the identity of two tyrosine decarboxylase genes (33).

The location and quantification of biogenic amines within the brain of genetic fly mutants has been further investigated by the Meinertzhagen laboratory. They developed

11 a method in the fly to quantify histamine (34), a transmitter known to be located in the eyes of the fly, and compared it, along with the amount of dopamine and serotonin, in white, brown, and scarlet mutants which are flies with three different eye-pigment mutations (35). Since scarlet and brown are the two pigments that control fly eye color, knocking out one pigment results in a fly with the other eye color, and a knockout of both pigments results in no eye pigment, white mutants. They measured a significant decrease

(in some cases over 50%) in the neurotransmitters of all three Drosophila mutants when compared to wild-type flies. Similar trends were observed in comparisons of wild-type vs. white mutant houseflies, blowflies, and two species of the flesh fly, signifying that many effects attributed to a mutant gene isolated in a white fly might be from the loss of pigment itself and not the mutated gene. They also noted that in separations of wild-type fly head homogenates, 71% of the total dopamine in the head was found in the brain, in contrast to the results reported by Hardie and Hirsh for white mutant flies.

Mass spectrometry to study proteins and peptides. Mass-spectrometric studies of the

Drosophila proteome have used a variety of methods including matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (36, 37) and ion- trap mass spectrometry (38). In addition, a separation step is often added to the analysis including reverse-phase liquid chromatography (38, 39), ion-mobility spectrometry (40-

42), or strong-cation-exchange chromatograpy (39, 42). The number of genes, transcripts, and proteins that have been observed within the adult Drosophila are summarized in Figure 1.3A.

Initial proteomic methods have been used to understand the basic biology of

Drosophila. Figure 1.3B shows a map by Taraszka et al. of the proteomes from three

Figure 1.3. Mass spectrometric measurements of the Drosophila proteome. (A) Venn diagram of the known adult Drosophila genome (thin black circles and numbers), mRNA transcripts (thin grey circles and numbers), proteome (thick grey circles and bold numbers), and the overlap between mRNA transcripts and proteome (bold black italics numbers). Circle size corresponds to the number of known genes, transcripts, and proteins listed below the circle. (B) LC-IMS-MS analysis of three digested individual flies. Many of these features are common within all three individuals but some examples of the differences have been labeled. Circled features designate peptides found in all three individuals, boxes only two individuals, and triangles only one individual. (Reprinted from (40, 41), with permission from the American Chemical Society).

13 individual Drosophila heads identifying 197 proteins and found at least 101 proteins present in all three samples (40). The other 96 proteins might not be expressed in every sample, or the flies could have been using different proteins at the time of sacrifice.

More globally, differences have been observed in the Drosophila proteome lifespan. The fly proteome has been investigated over sixty days, at seven day increments (42).

Approximately 1700 different proteins were identified and their changes in regulation compared between three different age groups: young (1-21 day old flies), middle (22-42 day old flies), and old (43-60 day old flies). Of these comparisons, a significant difference in protein regulation was observed for the young vs. middle-aged groups.

When the proteins experiencing an order of magnitude change or more in abundance were considered, 30 proteins were down-regulated while 12 proteins were up-regulated in the middle-aged group. These proteins were found to be associated with metabolism, development, reproduction, or defense response.

Proteomic methods utilizing Drosophila have yielded insight into Parkinson’s disease. Flies expressing either mutated A30P (39), mutated A53T (43), or normal human α-synuclein genes (38) all display symptoms of Parkinson’s disease and have been investigated. Symptoms include decreased locomotor ability, formation of Lewy body-like inclusions in the brain, and degeneration of dopaminergic neurons with age.

The symptoms are most severe for the flies with the A30P point mutation, followed by the A53T point mutated flies, and lastly the normal human α-synuclein mutated flies.

The three mutant fly types have had their proteomes compared to wild-type flies. Of note, the levels of 49 proteins in the A30P flies and 24 proteins in the A53T flies were significantly altered. Most of these proteins are associated with the actin cytoskeleton,

14 mitochondria, and membrane. In the normal human α-synuclein mutant flies, only 12 protein changes were observed, mostly related to metabolism and cellular signaling.

These protein changes correlate with the severity of the Parkinson’s symptoms seen in the mutated flies and might lead to general insight about the protein alterations associated with this disease.

Complimentary to the genomic and proteomic work, Drosophila neuropeptides have been investigated with MALDI-TOF mass spectrometry. Predel et al. characterized the adult fly peptidome with this technique and were able to identify 32 neuropeptides in the Drosophila CNS (37). Not only did this reveal the occurrence of these neuropeptides, but it also depicted their morphological distribution. Recently, Kravitz and coworkers improved upon this method by combining both MALDI-TOF mass spectrometry and electrospray ionization quadrupole time-of-flight (QTOF) mass spectrometry. Using the

Drosophila GAL4-UAS system for targeted gene expression, subsets of cells were genetically labeled to aid in sample preparation. They were able to identify 42 neuropeptides encoded by 18 different genes in adult Drosophila brain extract (44).

The larval Drosophila peptidome has been investigated with both one- and two- dimensional (1D and 2D) capillary liquid chromatography (LC) followed by QTOF mass spectrometry. Baggerman et al. identified 38 peptides using the 2D technique vs. 28 peptides using the 1D technique (45, 46). Their results demonstrate the increased efficiency of 2D LC/QTOF over its 1D counterpart for Drosophila larvae.

Yew, Cody, and Kravitz studied Drosophila cuticular pheromones with real-time mass spectrometry using atmospheric pressure ionization (47). This technique can provide near instantaneous analysis of samples, and pheromones can be chemically

15 investigated over a long period of time from live, awake Drosophila. Flies were immobilized by a vacuum applied through a pipette tip and probed with a metal pin attached to a micromanipulator. This allowed the fly to interact behaviorally with surrounding flies. Pheromone levels were found to be increased in females vs. males, in females after courtship, and as one moves closer to the genitals of the male fly. While this work shows the spatial and temporal resolution of atmospheric pressure mass spectrometry, it does lack the ability to measure analytes from inside of the fly.

Analytical Techniques for Measuring the Physiology of Intact Flies

Recently, analytical methods have been developed to record chemical measurements in real-time from Drosophila larvae and from adult Drosophila. The ability to acquire direct physiological information will help bridge the gap between observed fly behavior and the chemical signaling pathways that underlie those behaviors.

Work has been done to develop technologies for manipulating individual Drosophila embryos to study development as well. These tools will enable questions about the functions of an individual organism to be addressed that whole-tissue homogenization and pooled sampling methods cannot address.

Controlling individual fly embryo development using microfluidics. There is an increasing interest in using Drosophila embryos to study mechanisms of development and gene function. One powerful method of silencing a gene of interest is called ribonucleic acid interference (RNAi). Cells are exposed to specifically designed double- stranded RNA (dsRNA) that, once inside the cell, is cleaved into smaller dsRNA pieces

(siRNAs) by endogenous enzymes. The siRNA then binds to a RNA-induced silencing

16 complex (RISC) where it becomes unwound. The unwound siRNA guides the RISC to the corresponding messenger RNA (mRNA) whereby the RISC destroys the mRNA, thus eliminating the coding of that particular gene and the gene’s subsequent function (48,

49). While using cells for high-throughput screens is useful, embryos are more ideal model systems for studying development and gene function because they possess physiological content with greater biological complexity; however, until recently, performing RNAi on embryos was a tedious process that required a skilled technician to individually inject each embryo by hand. Solgaard and colleagues have developed a microfluidic device coupled with a computer-controlled injection system to inject

Drosophila embryos with dsRNA for high-throughput RNAi screens (50). This microelectromechanical systems-based device has been automated to detect embryos on a glass slide, followed by rapid injection of 60 pL RNAi aliquots into each embryo with

98% reliability. Although preliminary prototypes require initial manual injector alignment to the device, it has potential for future development into a fully automated process and has already been adapted for various embryo applications where controlled microinjections of small molecules, such as drugs or proteins, are necessary (51, 52).

Microfluidic technologies have also been utilized to fabricate devices capable of spatial and temporal control of developing Drosophila embryos. Ismagilov and coworkers have used a ‘Y’ junction device to investigate a compensatory regulation mechanism displayed by developing embryos towards external perturbations in temperature (Figure 1.4 top) (53, 54). When the anterior and posterior sides of an embryo were exposed to an extreme temperature gradient using two laminar streams held at different temperatures, the warmer half of the embryo had a higher number of nuclei,

Figure 1.4. Microfluidic device for the analysis of Drosophila embryos. The rate of development in each half of the embryo exposed to a T-step is affected by temperature, as demonstrated by the difference in nuclear density (number of nuclei in enlarged areas shown underneath in yellow numbering). (A, B) Embryos exposed to a T-step of 20 °C/27 °C for 140 min. (A) Anterior half 20 °C, posterior half 27 °C. (B) Anterior half 27 °C, posterior half 20 °C. (C, D) Identical set-up to A and B with embryos exposed to a greater T-step of 17 °C/27 °C for 150 min. In all images, higher nuclear density was observed in the warmer half of the embryo. (Reprinted from (53), with permission from Nature Publishing Group).

18 and therefore was developing more rapidly, than the cooler half (Figure 1.4A, B). When the temperature difference between the anterior and posterior sides was increased from

7°C to 10°C, the difference in the rate of development between the two sides increased as well (Figure 1.4C, D). Also, the Even-skipped gene (a gene that codes for segmentation during early embryonic development) was expressed sooner in the warmer region of the embryo causing the usual 7-stripe segmentation pattern to develop in the wrong order.

Interestingly, despite the different developmental rates forced upon the two regions of the embryo, when allowed to come to room temperature, the embryos displayed the completed stripe pattern correctly and developed into normal larvae suggesting

Drosophila have a compensation mechanism to counteract extreme environmental conditions during embryo development. This device has since been adapted to allow easier attachment of the embryos (55). Continued modifications of the device that enhance the ability to apply external gradients to an immobilized embryo will enable future studies on the mechanisms of biochemical networks during development.

Individual larva measurements. There has been recent progress in the development of techniques for measuring neurotransmitters from individual Drosophila larvae using a combination of electrochemical detection and optogenetic stimulation methods. Fast- scan cyclic voltammetry (FSCV) was employed because of its ability to measure rapid changes in electroactive species like serotonin (56). Channelrhodopsin-2 is a light- activated cation-selective ion channel that when placed under the control of a GAL4-

UAS system and crossed with flies of a driver line specific to serotonin (Tph-GAL4), will produce transgenic larvae that release serotonin upon exposure to blue light (24).

Recently, Venton and colleagues utilized the transgenic larvae to measure serotonin

19 release from neurons located in an isolated larva ventral nerve cord (VNC) using FSCV with a microelectrode (Figure 1.5A) (57). The extracellular serotonin concentration in the VNC was found to consistently vary between 280-640 nM during the duration of blue light exposure (Figure 1.5B, C). Inhibition of the serotonin transporter with cocaine and fluoxetine confirmed that the removal of serotonin from the extracellular space was due to transport, and demonstrated the potential use of this model system for studying basic serotonin signaling mechanisms.

The Drosophila larva model system has potential use in other areas as well. A novel sampling technique has been developed to obtain nanoliter volumes of hemolymph from individual Drosophila larvae for chemical analysis (58). Hemolymph contains amino acids such as glutamate and glutamine that are thought to play a role in neurodegeneration. This procedure extracts 50-300 nL of hemolymph from a single

Drosophila larva then, following derivatization with fluorescamine, its amino acid content is quantified using CE with laser-induced fluorescence detection. In a demonstration of this technique, Shippy and coworkers compared genderblind (gb) larvae, mutants developed previously by collaborators (59) that contain approximately half the normal extracellular glutamate concentration, to wild-type larvae. Overall the gb mutants were found to have 38% lower glutamate levels than the wild-type larvae with 13 amino acids in total successfully separated and quantified from each larva’s hemolymph

(n = 10-17). These initial findings support the continued development of this technique in quantifying amino acid levels from individual Drosophila hemolymph to better understand their role in human disease.

Figure 1.5. Measurements in Drosophila larvae following optogenetic stimulation. (A) Diagram of neuromuscular anatomy of a third-instar larva. (B) Representative traces of evoked peak serotonin concentration varying with blue light stimulus duration (2, 5, 10, and 30 s). (C) Pooled data (mean ± SEM, n = 6) shows an increase in peak height with increasing duration of blue light exposure. Peak height appears to plateau after 10 s; peak height at 30 s is not significantly different from that at 10 s (Student’s t-test, 2 tailed, p = 0.78). (Reprinted from (57, 59), with permission from the Society for Neuroscience and Elsevier).

21 Individual adult fly measurements. In addition to Drosophila embryos and larvae, measurements from intact, whole flies have been accomplished. Calcium imaging has been employed in conjunction with genetically encoded fluorescent proteins. Fluorescent proteins that measure calcium changes (an accepted indicator of electrical activity) can be genetically expressed in specific neurons to target a tissue of interest in the Drosophila brain using the GAL4-UAS system (60). This methodology has been used to explore several calcium-sensitive fluorescent proteins including cameleon 2.1, camgaroo 2, and

G-CaMP. Fiala and coworkers have labeled the mushroom body calyx and antennal lobe structures, which are brain regions in the Drosophila CNS, with cameleon 2.1 and measured odor-evoked calcium signals in vivo from both regions (61). Moreover, this technique can be altered to target any brain region of interest for which a GAL4 driver line exists (60).

Based upon previously published work on dissected mushroom bodies by Davis and coworkers (62), the GAL4-UAS system was used to label the mushroom bodies with camgaroo 2, and the intensity changes of the fluorescent Ca2+ reporter in response to acetylcholine application were recorded in an intact fly (63). In addition, Axel and colleagues employed two-photon calcium microscopy to image the antennae lobes of flies that expressed G-CaMP in their projection neurons (64). Using this technique, they were able to link odor-induced calcium changes to specific areas of the antennal lobe.

Each odor elicited a distinct pattern that appears to be conserved between different organisms of the same fly genotype. Calcium imaging techniques could potentially be used for quantitative investigation of olfactory learning and memory in the Drosophila mushroom bodies and antennae lobes.

22 Scope of the Thesis

Electrochemical detection has been used for in vivo measurements of dopamine in model systems such as rats, mice, and primates, but until very recently these measurements were not feasible in an organism as small as Drosophila. My thesis describes the development of electrochemical techniques for in vivo detection of dopamine in the nanoliter-sized brain of adult Drosophila.

A method for quantifying the uptake of exogenously applied dopamine by the

Drosophila dopamine transporter is described in Chapter 2 and used in much of the thesis. FSCV with a carbon-fiber microelectrode is used to monitor changes in dopamine concentration in the adult fly CNS. Figure 1.6A and Figure 1.6B show in vivo dopamine concentration traces demonstrating the change in extracellular dopamine before and after treatment with cocaine, which is known to block uptake by the dopamine transporter. A wild-type fly and a fumin (fmn) mutant fly that lacks a functional dopamine transporter have been compared. While the peak dopamine concentration, [DA]max, increased 3-fold in the wild-type fly following cocaine treatment, dopamine uptake remained unchanged in the fmn mutant fly. When the [DA]max observed in multiple flies is averaged (Figure

1.6C), the [DA]max of untreated wild-type flies is significantly lower than for fmn mutant flies. Interestingly, the [DA]max for cocaine treated wild-type flies is not significantly different from the untreated fmn mutant flies. These measurements support existing evidence that cocaine effectively blocks the Drosophila dopamine transporter and validate the use of this in vivo fly method as a model system to study drug addiction mechanisms.

Figure 1.6. Investigating dopamine transporter function in adult Drosophila. (A) Representative concentration trace of exogenously applied 1.0 mM dopamine in wild- type Drosophila before (black line) and after (red line) cocaine application. An increase in dopamine concentration in the adult wild-type fly was observed following a 5 min exposure to 1.0 mM cocaine. Black arrow corresponds to a 1.0 s dopamine application beginning at 5.0 s. (B) Representative concentration trace of exogenously applied 1.0 mM dopamine in the fmn mutant before (black line) and after (red line) cocaine application. No significant change was observed in the adult fmn mutant fly. (C) Comparison of baseline [DA]max for untreated wild-type and fmn mutant flies (mean ± SEM; Student’s t-test, p = 0.02 (*), n = 9) and the treated wild-type fly after application of 1.0 mM cocaine. The difference in [DA]max between wild-type flies treated with cocaine and untreated fmn mutants or untreated wild-type flies is not significantly different (mean ± SEM; Student’s t-test, p = 0.3 and 0.08, n = 6-9).

24 Chapter 3 describes the application of the in vivo microanalytical technique to investigate the effects of cocaine, amphetamines, and methylphenidate on dopamine clearance by the dopamine transporter in the fly. When under the influence of drugs of abuse, fruit flies exhibit behavioral responses that are amazingly comparable to human behaviors. The neurotransmitter dopamine has been shown to affect drug addiction mechanisms. Following drug treatments, elevated levels of extracellular dopamine are observed. This observation supports behavioral evidence that psychostimulants decrease dopamine transporter function in Drosophila and is similar to results obtained in mammalian systems. Furthermore, a study was developed to examine the effects of methylphenidate on the mechanism of cocaine in the brain using the Drosophila model system, and this is presented in Chapter 4.

Techniques for stimulating release of endogenous dopamine in the fly are discussed in Chapter 5 including chemical, electrical, and optogenetic methods. While the electroactive nature of dopamine makes in vivo electrochemistry an ideal approach for measuring dopaminergic transmission in the brain, pH fluctuations associated with dopamine release have been shown to interfere with electrochemically measured signals in the rat. The fabrication and characterization of a pH microelectrode sensor for use in the fly brain is described in Chapter 6. The ability of the pH sensor to monitor pH changes following neurotransmitter release in real-time has been demonstrated in the

Drosophila CNS.

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30 Chapter 2: In Vivo Electrochemical Measurements of Exogenously

Applied Dopamine in Drosophila melanogaster*

*Reproduced with permission from Makos, M. A., Kim, Y.-C., Han, K.-A., Heien, M. L., and Ewing, A. G. (2009) In Vivo Electrochemical Measurements of Exogenously Applied Dopamine in Drosophila melanogaster, Anal. Chem. 81, 1848-1854. © 2009 American Chemical Society.

31 Introduction

The field of in vivo electrochemistry in the brain began in the 1970’s with Ralph

Adams pioneering the detection of electroactive species. His group measured neurochemicals in the brains of anesthetized rats with carbon electrodes using cyclic voltammetry and chronoamperometry (1, 2). Subsequently, background-subtracted fast- scan cyclic voltammetry (FSCV) coupled with carbon-fiber microelectrodes has been developed and extensively used as an analytical technique for in vivo measurements of electroactive neurotransmitters (3-7). In vivo electrochemistry has mainly focused on the rat as the primary model system to address fundamental questions regarding neurotransmission mechanisms (8-10). While similar studies have been conducted in other model systems such as mice and primates, microanalytical methods for in vivo studies in a model organism as small as Drosophila melanogaster have remained undeveloped (11-14).

Drosophila has been traditionally used as a model organism for genetic research because its genetic manipulation is relatively straightforward, and the genome contains fewer genetic redundancies compared to the mammalian genome, facilitating the identification of functions of individual genes or molecules (15, 16). Drosophila has a short life cycle (12-14 days) and thus it is quite feasible to generate mutants that are genetically homogeneous more quickly in comparison to other model organisms used for in vivo electrochemistry including rats and mice. Although Drosophila has a relatively simple nervous system containing approximately 200,000 neurons, it exhibits many of the same higher-order brain functions as vertebrates at the molecular, cellular, and behavioral levels. Flies are capable of learning from prior experiences and storing

32 learned information (15, 16). Many monoamines including dopamine, serotonin, tyramine, and histamine that regulate human physiological processes are also found in the

Drosophila central nervous system (CNS). In addition, octopamine, specific to invertebrates, has similar roles to mammalian norepinephrine (17).

The neurotransmitter dopamine has been implicated in physiological human processes including attention, motivation, emotion, sleep, and addiction (18-21). In particular, the reinforcing properties of psychostimulants such as cocaine and amphetamine that block the dopamine transporter or other addictive substances such as ethanol and nicotine involve an elevated level of extracellular dopamine (18, 22-24).

However, the underlying neuronal mechanisms concerning how dopamine affects tolerance and addiction remain as yet poorly understood.

Constant-potential amperometry, chronoamperometry, and FSCV are the common electrochemical techniques that have been used to detect dopamine in vivo using model systems (25-27). While constant-potential amperometry has the advantage of excellent temporal resolution over most other electrochemical techniques, its lack of chemical specificity makes it useful only in a system where the identity of the analyte is known or when it is combined with a more chemically selective technique (10, 26, 28).

Voltammetry is one of the most widely accepted techniques used to identify single electrochemical substances. Specifically, background-subtracted FSCV is a dominant technique used for neurotransmitter detection in vivo because of its chemical selectivity, relatively high sensitivity, and sub-second temporal resolution (28-30).

This chapter reports on the development of these microanalytical techniques for in vivo electrochemical detection in the Drosophila CNS. A microsurgery procedure is

33 explained that allows an electrode to be inserted into an immobilized fly brain while the fly is kept viable for experimentation. Voltammetry has been carried out to monitor dopamine in the adult brain of the wild-type fly vs. the mutant fly lacking functional dopamine transporters. Significant differences are detectable for the clearance of exogenously applied dopamine by the transporter which supports the validity of the new method described here.

Methods

Chemicals. All chemicals were used as received and purchased from Sigma (St. Louis,

MO) unless otherwise stated. Adult-hemolymph like (AHL) saline (108 mM NaCl, 5 mM KCl, 2 mM CaCl2, 8.2 mM MgCl2, 4 mM NaHCO3, 1 mM NaH2PO4, 5 mM trehalose (Fluka BioChemika, Buchs, Switzerland), 10 mM sucrose, 5 mM Trizma base , pH 7.5) was made using ultrapure (18 MΩ·cm) water and filtered through a 0.2-

μm filter (31). All collagenase, KCl, propidium iodide (PI), dopamine, (+) cocaine, and tetrodotoxin (TTX) solutions were prepared using AHL saline.

In vivo Drosophila preparation. The Canton-S strain of Drosophila melanogaster was used for the wild-type fly in this chapter. The transgenic flies carrying tyrosine hydroxylase TH-GAL4 and UAS-mCD:GFP (membrane tethered green fluorescent protein) were used to visualize the dopamine neurons (32, 33). The fumin (fmn) mutant has a genetic lesion abolishing the dopamine transporter function. The genetic background of the w;fmn mutant was replaced with the Canton-S background (34). All flies were maintained at 25 °C on a standard cornmeal-agar medium, and 4 to 7 day-old male flies were used for experiments. For in vivo imaging and voltammetry, the flies

34 were immobilized on ice and mounted in a homemade collar (38.1 mm diameter concave plexiglass disk with a 1.0 mm hole in the center) with low melt agarose (Fisher Scientific,

Pittsburgh, PA). Microsurgery was performed on a stereoscope (Olympus SZ60,

Melville, NY) using small dissection scissors and forceps (World Precision Instruments,

Sarasota, FL). After the cuticle was removed from the top portion of the head to expose the brain, the head was covered with 0.1% collagenase solution for 30 min to relax the extracellular matrix in the brain and then rinsed and covered with AHL saline. The images were acquired using an Olympus SZX10 stereomicroscope and an Olympus DP71 digital camera (Figure 2.1A) or a Leica MZ16 stereomicroscope and a Leica DFC290 digital camera (Figure 2.1B and 2.1C; Mannheim, Germany).

Electrochemical measurements. Carbon-fiber microelectrodes were fabricated as previously described (6). Briefly, a single 5-μm diameter carbon fiber (Amoco,

Greenville, SC) was aspirated into a borosilicate glass capillary (B120-69-10, Sutter

Instruments, Novato, CA), and the capillary was pulled using a regular glass capillary puller (P-97, Sutter Instruments). Electrical contact was made by back-filling the capillary with silver paint (4922N DuPont, Delta Technologies Ltd., Stillwater, MN) and inserting a tungsten wire. To form a cylindrical electrode, the carbon fiber was cut to a length of 40-50 μm, as measured from the glass junction. Electrode tips were dipped into epoxy (Epo-Tek, Epoxy Technology, Billerica, MA) for 30 s to ensure a good seal between the fiber and the glass and then dipped into acetone for 15 s to remove epoxy from the exposed carbon fiber. A Ag/AgCl reference electrode was made by chlorodizing a silver wire (0.25 mm diameter, 99.999% purity, Alfa Aesar, Ward Hill,

35 MA) in bleach overnight. Micropipet injectors were fabricated by pulling glass capillaries in a glass capillary puller to an opening of approximately 5 μm.

Electrochemical data were collected using an Axopatch 200B Amplifier (Axon

Instruments, Foster City, CA) and two data acquisition boards (PCI-6221, National

Instruments, Austin, TX) run by the TH 1.0 CV program (ESA, Chelmsford, MA) (35).

For amperometric experiments, a constant potential (+750 mV) was first applied to the working electrode with respect to the reference electrode for at least 15 min to stabilize the background current. All cyclic voltammograms were obtained using a triangular waveform (scanned -0.6 V to +1.0 V vs. Ag/AgCl at 200 V/s) repeated every 100 ms

(low pass Bessel filter at 5 kHz). Prior to voltammetric experiments, all electrodes were cycled (-0.6 V to +1.0 V at 200 V/s) for at least 15 min to stabilize the background current. Electrochemical responses were plotted and statistical analysis performed using

Prism 3.0 (GraphPad Software, La Jolla, CA).

All electrodes were positioned under a Leica MZ16 stereomicroscope using micromanipulators (421 series, Newport, Irvine, CA) on top of a Newport BenchTop

Vibration Isolation System. Either a single-barrel glass micropipet or a three-barrel glass micropipet (3B120F-6, World Precision Instruments) was used to exogenously apply the dopamine solutions. For the three-barrel micropipet, each barrel was individually coupled to a microinjection system (Picospritzer II, General Valve Corporation, Fairfield,

NJ) using a PolyFil apparatus (World Precision Instruments).

Sample preparation for confocal imaging. Transgenic TH-GAL4/UAS-GFP flies were used to visualize the dopamine neurons. The TH-GAL4/UAS-GFP fly brain was exposed as described above then stained with PI (100 μg/mL) for 20 min. Prior to treatment with

36 PI, cell death control flies were treated with 1.0 M KCl for 10 min to model the fluorescence that would occur from PI in a fly brain containing cells that were no longer viable. After three washes in AHL saline (10 min each), phosphate-buffered saline (PBS) containing 4% paraformaldehyde was applied for 20 min. Three more washes were done in PBS only (10 min each) before the brain was dissected out and mounted in Vectashield mounting media (Vector Laboratories, Burlingame, CA). Fluorescence images of

Drosophila brains stained with PI (λex 536 nm, λem 617 nm) and labeled with GFP (λex

488 nm, λem 507 nm) were acquired using a Leica TCS SP5 laser-scanning confocal microscope with a 20x objective lens (Figure 2.2).

Results and Discussion

Drosophila preparation and set-up for in vivo measurements. Electrochemical methods provide a new tool for studying electroactive neurotransmitters in Drosophila. I am particularly interested in studying dopamine neurotransmission since it plays crucial roles in numerous CNS functions in Drosophila as in mammals (17). In the Drosophila brain, multiple clusters of dopamine neuronal cell bodies are spread throughout the outer layer of the brain cortex and innervate many brain regions. In particular, the dopamine neuronal cluster in the protocerebral anterior medial (PAM) brain area project to the nearby mushroom body structure that is crucial for many higher-order neuronal functions including learning and memory (36-38). Thus, I focused on the PAM neurons for in vivo analysis of dopamine neurotransmission. To place microelectrodes in the area where the

PAM neurons are located, a microsurgery procedure was developed. A single adult fly was immobilized in a homemade fly collar using agarose applied to the body and the

37 bottom portion of the head (Figure 2.1A), leaving the upper portion of the head uncovered and positioned for dissection. The cuticle was then removed, and the brain was kept bathed in AHL saline (Figure 2.1B). The salts in the AHL solution were at physiological concentrations, keeping the immobilized fly viable for 1.5 - 2.5 h which is sufficient time to perform electrochemical measurements (31). A micromanipulator was used to guide the cylindrical working electrode into the PAM region. The micropipets used for dopamine application throughout this chapter were positioned above the PAM area, approximately 10 μm from the working electrode (Figure 2.1B inset). The reference electrode was submerged in the AHL saline. Fluorescence microscopy was used to visualize the location of the PAM dopamine neurons in the brain of the transgenic

TH-GAL4/UAS-GFP fly which expresses GFP in dopamine neurons. The PAM area represents the largest cluster of dopamine neurons and is easily identifiable (36). Figure

2.1C shows a representative fluorescence image of a dissected brain with GFP-labeled dopamine neurons. The white box outlines the exposed brain regions where PAM neurons are clearly visible in green, while the fluorescent cells below the box represent other dopamine neuronal clusters. Experiments to investigate dopamine uptake were performed in the PAM dopamine neuronal area.

Viability of Drosophila following microsurgery preparation. Confocal fluorescence microscopy was used to verify that cells in the brain remain viable following the microsurgery preparation described above for in vivo electrochemistry in Drosophila.

Following microsurgery, brains were prepared by incubation in PI, a fluorescent dye which indicates damaged cell membranes. For comparison, control flies were treated with 1.0 M KCl prior to PI incubation to initiate cell death. A fluorescence image of the

Figure 2.1. Images of Drosophila taken during microsurgery. (A) Fly immobilized in a homemade fly collar (Scale bar = 500 µm). (B) Fly after cuticle has been removed. The exposed brain area with the PAM dopamine neurons is outlined by the black box (Scale bar = 100 µm, electrode and injector not to scale). Inset: Schematic showing relative electrode and micropipet injector placement for experiments. (C) Fluorescence image highlighting GFP-labeled dopaminergic neurons. White box outlines the PAM region (Scale bar = 100 µm).

39 brain of an adult TH-GAL4/UAS-GFP fly (Figure 2.2A) contains very little red fluorescence compared to the image of the apoptotic control fly (Figure 2.2B) which provides one indication that the Drosophila brain does remain viable following the dissection preparation for in vivo measurements.

Measuring exogenously applied dopamine in Drosophila. In previous studies, electrochemical detection with FSCV has been used to monitor in vivo dopamine concentrations in rats (3). Exogenously applied dopamine can be measured at the surface of a carbon-fiber microelectrode inserted into the PAM area of the Drosophila system.

To further characterize dopamine detection in the PAM area, color plots were used to display FSCV data. In these experiments, small amounts of a dopamine solution were ejected in the area near the electrode, and voltammetry was used to quantify the dopamine changes in the brain and to track its temporal characteristics. Here, 1.0 mM dopamine was exogenously applied to the adult wild-type brain using a single micropipet injector, and a microelectrode was used for dopamine detection in the PAM area. A false-color representation of current (Figure 2.3A) allows one to visualize cyclic voltammograms over time. The oxidation of dopamine is represented in green while blue corresponds to the reduction of the orthoquinone, allowing discrimination of a particular analyte from other species that may be present in the same brain region. Cyclic voltammetry can be used to identify electroactive species based on the potential at which oxidation occurs and the overall shape of the wave (10, 28, 29). For example, the cyclic voltammogram in Figure 2.3B is a background-subtracted average of ten successive cyclic voltammograms taken at the peak current from the color plot (Figure 2.3A). By

Figure 2.2. Confocal fluorescence micrographs of intact brains from adult transgenic TH-GAL4/UAS-GFP flies. GFP (green) was used to visualize dopamine neurons; PI (red) was used to stain damaged cells. Scale bar = 100 μm. (A) Brain demonstrating cell viability following microsurgery procedure. (B) Brain incubated with 1.0 M KCl as control model of a brain containing cells that are no longer viable for comparison.

41 inspection, the shape of the voltammogram and peak potential leads us to conclude that the increase in current in Figure 2.3A corresponds to the measurement of dopamine.

Finally, the current can be converted to dopamine concentration using in vitro electrode calibration (Figure 2.3C), and the time required for the concentration to decrease to half of its maximum value, t1/2, determined. The difference in applied dopamine concentration vs. that detected at the electrode (millimolar vs. micromolar) is attributed to reuptake and diffusion of the analyte into the surrounding tissue and solution.

Importantly, the time course of the uptake monitored in the fly brain following application of exogenous dopamine solution (t1/2 ~ 50 s) is consistent with measurements of clearance from tissue in other model systems like the rat following exogenous application of dopamine solution (39). Thus, this method is a valid approach to measure changes in exogenously applied dopamine concentration occurring in vivo in the adult fly brain.

Voltammetric vs. amperometric detection of dopamine in vivo. Oxidation of dopamine produces a current which is dependent on the concentration of applied dopamine and its diffusion, uptake, and metabolism as it traverses through tissue.

However, the local geometry and position of the micropipet injector also influence the signal. Specifically, the relative distance of the micropipet to the electrode in the PAM area (Figure 2.1B) affects the amplitude of the current measured. Because a single micropipet is difficult to position the same distance from the electrode multiple times, a pulled triple-barrel capillary was used to exogenously apply three different concentrations of dopamine to the PAM area in series. The current response from 1.0 mM dopamine, approximately 150 pmol (Appendix), applied to the PAM region was measured over

Figure 2.3. Exogenously applied 1.0 mM dopamine detected in vivo in an adult wild- type fly. (A) Successive voltammograms plotted as applied potential vs. time with false color representation showing current. (B) Background-subtracted fast-scan cyclic voltammogram of dopamine application (200 V/s, repetition frequency = 10 Hz). (C) Changes in dopamine concentration over time. Black arrow corresponds to a 1.0 s dopamine application beginning at 5.0 s. Dopamine concentration was determined by converting the maximum current from the sampled amperometry plot using the in vitro calibration average of three electrodes.

43 time, and repeated with 2.0 mM and 5.0 mM dopamine solutions, with each solution loaded into a separate barrel of the triple-barrel micropipet injector. Results obtained using amperometry to measure the dopamine concentration in vivo proved to be variable.

Indeed, the measured concentration at the electrode does not increase linearly with the applied concentration (r2 = 0.36, n = 4). Hence, FSCV was used for analysis.

Representative data collected using FSCV are shown in Figure 2.4. The measured peak currents were converted to dopamine concentration by in vitro calibration of the electrode using standard solutions (Appendix). The plot of normalized measured dopamine concentration vs. injected dopamine concentration constructed using FSCV measurements has a slope of 0.73 ± 0.08 (r2 = 0.84, n = 6), close to the expected value of

1. Thus, controlled concentrations of dopamine solutions can be applied locally to the fly

CNS and measured with voltammetry.

The differences observed between amperometry and FSCV are not surprising when one takes into account the limited sample volume of the Drosophila PAM region.

During amperometric measurements, I hypothesize that local dopamine is “consumed” by oxidization to the orthoquinone, and the local dopamine concentration is altered, making the dopamine unavailable for repeated measurements. The orthoquinone might also be involved in mechanisms of oxidative stress that could affect surrounding tissue in the local environment. In contrast, voltammetric measurements regenerate the measured analyte, minimizing the effect on surrounding tissue. Additionally, the diffusion layer, and thus the volume sampled, with FSCV is smaller than that sampled using amperometry (~3 pL vs. ~50 pL based on the parameters used in these experiments,

Appendix). Amperometry effectively measures dopamine changes that are averaged over

Figure 2.4. Voltammetric detection of exogenously applied dopamine solutions in the PAM area of an adult Drosophila brain. A triple-barrel micropipet was used to apply 1.0 mM (black line), 2.0 mM (red line), and 5.0 mM (blue line) dopamine solutions in series for 1.0 s beginning at 5.0 s (black arrow). Dopamine concentration was determined by converting the maximum current from the sampled amperometry plot using the in vitro calibration average of three electrodes.

45 a larger tissue volume, whereas FSCV measures the dopamine concentration locally around the electrode. This apparently leads to a more accurate measurement of dopamine concentration in this system.

Comparison of dopamine uptake in wild-type vs. fmn mutant flies. The fmn mutants are a Drosophila line where dopamine transporter function has been eliminated through genetic mutation. Thus, the cells that normally remove dopamine from the extracellular fluid after it is released cannot do so, or at least not by the normal mechanism, in fmn mutant flies. I used in vivo voltammetry to investigate the relative magnitude of uptake of dopamine in the fly brain by comparing the fmn mutants to wild-type flies.

Using the same FSCV parameters described in a previous section, differences in uptake between the wild-type and fmn mutant brains were first investigated. Dopamine was exogenously applied to the PAM area (1.0 mM) with a single micropipet injector, and the current response recorded (baseline measurement). Two baseline measurements were taken, and the maximum currents averaged together and converted to dopamine concentration for each fly. Interestingly, comparison of the black traces in Figure 2.5A and 2.5B shows that the peak dopamine concentration observed after injection, [DA]max, is considerably smaller in the wild-type fly compared to the fmn mutant fly. When the average baselines for signals in multiple flies are considered (Figure 2.5C), the [DA]max was significantly higher in fmn flies compared to wild-type flies (9.5 ± 2.4 µM vs. 3.1 ±

0.8 µM; Student’s t-test, p = 0.02, n = 9). This indicates that less dopamine is detected at the electrode after exogenous application in the wild-type flies and is likely due to a high rate of dopamine uptake via the functional dopamine transporter in the PAM neurons in these flies vs. the nonfunctional dopamine transporter in the fmn flies. Therefore,

Figure 2.5. Effect of cocaine on dopamine uptake. (A) Representative concentration trace of exogenously applied 1.0 mM dopamine in wild-type Drosophila before (black line) and after (red line) cocaine application. An increase in dopamine concentration in the adult wild-type fly was observed following a 5 min exposure to 1.0 mM cocaine. Black arrow corresponds to a 1.0 s dopamine application beginning at 5.0 s. (B) Representative concentration trace of exogenously applied 1.0 mM dopamine in the fmn mutant before (black line) and after (red line) cocaine application. No significant change was observed in the fmn mutant fly. (C) Baseline comparison of [DA]max for wild-type and fmn mutant flies (mean ± SEM; Student’s t-test, p = 0.02 (*), n = 9). (D) Comparison of adult wild-type vs. fmn mutant flies when 1.0 mM dopamine is exogenously applied after application of 1.0 mM cocaine. The increase in [DA]max is significantly higher in wild-type flies compared to fmn flies when treated with cocaine (mean ± SEM; Student’s t-test, p = 0.01 (*), n = 6).

47 [DA]max can be used to measure changes in dopamine uptake. It is important to point out that the measurements reported here are highly dependent on electrode and injector placement, resulting in some variation in the values in different flies of the same genotype. However, experiments comparing the relative amount of dopamine in different flies can be carried out by normalization to baseline signals following initial dopamine application, and temporal changes of uptake in the same fly with different conditions can be carried out.

The validity of this theory is demonstrated by using a known dopamine uptake inhibitor, cocaine, to block reuptake of exogenously applied dopamine. To account for differences in the injector positioning and fly-to-fly variability, the maximum currents of two baseline measurements were averaged for each fly, and all measurements for that particular fly were normalized to it. After the baseline measurements, the fly brain was bathed with 1.0 mM cocaine in AHL saline, and a voltammogram was obtained for exogenously applied dopamine after five minutes. Representative traces for wild-type and fmn mutant flies are shown in Figure 2.5A and 2.5B. After the cocaine application, higher dopamine concentrations were detected at the electrode compared to baseline in wild-type flies (Figure 2.5A). fmn mutants lacking functional dopamine transporters showed no change from baseline following the cocaine incubation (Figure 2.5B). When multiple cocaine-treated flies were considered (Figure 2.5D), the wild-type flies had significantly increased normalized [DA]max compared to the cocaine-treated fmn mutant flies (Student’s t-test, p = 0.01, n = 6). This data supports existing evidence that cocaine blocks dopamine transporter function in Drosophila (24).

48 The effect of tetrodotoxin (TTX) on dopamine uptake. The effect of neuronal activities on dopamine uptake was investigated by treating the brains of the two fly genotypes with TTX. TTX is a neurotoxin that blocks action potentials through the blockade of voltage-sensitive sodium channels (40-42).

To examine the effects of TTX, the fly brain was bathed with 1.0 μM TTX in

AHL saline after the baseline dopamine measurements, and voltammograms were obtained for injections of dopamine every five minutes. Representative traces for wild- type and fmn mutant flies are shown in Figure 2.6A and 2.6B. The fmn mutant clearly exhibited a different response than the wild-type flies following incubation with TTX.

After TTX treatments in wild-type flies, higher dopamine concentrations were detected at the electrode compared to baseline (Figure 2.6A). This could be due to several factors.

For example, dopamine uptake in the fly brain may depend on neuronal activity in which case inhibition of the action potential by TTX would abolish the uptake. Alternatively,

TTX might directly inhibit the uptake process. Both possibilities are supported by the result that fmn mutants lacking functional dopamine transporters showed no significant change from baseline following TTX incubation (Figure 2.6B).

Interestingly, the TTX-treated wild-type flies contained significantly increased normalized [DA]max and t1/2 compared to the TTX-treated fmn mutant flies (Figure 2.6C; two-way analysis of variance (ANOVA), p < 0.0001 for genotype for [DA]max, p = 0.04 for genotype for t1/2, n = 3). It is possible that the fmn mutant may have a compensatory increase in the transporter-independent process (i.e., an increased N-methylation) for inactivating endogenously released as well as exogenously applied dopamine, leading to decreased dopamine concentrations detected at the electrode. Previous studies have

Figure 2.6. Effect of TTX on dopamine uptake. (A) Representative concentration trace of exogenously applied dopamine in wild-type Drosophila before and after 1.0 µM TTX application. An increase in dopamine concentration in the adult wild-type fly was observed following exposure to TTX. Black arrow corresponds to a 1.0 s dopamine application beginning at 5.0 s. Baseline 2, 10 min, and 20 min traces were omitted for clarity. (B) Representative concentration trace of exogenously applied dopamine in the fmn mutant before and after TTX application. No significant change was observed in the adult fmn mutant fly. (C) Comparison of adult wild-type vs. fmn mutant flies when 1.0 mM dopamine is exogenously applied before and after application of 1.0 μM TTX. The increases in [DA]max are significantly higher in wild-type flies compared to fmn flies when treated with 1.0 μM TTX (mean ± SEM; two-way ANOVA, p < 0.0001 (***) for genotype, n = 3; SEMs for the baseline bars are too small to see).

50 reported the activity of the dopamine transporter to be dependent on membrane potential

(43). TTX blocks voltage-gated sodium channels, thereby reducing the activity of neurons via action potentials. Thus, the data suggest that the dopamine transporter is activity-dependent, as uptake is reduced in the wild-type flies with TTX.

Conclusions

Microanalytical tools have been developed for in vivo electrochemical measurements in the adult Drosophila CNS. Exogenously applied dopamine is detected using a cylindrical carbon-fiber microelectrode inserted into the dopamine neuronal cluster projecting to the mushroom bodies. The signal has been characterized using

FSCV. A known dopamine uptake blocker, cocaine, was used to validate this method for in vivo measurement of Drosophila dopamine transporter function. Electrochemical detection with FSCV was used to investigate the effect of TTX on the dopamine transporter and the peak dopamine concentration measured which is dependent on uptake.

This work presents a new in vivo method for studying electroactive neurotransmitters in

Drosophila which can be used to measure changes in dopamine uptake.

51 References

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53 33. Friggi-Grelin, F., Coulom, H., Meller, M., Gomez, D., Hirsh, J., and Birman, S. (2003) Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase, J. Neurobiol. 54, 618-627. 34. Kume, K., Kume, S., Park, S. K., Hirsh, J., and Jackson, F. R. (2005) Dopamine is a regulator of arousal in the fruit fly, J. Neurosci. 25, 7377-7384. 35. Heien, M. L., Phillips, P. E. M., Stuber, G. D., Seipel, A. T., and Wightman, R. M. (2003) Overoxidation of carbon-fiber microelectrodes enhances dopamine adsorption and increases sensitivity, Analyst 128, 1413-1419. 36. Nassel, D. R., and Elekes, K. (1992) Aminergic neurons in the brain of blowflies and Drosophila: dopamine- and tyrosine hydroxylase-immunoreactive neurons and their relationship with putative histaminergic neurons, Cell Tissue Res. 267, 147-167. 37. Davis, R. L. (2005) Olfactory memory formation in Drosophila: from molecular to systems neuroscience, Annu. Rev. Neurosci. 28, 275-302. 38. Kim, Y.-C., Lee, H.-G., and Han, K.-A. (2007) D1 dopamine receptor dDA1 is required in the mushroom body neurons for aversive and appetitive learning in Drosophila, J. Neurosci. 27, 7640-7647. 39. Sabeti, J., Adams, C. E., Burmeister, J., Gerhardt, G. A., and Zahniser, N. R. (2002) Kinetic analysis of striatal clearance of exogenous dopamine recorded by chronoamperometry in freely-moving rats, J. Neurosci. Methods 121, 41-52. 40. Narahashi, T., Moore, J. W., and Scott, W. R. (1964) Tetrodotoxin blockage of sodium conductance increase in lobster giant axons, J. Gen. Physiol. 47, 965-974. 41. Takata, M., Moore, J. W., Kao, C. Y., and Fuhrman, F. A. (1966) Blockage of sodium conductance increase in lobster giant axon by tarichatoxin (tetrodotoxin), J. Gen. Physiol. 49, 977-988. 42. Moore, J. W., Blaustein, M. P., Anderson, N. C., and Narahashi, T. (1967) Basis of tetrodotoxin's selectivity in blockage of squid axons, J. Gen. Physiol. 50, 1401- 1411. 43. Sonders, M. S., Zhu, S. J., Zahniser, N. R., Kavanaugh, M. P., and Amara, S. G. (1997) Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants, J. Neurosci. 17, 960-974.

54 Chapter 3: Using In Vivo Electrochemistry to Study the

Physiological Effects of Cocaine and Other Stimulants on the

Drosophila melanogaster Dopamine Transporter*

*Reproduced with permission from Makos, M. A., Han, K.-A., Heien, M. L., and Ewing, A. G. (2010) Using In Vivo Electrochemistry to Study the Physiological Effects of Cocaine and Other Stimulants on the Drosophila melanogaster Dopamine Transporter, ACS Chem. Neurosci. 1, 74-83. © 2010 American Chemical Society.

55 Introduction

The psychomotor stimulant drugs cocaine, amphetamine, and methylphenidate bind to the dopamine transporter and alter its function, increasing extracellular dopamine levels in the brain. The dopamine transporter is the plasma membrane protein primarily responsible for clearing dopamine from the extracellular space, which leads to the termination of dopamine neurotransmission (1, 2). Several lines of evidence have demonstrated that increased extracellular dopamine levels are central to the reinforcing and addictive properties exhibited by drugs of abuse (3, 4). It is well established that cocaine blocks dopamine uptake via the dopamine transporter to elevate the extracellular dopamine concentration (5, 6), and more recently it has been thought to affect the serotonin and norepinephrine transporters as well (7, 8). Amphetamine has dual effects on dopamine transport activity, both inhibiting dopamine uptake and inducing reverse transport through the dopamine transporter (9-11). Methylphenidate, a commonly prescribed medication for the treatment of attention deficit hyperactivity disorder (12), blocks the dopamine transporter and increases the synaptic dopamine concentration (13,

14). While methylphenidate is abused by humans and has a similar affinity for the dopamine transporter as cocaine (3, 6), abuse is not as widespread as that of cocaine. The pharmacokinetics of the two drugs is thought to contribute to the difference observed in their addictive properties (15). Neurochemicals in the central nervous system (CNS) associated with addiction have been investigated for several decades; however, the mechanisms underlying stimulant addictions and the behaviors they elicit are still not fully understood.

56 While animal model systems including rats, mice, and primates have been used for several decades to study the effects of psychostimulants on dopamine transporter function (7, 16, 17), recently, there is accumulating evidence for the validity of using

Drosophila melanogaster as a model system for neurotransmission (18, 19). In humans, dopamine and serotonin play significant roles in regulating diverse physiological processes including attention, motivation, and addiction, and these two monoamines have been found to exert similar functions in the fly (20-23). When exposed to cocaine, nicotine, or ethanol, Drosophila exhibits behavioral responses akin to those displayed by mammals (24-28). In addition to the above mentioned monoamines, octopamine is a major neurotransmitter in the CNS of invertebrates. Similar to norepinephrine in mammals, octopamine dynamics in Drosophila are affected by exposure to cocaine (29).

While behavioral studies are a crucial aspect of investigating psychostimulant actions in

Drosophila, the ability to quantify neurochemicals in vivo would greatly improve understanding of the molecular and cellular pathways behind the reinforcing and addictive effects of a drug.

The electroactive nature of several neurotransmitters makes in vivo electrochemistry an ideal approach for measuring chemical changes in the brain. Uptake studies on both exogenously applied dopamine and stimulated dopamine release have been characterized in vivo using voltammetry and chronoamperometry techniques in rats

(30-32). In particular, fast-scan cyclic voltammetry (FSCV) coupled with carbon-fiber microelectrodes is a valuable method for quantification of biogenic amines in the CNS because of its chemical selectivity and subsecond temporal resolution (33-35), and it has been used previously in rats, mice, Drosophila flies, and Drosophila larvae (5, 36-38).

57 Here, I utilized recently developed microanalytical techniques introduced in Chapter 2 to measure changes in the uptake of exogenously applied dopamine in the CNS of adult

Drosophila with treatments of cocaine, amphetamine, methamphetamine, or methylphenidate. The physiological stimulant concentration necessary to significantly block uptake by the dopamine transporter was approximately 10 µM.

Methods

Chemicals. All chemicals were used as received and purchased from Sigma (St. Louis,

MO) unless otherwise stated. Adult-hemolymph like (AHL) saline (108 mM NaCl, 5 mM KCl, 2 mM CaCl2, 8.2 mM MgCl2, 4 mM NaHCO3, 1 mM NaH2PO4, 5 mM trehalose (Fluka BioChemika, Buchs, Switzerland), 10 mM sucrose, 20 mM Trizma base , pH 7.5) was made using ultrapure (18 MΩ·cm) water and filtered through a 0.2-

μm filter (18). All collagenase, KCl, dopamine, N-acetyl-p-aminophenol (APAP, acetaminophen), (+) cocaine, (+) amphetamine, (+) methamphetamine, and methylphenidate solutions were prepared using AHL saline.

In vivo Drosophila preparation. The Canton-S strain of Drosophila melanogaster was used for the wild-type fly in this chapter. The fumin (fmn) mutant has a genetic lesion abolishing the dopamine transporter function. The genetic background of the w;fmn mutant was replaced with the Canton-S background. All flies were maintained at 25 °C on a standard cornmeal-agar medium, and 4 to 10 day-old male flies were used for experiments. The flies were prepared for in vivo voltammetry as described in Chapter 2.

Briefly, flies were immobilized on ice and mounted in a homemade collar (38.1 mm diameter concave plexiglass disk with a 1.0 mm hole in the center) with low melting

58 agarose (Fisher Scientific, Pittsburgh, PA). Microsurgery was performed on a stereoscope (Olympus SZ60, Melville, NY). After the cuticle was removed from the top portion of the head to expose the brain, the head was covered with 0.1% collagenase solution for 30 min to relax the extracellular matrix in the brain. The head of the immobilized fly was then rinsed and bathed with AHL saline (“bath application method”) with the preparation maintaining its viability for 1.5 - 2.5 h.

Electrochemical measurements. Carbon-fiber microelectrodes were fabricated as described in Chapter 2 (38). Briefly, a single 5-μm diameter carbon fiber (Amoco,

Greenville, SC) was aspirated into a borosilicate glass capillary (B120-69-10, Sutter

MN) and inserting a tungsten wire. To form a cylindrical electrode, the carbon fiber was cut to a length of 40-50 μm, as measured from the glass junction. Electrode tips were dipped into epoxy (Epo-Tek, Epoxy Technology, Billerica, MA) for 30 s to ensure a good seal between the fiber and the glass and then dipped into acetone for 15 s to remove epoxy from the exposed carbon fiber. Standard dopamine solutions were used for in vitro electrode calibration (Appendix). A Ag/AgCl reference electrode was made by chlorodizing a silver wire (0.25 mm diameter, 99.999% purity, Alfa Aesar, Ward Hill,

MA) in bleach overnight. All electrodes were positioned using micromanipulators (421 series, Newport, Irvine, CA). Micropipet injectors were fabricated by pulling glass capillaries in a glass capillary puller to an opening of approximately 5 μm. Micropipet

59 injectors were coupled to a microinjection system (Picospritzer II, General Valve

Corporation, Fairfield, NJ) and used to exogenously apply dopamine solutions.

Electrochemical data were collected using an Axopatch 200B Amplifier (Axon

Instruments, Foster City, CA) or a Dagan Chem-Clamp potentiostat (Dagan Corporation,

Minneapolis, MN) and two data acquisition boards (PCI-6221, National Instruments,

Austin, TX) run by the TH 1.0 CV program (ESA, Chelmsford, MA) (36). All cyclic voltammograms were obtained using a triangular waveform (scanned -0.6 V to +1.0 V vs.

Ag/AgCl at 200 V/s) repeated every 100 ms (low pass Bessel filter at 3-5 kHz). Prior to voltammetric experiments, all electrodes were cycled (-0.6 V to +1.0 V at 200 V/s) for at least 15 min to stabilize the background current. Electrochemical responses were plotted and statistical analysis performed using Prism 5.0 (GraphPad Software, La Jolla, CA).

Results and Discussion

The effect of 1.0 mM cocaine treatment on dopamine uptake. Microanalytical techniques developed for in vivo electrochemical detection in Drosophila provide a method for studying the physiological effects of drug treatments on redox-active neurotransmitters. In Chapter 2, I characterized exogenously applied dopamine uptake using electrochemical detection with a carbon-fiber microelectrode inserted into the protocerebral anterior medial (PAM) area of an adult Drosophila brain (38). In this chapter, I utilize this procedure to explore dopamine neurotransmission in the Drosophila

CNS. Dopamine neuronal cell bodies are clustered together in several distinct areas throughout the Drosophila brain with the largest neuronal cluster located in the PAM region projecting to the nearby mushroom body (39-41), a key brain structure for learning

60 and memory (42). Octopamine levels in this particular brain region are insignificant, simplifying measurements of dopamine. Thus, my in vivo investigation of dopamine uptake in Drosophila is focused on the PAM area.

Following microsurgery, a micromanipulator was used to insert the cylindrical working electrode into the PAM region while the reference electrode was submerged in the AHL saline bath covering the exposed fly brain. Small amounts of dopamine were ejected just above the PAM area, approximately 10 μm from the working electrode, with a single micropipet injector. FSCV was used to monitor changing dopamine levels in the

CNS of both wild-type and fmn mutant flies over time. Voltammetry was performed by applying potential in a triangular waveform to the electrode while the current response was recorded. To visualize changes over time, a false-color representation of current is used (Figure 3.1A) where the green corresponds to the oxidation of dopamine, and the reduction of the orthoquinone is represented in blue (33). The current response was converted to dopamine concentration using in vitro electrode calibration (Appendix).

The peak dopamine concentration measured is referred to as [DA]max which is an established parameter for measuring changes in uptake of extracellular dopamine (17). In addition to [DA]max, another parameter used to compare dopamine clearance between the two fly genotypes is t1/2, the full width of time at half maximum of the dopamine concentration (Figure 3.1B).

The validity of using [DA]max to compare changes in dopamine uptake via the functional dopamine transporter in wild-type flies vs. the nonfunctional dopamine

Figure 3.1. In vivo detection of exogenously applied 1.0 mM dopamine in the adult Drosophila brain. (A) Applied potential vs. time gives a visual representation of successive voltammograms with current viewed in false color. (B) Dopamine concentration plotted over time. Dopamine concentration was determined from the measured current using an in vitro calibration average of three electrodes. The black arrow corresponds to a 1.0 s dopamine application beginning at 5.0 s.

62 transporter in fmn flies has been demonstrated (38). Here, [DA]max was used to investigate the effectiveness of a known dopamine uptake inhibitor, cocaine, on blocking uptake by the Drosophila dopamine transporter in vivo. A 1.0 mM dopamine solution was exogenously applied to the PAM area for 1.0 s (corresponding to ~150 pmol dopamine ejected, Appendix), and the current response was recorded for 3 min (Figure

3.2A, B: “baseline 1”). Following three baseline measurements, the fly brain was bathed with 1.0 mM cocaine in AHL saline for 5 min and then the current response was recorded over time following dopamine injection (“5 min cocaine”). Cocaine treatment was continued and dopamine injections were repeated every 5 min while the current response was recorded.

The representative cyclic voltammogram in Figure 3.2C is a background- subtracted average of ten successive cyclic voltammograms acquired during an in vivo dopamine baseline measurement from an adult wild-type fly brain (dashed red line). A background-subtracted average of ten successive cyclic voltammograms of exogenously applied dopamine following 15 min of 1.0 mM cocaine treatment is plotted for comparison (solid black line). Both voltammograms are from the time period when

[DA]max was measured, and by inspection, the voltammetric peaks correspond to the electrochemical signature of dopamine (35, 43). After a 1.0 mM cocaine treatment, a 3- fold increase in [DA]max was observed for the adult wild-type fly (Figure 3.2A) while the

[DA]max of the fmn mutant fly (Figure 3.2B) remained unchanged. Notably, comparison of the baseline measurements in Figure 3.2A, B shows a significant difference between the two fly types following exogenous dopamine application. Less dopamine is detected in the wild-type fly vs. the fmn fly, which is likely due to dopamine uptake by the

Figure 3.2. Effect of 1.0 mM cocaine treatment on uptake of an exogenously applied 1.0 mM dopamine solution. (A) Representative concentration trace in the wild-type fly before (baseline 1, 2) and after cocaine treatment. A significant increase in dopamine concentration was observed. (B) Representative concentration trace in the fmn mutant fly before (baseline 1, 2) and after cocaine treatment. Dopamine concentration was determined by converting the measured current using in vitro electrode calibration. The black arrow corresponds to a 1.0 s dopamine application beginning at 5.0 s. (C) Background-subtracted fast-scan cyclic voltammogram of baseline extracellular dopamine (dashed red line) and extracellular dopamine after 15 min of cocaine treatment (solid black line) in a wild-type fly (200 V/s, average of 10 scans each).

64 functional transporter that is present only in the wild-type fly. When the average [DA]max of multiple flies was considered (Figure 3.3), the [DA]max of untreated wild-type flies

(“baseline 1, 2”) was significantly lower than for fmn mutant flies. Interestingly, the

[DA]max for cocaine treated wild-type flies was not significantly different from the untreated fmn mutant flies (“baseline 1, 2”) which supports existing evidence that cocaine blocks the dopamine transporter in Drosophila (44). Upon comparison of the two genotypes, wild-type flies exhibited a significantly increased normalized [DA]max with

1.0 mM cocaine treatment compared to fmn mutant flies under the same treatment (Figure

3.4; two-way analysis of variance (ANOVA), p < 0.0001 for genotype, p = 0.0008 for time, p = 0.0002 for interaction, n = 6). To account for slight differences in dopamine injector positioning between flies, the [DA]max from two of the dopamine baseline measurements for a fly were averaged together, and all measurements for that fly were calculated as a percent of the average baseline measurement (i.e., [DA]max normalized)

(38, 45, 46). The maximum effect of the cocaine treatment on the wild-type flies was observed within 10 min and remained fairly constant for over 20 min of cocaine treatment while neither genotype experienced a significant change in t1/2. These observations indicate that cocaine effectively blocks the Drosophila dopamine transporter function in vivo.

Determining the physiological cocaine concentration in the Drosophila brain. To estimate the concentration of the 1.0 mM cocaine solution in the PAM area, APAP was used to mimic the bath application method of the cocaine treatment. APAP was selected because it is an electroactive molecule that is thought to undergo neither rapid metabolism nor uptake by monoamine transporters, thus allowing only the oxidation

Figure 3.3. Investigating dopamine transporter function. Comparison of baseline [DA]max for untreated (no cocaine) wild-type and fmn mutant flies (mean ± SEM; Student’s t-test, p = 0.02 (*), n = 9) and wild-type flies after 15 min of 1.0 mM cocaine treatment. The difference in [DA]max between untreated fmn mutants and wild-type flies treated with cocaine is not significantly different (mean ± SEM; Student’s t-test, p = 0.3, n = 6-9).

Figure 3.4. Comparison of wild-type and fmn mutant flies when 1.0 mM dopamine was exogenously applied before and after 1.0 mM cocaine treatment. There is a significant increase in normalized [DA]max for wild-type flies vs. fmn flies with cocaine treatment (mean ± SEM; two-way ANOVA, p < 0.0001 (***) for genotype, p = 0.0008 (***) for time, p = 0.0002 (***) for interaction, n = 6). The black arrow corresponds to the beginning of the cocaine treatment.

67 current from diffusion of the 1.0 mM bath solution into the brain region to be measured

(47). Furthermore, detection of APAP using voltammetry is well documented (48, 49).

To determine the physiological drug concentration in the Drosophila brain region from a

1.0 mM bath application over the experimental time period, a carbon-fiber microelectrode was placed in the PAM region of Drosophila, and the fly head was bathed in 1.0 mM APAP in AHL saline solution. Background-subtracted FSCV was employed to measure the current in vivo from oxidation of APAP at the surface of the implanted electrode (Figure 3.5A). The peak oxidation current was converted to APAP concentration, [APAP], using in vitro electrode calibration with APAP (Figure 3.5B).

The actual [APAP] in the Drosophila brain, or the physiological [APAP], is approximately 2 orders of magnitude lower (12 ± 5 µM, n = 3 flies) than the applied 1.0 mM bath [APAP]. While the concentration that diffuses into the tissue might differ slightly between cocaine and APAP due to the distinct properties of the two species, such as diffusion rate, relative permeability into the tissue, and size (Figure 3.5C, D) this difference is insignificant compared to the high resistance to diffusion of the brain tissue.

When these calculations are applied to the cocaine solutions, a 1.0 mM cocaine bath application corresponds to approximately a 12 µM or 0.004 mg/mL cocaine concentration in the PAM area. This is significantly lower than that used in a study by

Hirsh and colleagues where 0.5 mg/mL cocaine was applied directly to Drosophila nerve cords (20). Interestingly, my physiological cocaine concentration is consistent with a recent report by Venton and coworkers that found 10 µM cocaine was sufficient to effectively block serotonin reuptake by serotonin transporters located in the ventral nerve cords of Drosophila larvae (37).

Figure 3.5. Determining the physiological APAP concentration in the Drosophila CNS from a 1.0 mM APAP bath application. (A) Background-subtracted fast-scan cyclic voltammogram of APAP measured in vivo of an adult wild-type fly with a bath application of 1.0 mM APAP (average of 10 successive scans). (B) Electrode calibration plot in standard APAP solutions (mean ± SEM; n = 5). The physiological APAP concentration in the Drosophila CNS is approximately 2 orders of magnitude lower than the concentration of the applied bath solution. (C) Structure of APAP. (D) Structure of cocaine.

69 Drosophila dopamine transporter inhibition as a function of cocaine concentration.

Electrochemical detection with FSCV was used to investigate the effect of three different concentrations of cocaine (0.05, 0.5, or 1.0 mM) on dopamine uptake by the Drosophila dopamine transporter. The fly was prepared for in vivo electrochemical measurements and bathed with 0.05 mM cocaine in AHL saline after the baseline dopamine measurements were acquired. Voltammograms of 1.0 mM dopamine injections were obtained every 5 min.

Figure 3.6 is a comparison of the normalized [DA]max for wild-type vs. fmn mutant flies after separate treatments for 10 min with either 0.05, 0.5, or 1.0 mM cocaine.

A two-way ANOVA was used to analyze the comprehensive data at all doses and a significant difference in normalized [DA]max was observed for the two fly types (two-way

ANOVA, p < 0.0001 for genotype, concentration, and interaction, n = 6 for each concentration and genotype). In addition, wild-type flies incubated with 1.0 mM cocaine had significantly increased normalized [DA]max compared to control measurements of

AHL saline only (one-way ANOVA, p < 0.0001; post hoc Tukey pairwise comparisons, p < 0.0001, n = 6). Higher dopamine concentrations were detected in wild-type flies treated with 0.5 mM cocaine as well; however, the effect was not as robust as that observed with the 1.0 mM cocaine treatment ([DA]max increased ~20% vs. ~125% compared to AHL treatments). When the applied cocaine concentration was further decreased to 0.05 mM, there was no significant difference in the normalized [DA]max for wild-type flies from AHL saline measurements. Neither fly genotype exhibited a significant change in [DA]max from baseline dopamine measurements when only AHL saline (no cocaine) was applied in a control experiment (one-way ANOVA, p > 0.05 for

Figure 3.6. Comparison of wild-type and fmn mutant flies when 1.0 mM dopamine was exogenously applied before (baseline) and after 10 min of various concentrations of cocaine treatments. One of the following treatments was applied: AHL saline only, 0.05 mM, 0.5 mM, or 1.0 mM cocaine solution (mean ± SEM; two-way ANOVA, p < 0.0001 (***) for genotype, concentration, and interaction, n = 6; SEMs for the baseline bars are too small to see). The bath solutions for the baseline and AHL saline treatment were identical. The AHL saline treatment was included as a control to ensure the [DA]max response did not increase from a temporal effect owing to the AHL solution. There is a significant increase in normalized [DA]max for wild-type flies after cocaine treatments compared to AHL saline (no cocaine) treatment (one-way ANOVA, p < 0.0001; post hoc Tukey pairwise comparisons, p < 0.0001 (***) for the 1.0 mM cocaine treatment, n = 6). No significant change was observed in the fmn mutant flies between AHL saline (no cocaine) treatment and the three cocaine treatments (one-way ANOVA, p = 0.9, n = 6).

71 both fly genotypes, n = 6). Only the baseline measurements from these AHL saline control experiments are plotted for simplicity. There was no significant difference between baseline measurements for wild-type flies that were later treated with cocaine vs. baseline measurements for wild-type flies just treated with AHL saline (one-way

ANOVA, p = 0.8, n = 6). Similarly, there was no significant difference between baseline measurements for fmn flies that were later treated with cocaine vs. baseline measurements for fmn flies just treated with AHL saline (one-way ANOVA, p = 0.09, n =

6). The fmn mutant flies lacking the dopamine transporter exhibited no change in extracellular dopamine concentration after 0.05, 0.5, or 1.0 mM cocaine treatment (one- way ANOVA, p = 0.9, n = 6).

Therefore, at the 1.0 mM concentration, cocaine appears to overcome a threshold concentration and significantly blocks the Drosophila dopamine transporter in vivo.

These data are consistent with the effect of cocaine on mammalian dopamine transporter function (5, 8) and with observations previously made with this technique (38). These findings support the use of Drosophila as a model system for studying pharmacological effects in vivo. Although the effect of volatilized cocaine on Drosophila behavior has previously been demonstrated (20), the findings presented here provide the first in vivo investigation of the effective cocaine concentration needed to block uptake of exogenously applied dopamine by the dopamine transporter in the adult fly.

The effect of other stimulant treatments on dopamine uptake. In addition to cocaine, the effects of three other stimulants on Drosophila dopamine transporter function were investigated. Flies were prepared as for cocaine experiments and then treated with either

1.0 mM amphetamine, methamphetamine, or methylphenidate in AHL saline. Figure 3.7

72 contains a summary of the normalized [DA]max for adult wild-type flies compared to fmn mutant flies following each of these drug treatments. When dopamine levels in the flies treated with amphetamine were examined over time, there was a small, but significant difference in the amount of dopamine detected in the PAM region of the wild-type brain compared to the same region in the fmn mutant (Figure 3.7A; two-way ANOVA, p =

0.005 for genotype, n = 5). However, even after 30 min of treatment, the observed

[DA]max for the amphetamine-treated wild-type flies was lower than that of wild-type flies treated with 1.0 mM cocaine (~25% increase vs. ~125% increase). This finding is consistent with in vitro inhibition studies demonstrating amphetamine is a less potent inhibitor of the Drosophila dopamine transporter than cocaine (44).

The Drosophila dopamine transporter was significantly affected by treatment with

1.0 mM methamphetamine as well (Figure 3.7B; two-way ANOVA, p = 0.01 for genotype, n = 5-6). Methamphetamine-treated wild-type flies exhibited a similar increase in [DA]max compared to the amphetamine-treated wild-type flies (~30% increase vs.

~25% increase). Interestingly, the trend in time until maximum blocking of dopamine uptake occurs is later with methamphetamine treatment than with amphetamine or cocaine treatment. Although the difference between the normalized [DA]max after 5 min and 20 min of methamphetamine treatment in wild-type flies is not significantly different

(Student’s t-test, p = 0.4, n = 6), the kinetics of the action of methamphetamine on the fly dopamine transporter could be of interest in future investigations. There is in vitro evidence that methamphetamine and amphetamine cause internalization of the mammalian dopamine transporter. These data suggest an additional mechanism that contributes to the decrease in transporter activity by amphetamines, in addition to

Figure 3.7. Comparison of wild-type and fmn mutant flies when 1.0 mM dopamine was exogenously applied before and after 1.0 mM stimulant treatment. (A) Following amphetamine treatment, the increases in normalized [DA]max are significantly higher in wild-type flies compared to fmn mutant flies (mean ± SEM; two-way ANOVA, p = 0.005 (**) for genotype, n = 5). Additionally, the 30 min treatment is significantly different from baseline 2 for the wild-type flies (one-way ANOVA, p = 0.03 (*); post hoc Tukey pairwise comparisons, p < 0.05 (*)). (B) The increases in normalized [DA]max are significantly higher in wild-type vs. fmn flies following methamphetamine treatment (mean ± SEM; two-way ANOVA, p = 0.01 (*) for genotype, n = 5-6). (C) Following methylphenidate treatment, the increases in normalized [DA]max for wild-type compared with fmn flies are significantly higher (mean ± SEM; two-way ANOVA; p = 0.03 (*) for interaction; p < 0.0001 (***) for genotype, n = 5).

74 blocking and inducing reverse transport of dopamine through the dopamine transporter

(50-52). While in vitro model systems are often used to predict the effects of psychostimulants on monoamine uptake, in vitro results are not always an accurate reflection of the potential of a compound to modulate in vivo function (53-55). Thus, development of analytical methods capable of in vivo evaluation of drug efficacy plays a critical role in the neuroscience field. These in vivo measurements confirm amphetamines do indeed alter Drosophila dopamine transporter function; however, with the current experimental set-up, it is not possible to speculate on the exact mechanisms of action occurring in the fly CNS.

Although methylphenidate is commonly studied in mammalian systems, very little, if any, literature is available on the efficacy of this drug in Drosophila. Because the fruit fly is becoming a more widely used model system for studying the neurochemical basis for human behaviors and addictions (19), I chose to examine the effect of this commonly prescribed drug on dopamine uptake using our in vivo method. Following methylphenidate treatment, wild-type flies displayed a significantly higher extracellular dopamine concentration compared to baseline dopamine measurements and the treated fmn mutant flies (Figure 3.7C; two way ANOVA, p = 0.03 for interaction, p < 0.0001 for genotype, n = 5). This indicates that methylphenidate blocks dopamine uptake occurring via the Drosophila dopamine transporter. This finding correlates with the proposed mechanism of methylphenidate in the human brain (13, 14) and supports the use of

Drosophila in future studies on methylphenidate. Of the four stimulants investigated, cocaine and methylphenidate displayed the greatest effect on Drosophila dopamine transporter function in vivo (Table 3.1).

75 a Table 3.1. Change in [DA]max for four drugs of abuse .

cocaine amphetamine methamphetamine methylphenidate

IC (µM) 50 6.0b 4.9b Drosophila 4.5b 6.8b 2.7c (+) 6.6; (-) 34.0c dopamine transporter

wild type: [DA]max 223 ± 40 % 117 ± 8 % 129 ± 22 % 174 ± 31 % normalized (20 min treatment)

fmn mutant: [DA]max 91 ± 8 % 102 ± 8 % 99 ± 4 % 102 ± 11 % normalized (20 min treatment)

a Maximum changes for dopamine ([DA]max) values are for (+) amphetamine and (+) methamphetamine. [DA]max values are mean ± SEM for 1.0 mM drug concentrations (n b c = 5-6). Literature IC50 values are included for comparison. Reference (56). Reference (44).

76 In these experiments, exogenously applied dopamine is cleared primarily through diffusion, metabolism, and uptake by the dopamine transporter. By comparing two fly genotypes whose diffusion and metabolism are presumably similar since they only differ in dopamine transporter function, I was able to investigate the uptake component of dopamine clearance in the presence of various stimulants. All four stimulants tested caused significantly increased dopamine signal amplitudes ([DA]max), which has been observed in the cocaine-treated rat CNS where chronoamperometry was employed to measure exogenously applied dopamine concentrations in vivo (17, 45). In these studies,

Gerhardt and coworkers also reported an increase in the time course of the enhanced dopamine signal amplitudes, which was not observed in Drosophila. I speculate that diffusion plays a prominent role in the clearance of dopamine from the Drosophila CNS due to its reduced size (~ 5 nL) in comparison to the rat CNS which might experience less diffusion of dopamine away from the electrode (32). A change in t1/2 could be too minor to observe in the fly system relative to this diffusion factor.

Conclusions

This chapter presents in vivo measurements of dopamine uptake using exogenously applied dopamine as a function of cocaine concentration in Drosophila. In addition, physiological effects of amphetamine, methamphetamine, and methylphenidate are also reported for the adult fly. Cocaine and methylphenidate were found to be more potent at inhibiting dopamine uptake in vivo by the Drosophila dopamine transporter than amphetamine and methamphetamine. It is most likely that the variation in the dose- response results among the four stimulants tested reflects different interactions of the

77 drugs with the dopamine transporter. Little is known about the in vivo nature of drug interactions with invertebrate transporters, mainly because of the lack of tools heretofore available for quantifying neurotransmitters in such small native environments. These data support continued use of this in vivo Drosophila model system to further investigate dopamine neurotransmission and enhance understanding of the physiological mechanisms that underlie human behaviors and addictions.

78 References

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82 Chapter 4: Oral Administration of Methylphenidate Blocks the

Effect of Cocaine on Uptake at the Drosophila Dopamine

Transporter*

*In preparation for submission to ACS Chem. Neurosci.

83 Introduction

Cocaine addiction is a disease currently estimated to affect 2.1 million users in the

United States alone (1). The molecular and cellular actions of cocaine in the brain are complex and affect the neurotransmission of several chemicals including dopamine, serotonin, and norepinephrine through alteration of their transporter function (2-5).

Voltage-gated sodium channels are also blocked by cocaine (6), further supporting the idea that cocaine works as a nonselective drug in the central nervous system (CNS), and making it difficult for scientists to develop a suitable drug treatment to combat cocaine addiction (7-9). The reinforcing and addictive properties of cocaine have been linked to an increase in extracellular dopamine levels which are caused by blocking the dopamine transporter (10, 11). It is widely accepted that cocaine decreases dopamine uptake through binding of the dopamine transporter (12, 13), and it has been demonstrated that the euphoric feeling experienced by cocaine abusers is associated with the blockade and subsequent increase in extracellular dopamine (14).

Methylphenidate (Ritalin®), a commonly prescribed medication for the treatment of attention deficit hyperactivity disorder (ADHD) (15), blocks the dopamine transporter with a binding affinity similar to that of cocaine and increases the extracellular dopamine concentration in the human brain (16-18). Methylphenidate has been shown to increase extracellular norepinephrine by blocking the norepinephrine transporter as well (19).

Although cocaine and methylphenidate undergo similar binding to the dopamine transporter in the CNS, the abuse potential of the two psychostimulants is different.

Typically, drugs that demonstrate reinforcing effects in laboratory animals are abused by humans (20), and monkeys will self-administer methylphenidate and cocaine

84 intravenously at similar rates (21, 22). However, studies have demonstrated that adult humans do not consistently choose oral methylphenidate over placebo (23-25). While methylphenidate is abused by humans, its abuse is much more limited than that of cocaine which is considered one of the most addictive drugs known (26-28).

The route of administration of psychostimulants alters their pharmacokinetic properties which can influence the abuse potential of a particular drug (29). When three cocaine administration routes were compared, including smoked (crack), intravenous, and intranasal, the length of time for each preparation to reach the CNS and for the euphoric feeling to be experienced by the user was different (30, 31). Although the administered doses of cocaine caused equivalent levels of blockade of the dopamine transporter with all three routes, smoked cocaine was found to have a higher abuse potential, greater reinforcing properties, and to be more addictive than the other two routes. When the length of time for different routes of methylphenidate administration was investigated, orally administered methylphenidate took approximately eight times longer than intravenous administration to reach maximum blockade of the dopamine transporter (17,

32). Indeed, the observation that the shorter the time interval between intake of a drug and the perceived affects of a drug, the greater the reinforcing properties and therefore addictive potential of that drug has been documented (33, 34). The slow adsorption of oral methylphenidate is believed to be an important factor in its limited abuse.

While orally administered methylphenidate in humans has been found to cause little, if any, euphoric feelings (17, 35), intravenous administration of methylphenidate by cocaine abusers causes feelings that are similar to those experienced with intravenous cocaine use (36, 37). Both drugs have a fast adsorption rate in the brain (maximum

85 concentration occurs in 2-8 min for cocaine and 4-10 min for methylphenidate, respectively) which is thought to elicit the hedonic feeling associated with drug abuse

(37). The uptake rate of intravenous administration of cocaine and methylphenidate is similar; however, the clearance rate of the two stimulants differs significantly. The half- life of methylphenidate in the brain, based on the duration of dopamine transporter blockade, is longer than that of cocaine (75-90 min vs. 15-25 min, respectively) even though the initial reinforcing feeling it gives the user disappears just as quickly (~10 min) as with cocaine use (37). The clearance of methylphenidate from the brain is necessary before it is possible for an individual to fully experience the reinforcing effects of the drug again, thus it is speculated that frequent repeated administration and overall abuse of intravenous methylphenidate is limited in comparison to cocaine.

Over the last decade, methylphenidate has been investigated as a potential agonist, or replacement medication, for cocaine addiction treatment as a similar approach has been successful where methadone is used for treating opiate addiction (38, 39).

Several studies have investigated the effects of oral methylphenidate on cocaine users, and mixed results have been found. Individuals experiencing fewer cravings and reduced cocaine use with methylphenidate treatment have been reported (40), while other studies have found no change in cocaine use or cravings for cocaine users after taking methylphenidate (35, 41). Studies involving the subset of cocaine users who also had symptoms of ADHD have reported more promising results (42-47). The ADHD cocaine users experienced fewer cocaine cravings, decreased their cocaine use, and felt some degree of improvement in their ADHD symptoms. These data suggest methylphenidate could be a successful agonist medication for cocaine users with ADHD, but they do not

86 explain the conflicting results for cocaine users without ADHD. A better understanding of the chemical mechanisms in the CNS during co-administration of methylphenidate and cocaine is needed to shed light on this potential treatment for cocaine addiction.

Animal models including rats, mice, and primates have been used to investigate neurochemicals in the CNS associated with drug addiction (10, 13, 21, 22). Techniques that use invertebrates, such as Drosophila melanogaster (fruit fly) and Apis mellifera

(honey bee), for research involving drugs of abuse have been established as well (48-50).

A method recently developed by the Ewing laboratory (51, 52) utilizes fast-scan cyclic voltammetry (FSCV) coupled with carbon-fiber microelectrodes to quantify dopamine, an electroactive neurotransmitter, in the CNS of Drosophila. Here, I apply this microanalytical technique to study the efficacy of orally consumed methylphenidate on dopamine uptake in Drosophila and its effect on preventing the actions of cocaine on the dopamine transporter in vivo.

Methods

Chemicals. All chemicals were used as received and purchased from Sigma (St. Louis,

MO) unless otherwise stated. Adult-hemolymph like (AHL) saline (108 mM NaCl, 5 mM KCl, 2 mM CaCl2, 8.2 mM MgCl2, 4 mM NaHCO3, 1 mM NaH2PO4, 5 mM trehalose (Fluka BioChemika, Buchs, Switzerland), 10 mM sucrose, 20 mM Trizma base , pH 7.5) was made using ultrapure (18 MΩ·cm) water and filtered through a 0.2-

μm filter (53). All collagenase, dopamine, (+) cocaine, and methylphenidate bath treatment solutions were prepared in AHL saline.

87 Drosophila rearing and in vivo preparation. Male flies, 4 to 10 days old, of the

Canton-S strain of Drosophila melanogaster were used for all experiments. Flies were maintained at 25 °C on a standard cornmeal-agar medium. Some flies received an additional food supplement consisting of a yeast paste containing 10 mM methylphenidate aqueous solution that was prepared fresh daily. The flies were reared on the methylphenidate yeast paste for 3-5 days prior to experimentation. All flies were prepared for in vivo voltammetry as described in Chapters 2 and 3 (51, 52). Briefly, flies were mounted in a homemade collar (38.1 mm diameter concave plexiglass disk with a

1.0 mm hole in the center) with low melting agarose (Fisher Scientific, Pittsburgh, PA) following immobilization with ice. Under a stereoscope (Olympus SZ60, Melville, NY) the cuticle was removed from the top portion of the head using dissection forceps and scissors (World Precision Instruments, Sarasota, FL) to expose the brain. Following microsurgery, 0.1% collagenase solution was applied to the head for 30 min to relax the extracellular matrix in the brain. The immobilized fly head was then rinsed and bathed with AHL saline, allowing the preparation to remain viable for 1.5 - 2.5 h.

Electrochemical measurements. The fabrication of the cylindrical carbon-fiber microelectrodes used for this study has been described in detail previously (51, 54). The exposed carbon fiber portion of the cylindrical electrodes was 40-50 μm in length. The in vitro electrode calibration with standard dopamine solutions is shown in the Appendix.

In all experiments, the Ag/AgCl reference electrode used was made by chloridizing a silver wire (0.25 mm diameter, 99.999% purity, Alfa Aesar, Ward Hill, MA) in bleach overnight. Electrodes were positioned using micromanipulators purchased from Newport

(421 series, Irvine, CA). Glass capillaries (B120-69-10, Sutter Instruments, Novato, CA)

88 were pulled using a glass capillary puller (P-97, Sutter Instruments) and cut to an opening of ~5 μm to form micropipet injectors. The injectors were used to exogenously apply 1.0 mM dopamine solution by coupling them to a microinjection system (Picospritzer II,

General Valve Corporation, Fairfield, NJ).

A Dagan Chem-Clamp potentiostat (Dagan Corporation, Minneapolis, MN) and two data acquisition boards (PCI-6221, National Instruments, Austin, TX) run by the TH

1.0 CV program (ESA, Chelmsford, MA) were used to collect all electrochemical data

(55). Cyclic voltammograms were obtained by applying a triangular waveform potential

(-0.6 V to +1.0 V vs. a Ag/AgCl reference electrode) repeated every 100 ms at a scan rate of 200 V/s (low pass Bessel filter at 3 kHz). All electrodes were allowed to cycle for at least 15 min prior to recording to stabilize the background current. The recorded current response was converted to dopamine concentration via in vitro electrode calibration

(Appendix). Statistical analysis was accomplished using Prism 5.0 (GraphPad Software,

La Jolla, CA).

Results and Discussion

Dopamine uptake in the Drosophila CNS following cocaine bath treatment. In

Chapter 2, I described the development of a procedure for in vivo electrochemical detection in adult Drosophila (51), and I demonstrated its use to study the effects of cocaine and methylphenidate on the clearance of the redox-active neurotransmitter dopamine in Chapter 3 (52). The Drosophila brain contains dopaminergic neurons clustered together in several distinct locations with the largest neuronal cluster located in the protocerebral anterior medial (PAM) region (56). By inserting a cylindrical carbon-

89 fiber microelectrode into the PAM area of a Drosophila brain, changes in uptake of exogenously applied dopamine can be quantified. This method is used in this chapter to monitor the effects of cocaine and methylphenidate on dopamine clearance in the

Drosophila CNS.

Following fly microsurgery (see Methods), a carbon-fiber working electrode was placed in the PAM region. Dopamine was exogenously applied just above the fly brain tissue with a micropipet injector, and background-subtracted FSCV was used to measure the current response in the extracellular fluid of the CNS over time. Using the peak dopamine concentration, [DA]max, to monitor changes in the uptake of extracellular dopamine in the CNS has been established (52, 57), and this parameter is utilized here.

Initially, the in vivo baseline current response was recorded for 3 min after a 1.0 mM dopamine solution was exogenously applied to the PAM area for 1.0 s (~150 pmol dopamine applied). Following three baseline measurements, the fly brain was bathed in

1.0 mM cocaine, which has been shown to inhibit dopamine uptake by the Drosophila dopamine transporter (52), for 5 min and then dopamine was applied again while the current response was recorded. Dopamine injections were repeated every 5 min throughout the 25 min cocaine treatment. Figure 4.1A compares a baseline concentration trace of dopamine (black line) with a concentration trace obtained after cocaine treatment

(red line). The representative traces demonstrate the effectiveness of a bath application of cocaine in blocking dopamine uptake via the dopamine transporter.

Although the effect of different administration routes of methylphenidate has been studied in mammalian systems, to my knowledge no reports have been published on the efficacy of orally consumed methylphenidate in Drosophila. Flies were orally fed a paste

Figure 4.1. Effect of orally consumed methylphenidate on cocaine inhibition of the dopamine transporter in the adult Drosophila brain. (A) Representative concentration traces (taken from the maximum anodic peak potential) of exogenously applied 1.0 mM dopamine in a wild-type fly that did not consume methylphenidate before (baseline, black line) and after 1.0 mM cocaine bath treatment (red line). A significant increase in dopamine concentration was observed following cocaine application. Dopamine concentration was determined from conversion of the measured current using in vitro electrode calibration. The black arrow corresponds to a 1.0 s dopamine application beginning at 5.0 s. (B) Representative concentration traces of exogenously applied 1.0 mM dopamine in a wild-type fly that consumed methylphenidate before (baseline, black line) and after 1.0 mM cocaine bath treatment (red line). No change in dopamine concentration was observed following cocaine application. (C) Applied potential vs. time gives a visual representation of successive voltammograms that correspond to the baseline current (black line) in (A) with current viewed in false color. (D) Applied potential vs. time gives a visual representation of successive voltammograms that correspond to the baseline current (black line) in (B) with current viewed in false color.

91 consisting of a 10 mM methylphenidate solution mixed with yeast for 3-5 days prior to the cocaine bath application experiment described above. The 1.0 mM cocaine bath application treatment did not affect the [DA]max following dopamine injection for the flies that consumed the methylphenidate paste (Figure 4.1B). To visualize changes over time, a false-color representation of current is used where the green corresponds to the oxidation of dopamine, and the reduction of the orthoquinone is represented in blue (58).

The color representation for a baseline measurement of current for a fly that did not consume methylphenidate is shown in Figure 4.1C while Figure 4.1D corresponds to a fly that consumed methylphenidate.

Effect of orally consumed methylphenidate on dopamine uptake in Drosophila. In

Chapter 3, it was shown that a 1.0 mM bath application of methylphenidate is sufficient to effectively block dopamine uptake occurring via the dopamine transporter in

Drosophila wild-type flies (52). Here, the results are compared to wild-type flies that orally consumed methylphenidate prior to administration of the 1.0 mM bath application of methylphenidate to determine if oral administration of methylphenidate is capable of blocking the Drosophila dopamine transporter in vivo to a similar degree as the bath administration.

Flies that consumed a paste consisting of a 10 mM methylphenidate solution mixed with yeast for 3-5 days prior to the methylphenidate bath treatment were compared to flies that did not consume the methylphenidate paste. The 1.0 mM methylphenidate bath application treatment had no effect on the peak current response following dopamine injection for the flies that consumed methylphenidate (Figure 4.2A). To eliminate systematic effects, such as slight differences in dopamine injector positioning between

Figure 4.2. Effect of orally consumed methylphenidate on Drosophila dopamine transporter function. (A) The uptake of exogenously applied 1.0 mM dopamine by flies that orally consumed 10 mM methylphenidate (black) was compared with flies that did not consume methylphenidate (green). After baseline dopamine measurements, both groups of flies were treated with bath-applied 1.0 mM methylphenidate for 25 min. There was a significant increase in normalized [DA]max for the flies that did not consume methylphenidate prior to the bath methylphenidate treatment (mean ± SEM; two-way ANOVA, p = 0.05 for interaction, p < 0.0001 for two fly groups, p = 0.03 for bath treatment, n = 5-6). (B) After baseline dopamine measurements, both groups of flies were treated with bath-applied 1.0 mM cocaine for 25 min. There was a significant increase in normalized [DA]max for the flies that did not consume methylphenidate prior to the bath cocaine treatment (mean ± SEM; two-way ANOVA, p = 0.009 for interaction, p < 0.0001 for two fly groups, p = 0.002 for bath treatment, n = 6). SEMs for the baseline bars are too small to see.

93 flies, the [DA]max was normalized, and the averages of several flies were compared. For normalization, the [DA]max from two of the dopamine baseline measurements for a fly were averaged together, and all measurements for that particular fly were calculated as a percent of the average baseline measurement (i.e., [DA]max normalized). The normalized

[DA]max averages for the two groups of flies, flies that consumed the 10 mM oral methylphenidate paste and flies that did not, were compared. The flies that did not eat the methylphenidate paste displayed a significantly higher change in normalized [DA]max following the 1.0 mM bath application of methylphenidate compared to the flies that did consume the methylphenidate paste (two-way analysis of variance (ANOVA), p = 0.05 for interaction, p < 0.0001 for two fly groups, p = 0.03 for bath treatment, n = 5-6). A bath application of methylphenidate does not appear to change uptake by the dopamine transporter of flies that have previously consumed methylphenidate. This suggests oral consumption of methylphenidate blocks the Drosophila dopamine transporter in a manner similar to that of orally consumed methylphenidate in humans (17).

Cocaine effects are undetectable following the oral consumption of methylphenidate.

Bath application of 1.0 mM cocaine was shown to effectively block the Drosophila dopamine transporter in Chapter 3 (52). To investigate whether prior methylphenidate consumption is able to affect the action of cocaine on the dopamine transporter, flies were tested that had been fed the 10 mM methylphenidate paste. Electrochemistry was used to monitor exogenously applied dopamine clearance before and after application of a 1.0 mM cocaine bath. Voltammograms were obtained with dopamine exogenously applied every 5 min for 25 min. Figure 4.2B is a comparison of the normalized [DA]max for the two groups of flies. The flies that did not consume methylphenidate experienced a

94 significantly increased normalized [DA]max following the cocaine bath treatment, whereas flies that had consumed methylphenidate did not exhibit a change in dopamine uptake with the cocaine treatment (two-way ANOVA, p = 0.009 for interaction, p < 0.0001 for two fly groups, p = 0.002 for bath treatment, n = 6). Again, this indicates that orally consumed methylphenidate effectively blocks the Drosophila dopamine transporter function in vivo, and other effects from the addition of cocaine are not observed. This result is comparable to the mechanism of action that has been observed in baboons that were given methylphenidate prior to cocaine administration (37). In addition, the regional distribution patterns of methylphenidate and cocaine throughout the human brain have been found to be almost identical using positron emission tomography with similar in vivo potencies at the human dopamine transporter (37, 59). These data reinforce the validity of using Drosophila as a model system for studying mechanisms of cocaine addiction in humans.

Comparison of the extracellular dopamine concentration in the Drosophila CNS following drug treatments. To further investigate the functionality of the Drosophila dopamine transporter following drug treatments, non-normalized [DA]max data was considered. The non-normalized [DA]max was obtained from the cyclic voltammograms of multiple flies, and the averages for four different groups of flies were compared

(Figure 4.3). The average [DA]max of flies not treated with any form of cocaine or methylphenidate (“untreated”) was significantly lower than that of flies that had consumed oral methylphenidate (Student’s t-test, p = 0.009) and flies treated with bath- applied cocaine only (Student’s t-test, p = 0.006). This confirms the dopamine transporter in the untreated flies was more functional than that of flies treated with oral

Figure 4.3. Comparison of dopamine concentration in the Drosophila CNS following drug treatments. Untreated flies had a significantly lower extracellular dopamine concentration than either flies that orally consumed 10 mM methylphenidate (mean ± SEM; Student’s t-test, p = 0.009 (**)) or flies that were treated with 1.0 mM bath-applied cocaine for 20 min (mean ± SEM; Student’s t-test, p = 0.006 (**)). There was no significant difference between the flies that orally consumed methylphenidate and the flies treated with bath-applied cocaine (mean ± SEM; Student’s t-test, p = 0.9). Flies treated with 1.0 mM bath-applied methylphenidate for 20 min were not significantly different from the flies of the other three groups (mean ± SEM; Student’s t-test, p = 0.12- 0.17).

96 methylphenidate or bath-applied cocaine. The non-normalized data of flies subjected to only bath-applied methylphenidate were not significantly different from the flies of the other three groups. Of importance, there was no significant difference between the flies that had orally consumed methylphenidate and the flies that were treated with the bath- applied cocaine.

Conclusions

Although there has been improvement in understanding the actions of cocaine in the brain, an effective drug treatment has yet to be found for cocaine addiction.

Methylphenidate binds the dopamine transporter and increases extracellular dopamine levels in the CNS similar to cocaine without producing as many of the addictive and reinforcing properties. In this chapter the Drosophila model system was utilized to investigate the mechanism behind treating cocaine addiction with methylphenidate. In vivo electrochemical measurements suggest oral consumption of methylphenidate sufficiently blocks the Drosophila dopamine transporter thus preventing further inhibition of the transporter by cocaine applied directly to the CNS. This highlights the possibility of methylphenidate as a potential treatment for cocaine addiction and the value of Drosophila as a model system for future drug abuse research.

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102 Chapter 5: Methods for Stimulating Dopamine Release in the

Drosophila CNS

103 Introduction

Electrochemistry has been used to monitor neurotransmission in the brain for several decades. A voltammetric technique capable of measuring species in rat brain tissue was reported by Leland Clark and coworkers in 1965 (1). This was followed by in vivo cyclic voltammetry work completed in Ralph Adams’s laboratory. They used 1-2 mm diameter electrodes made from carbon paste or solidified graphite-epoxy resin to measure electroactive neurochemicals in the brains of anesthetized rats (2, 3). The fabrication and characterization of the carbon-fiber microelectrode by Wightman and coworkers in 1980 allowed more rapid voltammetric measurements to be carried out in comparison to the conventional electrodes used at that time (4-6). This tool led to the progression of electroanalytical techniques to monitor in vivo neurotransmitter dynamics in the central nervous system (CNS) (7-11). Several voltammetric techniques have been developed for in vivo measurements including differential pulse voltammetry, normal pulse voltammetry, linear sweep voltammetry, and cyclic voltammetry (12). Fast-scan cyclic voltammetry (FSCV) has become a widely used voltammetric technique for in vivo applications because it is capable of detecting neurotransmitter changes in real-time as well as providing chemical information regarding the identity of the electroactive species being measured (12-15).

The neurotransmitter dopamine is of interest because it is known to regulate several human physiological processes including motivation and addiction. Dopamine neurotransmission is believed to contribute to the reinforcing and addictive properties of drugs of abuse such as cocaine and amphetamines (16-18). Monitoring in vivo changes in dopamine uptake in the Drosophila CNS in the presence of psychostimulants has been

104 discussed in Chapters 2-4. In addition to uptake, dopamine release is an important component of neurotransmission. Various electrochemical techniques have been developed to detect in vivo dopamine release. New approaches to stimulate neurotransmitter release have been developed as well and used in conjunction with electrochemical detection including chemical, electrical, and most recently optogenetic stimulation.

A number of chemicals are capable of evoking neurotransmitter release from a neuron. A widely used approach is to depolarize the cell membrane using an elevated concentration of K+ ions (19, 20). This causes voltage-dependent ion channels to open, whereby an action potential is generated, and that causes neurotransmitter-filled vesicles to release their contents into the extracellular space. The neurotransmitter dopamine is released in both the rat and mouse CNS in response to K+ ion stimulation (21-23).

Veratridine is another depolarizing agent that effectively induces dopamine release in mammals (24-26). Caffeine and nicotine are two stimulants that have been shown to increase extracellular dopamine in the rat CNS through binding of their respective receptors, adenosine and nicotinic acetylcholine (27-31). In addition, Ba2+ ions have been reported to trigger dopamine release in mammalian neuronal preparations (32, 33).

The chemical stimulants listed above are summarized in Table 5.1.

While chemical stimulation is an efficient way to elicit dopamine release in vivo, a more controlled approach is to utilize the electrical properties of neurons. Brief electrical pulses generate action potentials which leads to the release of neurotransmitters

(34, 35). Stimulation of a particular pathway in regions where multiple pathways exist is possible with this method through specific electrode placement, which offers an

105

Table 5.1. Eliciting dopamine release via chemical stimulation.

Stimulant Action References

in vivo rat and mouse CNS (21, 22) potassium depolarizes cell membrane in vitro rat brain tissue (23)

in vivo rat CNS (24) depolarizing agent that acts as a veratridine in vitro rat brain tissue (25) sodium channel agonist in vitro guinea pig cochleae (26)

caffeine adenosine receptor antagonist in vivo rat CNS (27, 28)

nicotinic acetylcholine receptor nicotine in vivo rat CNS (29-31) agonist

thought to induce exocytotic bovine cell culture (32) barium release by a mechanism similar to rat neuronal preparation (33) calcium

106 advantage over chemical stimulation methods. Dopamine release induced by electrical stimulation was measured in vivo by Ewing et al. in the rat CNS (36). Since then electrical stimulation has been extensively used to elicit dopamine release in many applications involving in vivo dopamine neurotransmission (37-44). One drawback of electrical stimulation is that it causes most neurons within the stimulated region to undergo neurotransmitter release simultaneously, and this is not an accurate model for the timing of naturally occurring dopamine release events in the brain.

A targeted approach to manipulate neuronal function that has recently been investigated utilizes light in place of chemicals or electricity to stimulate cell activity (45,

46). Channelrhodopsin-2 (ChR2) is an ion channel found in the green alga

Chlamydomonas reinhardtii that can be genetically inserted into neurons and optically controlled. The ChR2 protein is composed of seven trans-membrane domains and contains the chromophore all-trans retinal. Upon exposure to blue light, all-trans retinal undergoes isomerization to 13-cis retinal, thus causing a conformational change that allows the trans-membrane protein to open (47, 48). Na+ ions flow through the channel and enter the cell as they travel down their electrochemical gradient (45). The increase in positive charge in the cell causes depolarization of the cell membrane and leads to vesicular neurotransmitter release into the extracellular space (Figure 5.1). By genetically expressing ChR2 in a specific type of neuron, blue light stimulation can be used to elicit release of a particular neurotransmitter of interest (49). Preliminary studies have demonstrated that ChR2 can be expressed in both adult Drosophila and larvae dopaminergic neurons and specific release of dopamine initiated with blue light (49-51).

107

Figure 5.1. Cartoon depiction of the effects of blue light exposure on neurons expressing Channelrhodopsin-2 (ChR2). Upon illumination with blue light, vesicular neurotransmitter release is stimulated in neurons (light green) genetically altered with ChR2. The lipid bilayer membrane (light purple) containing the ChR2 ion channel is enlarged for clarity.

108 In this chapter, I investigate chemical, electrical, and optogenetic methods to stimulate dopamine release in the Drosophila CNS with FSCV detection. Chemical and electrical stimulation tools successfully used in larger mammalian model systems were modified for and tested in the smaller fly CNS (~5 nL). In addition, I explore using blue light to noninvasively stimulate neurochemical release through the novel ChR2 ion channel by genetic insertion of the ChR2 channel in Drosophila dopamine neurons. The results suggest optogenetic stimulation initiates targeted neuronal release in the

Drosophila CNS.

Methods

Chemicals. All chemicals were used as received and purchased from Sigma (St. Louis,

MO) unless otherwise stated. Adult-hemolymph like (AHL) saline (108 mM NaCl, 5 mM KCl, 2 mM CaCl2, 8.2 mM MgCl2, 4 mM NaHCO3, 1 mM NaH2PO4, 5 mM trehalose (Fluka BioChemika, Buchs, Switzerland), 10 mM sucrose, 20 mM Trizma base , pH 7.5) was made using ultrapure (18 MΩ·cm) water and filtered through a 0.2-

μm filter. All collagenase, dopamine, KCl, veratridine (EMD Biosciences, Inc., La Jolla,

CA), caffeine, nicotine, BaCl2, and (+) cocaine solutions were prepared in AHL saline.

In vivo Drosophila preparation. The Canton-S strain of Drosophila melanogaster was used for the wild-type fly in this chapter. Using the Drosophila galactosidase-4-upstream activating sequences (GAL4/UAS) gene-targeting system explained in Chapter 1, mutant flies were bred by crossing female flies carrying ChR2 with male flies expressing tyrosine hydroxylase (TH) to produce mutant flies of the genotype TH-GAL4/UAS:ChR2

(49, 52). The dopaminergic neurons of the mutant flies can be controlled with blue light

109 stimulation. Wild-type flies were maintained on a standard cornmeal-agar medium, while mutant flies were maintained in the dark and fed yeast containing 10 mM all-trans retinal (light sensitive chemical necessary for ChR2 function) made fresh daily. All flies were kept at 25 °C, and 4 to 10 day-old male flies were used for experimentation. Flies were prepared for in vivo FSCV measurements as described in Chapter 2 (53). Briefly, flies were immobilized on ice and mounted in a homemade collar (38.1 mm diameter concave plexiglass disk with a 1.0 mm hole in the center) with low melt agarose (Fisher

Scientific, Pittsburgh, PA). Microsurgery was performed on a stereoscope (Olympus

SZ60, Melville, NY) using small dissection scissors and forceps (World Precision

Instruments, Sarasota, FL) to remove the cuticle. The head was covered with 0.1% collagenase solution for 30 min to relax the extracellular matrix in the brain and then rinsed and covered with AHL saline to maintain the viability of the preparation for 1.5 -

2.5 h.

Electrochemical measurements. Cylindrical carbon-fiber microelectrodes were fabricated as described in Chapter 2 (53). Briefly, a single 5-μm diameter carbon fiber

(Amoco, Greenville, SC) was aspirated into a borosilicate glass capillary, and the capillary was pulled using a regular glass capillary puller (P-97, Sutter Instruments,

Novato, CA). Electrical contact was made by back-filling the capillary with silver paint

(4922N DuPont, Delta Technologies Ltd., Stillwater, MN) and inserting a tungsten wire.

The carbon fiber was cut to a length of 40-50 μm, as measured from the glass junction.

Electrode tips were dipped into epoxy (Epo-Tek, Epoxy Technology, Billerica, MA) for

30 s to ensure a good seal between the fiber and the glass and then dipped into acetone for 15 s to remove epoxy from the exposed carbon fiber. A Ag/AgCl reference electrode

110 was made by chlorodizing a silver wire (0.25 mm diameter, 99.999% purity, Alfa Aesar,

Ward Hill, MA) in bleach overnight. All electrodes were positioned using micromanipulators (421 series, Newport, Irvine, CA).

Electrochemical data were collected using either an Axopatch 200B Amplifier

(Axon Instruments, Foster City, CA) or a Dagan Chem-Clamp potentiostat (Dagan

Corporation, Minneapolis, MN) and two data acquisition boards (PCI-6221, National

Instruments, Austin, TX) run by the TH 1.0 CV program (ESA, Chelmsford, MA) (54).

Cyclic voltammograms were obtained using a triangular waveform (scanned -0.6 V to

+1.0 or +1.2 V vs. Ag/AgCl at 200 V/s) repeated every 100 ms (low pass Bessel filter at

3-5 kHz). Prior to voltammetric experiments, all electrodes were cycled for at least 15 min to stabilize the background current. Electrochemical responses were plotted and statistical analysis performed using Prism 5.0 (GraphPad Software, La Jolla, CA). After collection, voltammograms were smoothed (nearest neighbor smooth) and filtered at 2.0 kHz using the TH 1.0 CV program. The current traces were filtered at 0.5-1.0 Hz.

Chemical stimulation equipment. A single-barrel borosilicate glass capillary (B120-

69-10, Sutter Instruments) was used to make injectors to apply KCl (100-500 mM), veratridine (100 µM), caffeine (1 mM), nicotine (100 µM), and BaCl2 (10 mM) stimulation solutions. Micropipet injectors were fabricated by pulling the capillaries in a glass capillary puller to an opening of approximately 5 μm. Stimulation solutions were pneumatically applied using a Picospritzer II (General Valve Corporation, Fairfield, NJ).

Electrical stimulation equipment. The first type of electrode used to electrically stimulate dopamine release in the fly was composed of two individually insulated, 75 µm diameter platinum electrodes (MS303/9-B/SPC, Plastics One Inc., Roanoke, VA). The

111 second type of electrode used was composed of two tungsten electrodes each with a 125

µm diameter shaft, but tapered at a 12° angle to a point (57720, A-M Systems Inc.,

Carlsborg, WA). A battery operated, constant current stimulus isolator (NeuroLogTM

System NL800, Digitimer Ltd., Holliston, MA) was used to deliver computer controlled stimulation through the electrodes. A range of monophasic pulses was tested (0.1-10.0

V, 0.5-8.0 ms per phase, 1-24 pulses, 10-60 Hz, 10 µA-10 mA).

Optogenetic stimulation equipment. Blue light was applied through computer control of a 3-W Luxeon Star LED with a peak intensity of ~470 nm (LXHL-LB3C, Newark,

Chicago, IL). Red light was applied in a similar manner with a 1-W Luxeon Star LED with a peak intensity of ~625 nm (LXHL-MD1B, Newark).

Results and Discussion

Chemical stimulation of dopamine release in Drosophila. In the fly brain, dopamine neurons project to the nearby mushroom body (MB) structure which is crucial for many higher-order functions including learning and memory (55, 56). The neuronal cluster in the protocerebral anterior medial (PAM) brain area is the largest group of dopamine neurons in the Drosophila CNS (57), and this is the region where I placed the working electrode in dopamine uptake experiments reported in Chapters 2-4. Dopamine release occurs in the region where the dopamine neurons project; therefore, the working electrode was placed in the MB in this chapter.

After the microsurgery procedure was performed (see Methods), a micromanipulator was used to guide a 5 µm diameter cylindrical carbon-fiber electrode into the MB area. FSCV was used to measure changes in current in this brain region, and

112 the reference electrode was submerged in the surrounding AHL saline. Micropipets were used to deliver chemical stimulation solutions to the MB area with pneumatic pressure

(Figure 5.2A). The chemicals investigated as potential stimulants in Drosophila included potassium, veratridine, caffeine, nicotine, and barium. Although all five of the solutions successfully stimulate dopamine release through a range of mechanisms in mammalian model systems (Table 5.1), dopamine release was not detected in Drosophila.

Various experimental parameters could account for the data obtained with the chemical stimulation method. One, diffusion causes a decrease in the concentration of the chemical stimulant that reaches the brain region surrounding the working electrode compared with the original concentration in the micropipet injector. However, the decrease in concentration can be approximated by the micropipet injection of dopamine described in Chapter 2 (Figure 2.4). The concentration that diffuses into the tissue depends on the diffusion rate, relative permeability into the tissue, and size of a particular chemical species. Here, the limiting factor is the high resistance to diffusion of the brain tissue. Pneumatically applying 1.0 mM dopamine for 1.0 s just above the fly brain results in a concentration of ~7 µM in the Drosophila tissue. Therefore, the concentration of the stimulant in the fly brain region is approximately three orders of magnitude lower than that of the applied solution.

Another possibility is that invertebrate systems respond to the chemical stimulants differently than mammals, but it is unlikely that the solutions tested would not stimulate the fly CNS and initiate dopamine release. Released dopamine dissipates from the extracellular space via uptake by the dopamine transporter (IC50 = 2.9 µM for

Drosophila), metabolism, and diffusion (58). It seems likely that released dopamine is

113

Figure 5.2. Schematic comparing three methods for stimulating neurotransmitter release in adult Drosophila. The position of the instruments used for chemical, electrical, and optogenetic stimulation is marked with respect to the cylindrical working electrode (not drawn to scale).

114 diluted by the surrounding chemical stimulation solution in the small volume of the fly brain, thus the concentration of dopamine at the electrode surface is lower than the limit of detection (LOD) of the working electrode (65 nM). The dopamine concentration inside vesicles is well above the LOD (~100 mM); however, vesicles are attoliters in volume. A 1 aL vesicle corresponds to ~0.1 amol of dopamine. Diffusion from the aL vesicle to the nL surrounding environment results in a decrease of 10-9. This dilution in concentration will affect measurements in the ~5 nL fly brain more significantly than a larger mammalian system, like the rat brain, and suggests an explanation as to why this method of stimulation was successful in other model systems, but not in the fly.

Electrical stimulation of dopamine release in Drosophila. Electrical stimulation is a method that has been extensively used to elicit neurotransmitter release in mammalian systems. Two electrodes are placed on either side of a neuronal pathway of interest (36).

Through application of a voltage, action potentials in neurons are initiated which results in neurotransmitter release. The dimensions of the fly CNS required modification of electrical stimulation procedures used in mammalian systems. Following the microsurgery procedure, I placed two stimulation electrodes on either side of the MB structure where the cylindrical working electrode was positioned with the tips of all three electrodes in the same horizontal plane (Figure 5.2B). The working electrode was ~50

µm from each stimulation electrode, and the reference electrode was submerged in the surrounding AHL saline outside of the fly head. Electrical pulses were applied through the stimulation electrodes, and a wide range of values for the pulse parameters was tested

(see Methods). In vivo fluctuations in current were measured in the Drosophila CNS with FSCV detection; however, after inspection of the voltammograms, the current

115 changes were not attributed to dopamine release. This could be due to several factors.

The parameters used for electrical stimulation must be sufficient to depolarize the dopamine neurons while not affecting neurotransmission of the entire fly CNS.

Additional neurochemical changes in the system from overstimulation might coincide with the time scale of dopamine release, which could alter the expected electrochemical voltammetric signature of dopamine.

Two types of commercially available stimulation electrodes were used for this investigation: platinum stimulation electrodes 75 µm in diameter and tungsten electrodes

125 µm in diameter but tapered to a sharp point. The size of a fly MB is ~100 µm in width, and the electrical stimulation electrodes cause damage to the area where they are placed. A potential solution to alleviating some of the physical destruction caused in the

CNS by the stimulation electrodes is to build an electrical stimulation set-up with smaller electrodes that have been fabricated by hand. Another approach is to use optogenetic stimulation of neurons, which eliminates the need to place any stimulation electrodes in or around the fly brain.

Optogenetic stimulation of dopamine release in Drosophila. Endogenous dopamine release was evoked in TH-GAL4/UAS:ChR2 mutant flies using optogenetic stimulation.

The dopamine neurons of the mutant flies were genetically altered to express ChR2, a cation-selective ion channel that can be activated with blue light on a millisecond time scale (59, 60). The ChR2 protein contains the chromophore all-trans retinal, which undergoes a conformation change upon exposure to blue light. This causes the ChR2 ion channel to open and allows Na+ ions to enter the cell. The increased positive charge in

116 the cell depolarizes the cell membrane and leads to vesicular dopamine release into the extracellular space (Figure 5.1).

Following fly microsurgery, a blue LED was positioned ~1.5 mm above the exposed Drosophila MB (Figure 5.2C). FSCV was used to monitor neurochemical release. A TH-GAL4/UAS:ChR2 mutant fly was illuminated with blue light for 10 s to stimulate dopamine release. As a control, the experiment was repeated with TH-

GAL4/UAS:ChR2 mutant flies that had not consumed all-trans retinal which is necessary for ChR2 ion channel function to occur. This is an effective way to eliminate the response of the ChR2 channel to blue light in the Drosophila CNS system (49, 61).

The results suggest an electroactive species is released in the MB region of

Drosophila with optical stimulation. Figure 5.3A compares the current measured during blue light stimulation of a mutant fly that consumed all-trans retinal (black line) with a mutant fly that did not consume all-trans retinal (gray line). While a significant difference in the two flies is observed, the ~0.07 nA increase in measured current from the fly that did not consume all-trans retinal is not anticipated. By inspection, the cyclic voltammogram for this current (Figure 5.3B, gray line) does not resemble a typical wave shape of dopamine. The non-dopamine like voltammogram confirms that dopamine is not released in the TH-GAL4/UAS:ChR2 mutant fly that did not consume all-trans retinal. There is apparently a contribution to the measured signal from factors other than dopamine oxidation. The voltammogram of the peak signal from the fly that consumed all-trans retinal (black line) is similar to the shape of a dopamine voltammogram, suggesting that dopamine is released and measured. However, the formal potential of the voltammogram is shifted +200 mV compared to a representative voltammogram of

117

Figure 5.3. Effect of blue light stimulation on flies with genetically altered dopamine neurons. (A) Representative current trace in a TH-GAL4/UAS:ChR2 mutant fly that consumed all-trans retinal (black line) vs. a mutant fly that did not consume all-trans retinal (gray line). The black arrow corresponds to a 10-s stimulation with blue light beginning at 5 s. (B) Background-subtracted fast-scan cyclic voltammograms (average of 2 scans each, 200 V/s) corresponding to the measured peak current during blue light simulation of a mutant fly that consumed all-trans retinal (black line) and a mutant fly that did not consume all-trans retinal (gray line). (C) Representative background- subtracted fast-scan cyclic voltammogram of exogenously applied dopamine measured in the fly CNS for comparison (average of 5 scans, 200 V/s).

118 exogenously applied dopamine that has been measured in the fly CNS (Figure 5.3C), thus necessitating further characterization of the signal. It is possible that another, as yet unidentified, electroactive compound is released in this brain region.

The possibility of electrical interference from the direct exposure of the fly to a

LED light was examined. A TH-GAL4/UAS:ChR2 mutant fly was exposed to a 10-s blue light stimulation then 15 min later a 10-s red light stimulation as a control as it has been shown it does not stimulate ChR2 function in mutant Drosophila larvae (49, 50).

Flies were also tested with the red light first followed by the blue light to ensure the order of exposure to the two wavelengths of light did not alter the response of the fly. The cylindrical electrode remained untouched between light stimulations. Figure 5.4 compares two voltammograms obtained in a TH-GAL4/UAS:ChR2 mutant fly that consumed all-trans retinal. The voltammogram observed following blue light stimulation

(blue line) resembles the wave shape of dopamine, but again the shift in formal potential is evident. Red light stimulation (red line) does not cause any significant change in the measured current. Thus the shift in formal potential and the shape of the voltammogram appear to correspond to dopamine release in the fly and are not due to an electrical alteration caused from LED illumination.

Of note, two peak currents were measured in a TH-GAL4/UAS:ChR2 mutant fly that had consumed all-trans retinal which were approximately three times higher than the typical peak current recorded for the mutant flies (Figure 5.5A). The two increases in current appear to correspond to spontaneous release as they did not occur during the stimulation time period with blue light. A 10-s blue light stimulation was applied starting at 5 s; however, the peaks were recorded 55 s and 105 s later. The height of the two

119

Figure 5.4. Voltammograms obtained during blue and red light stimulation of a TH- GAL4/UAS:ChR2 mutant fly. Background-subtracted fast-scan cyclic voltammograms (average of 2 scans each, 200 V/s) in the MB region of a TH-GAL4/UAS:ChR2 mutant fly that consumed all-trans retinal. A trace obtained during the measured peak current from a 10-s blue light simulation (blue line) was compared with a trace obtained during a 10-s red light stimulation (red line).

120 peaks in Figure 5.5A corresponds to ~725 nM and ~945 nM dopamine if compared to in vitro calibration of the electrode (Appendix). Figure 5.5B is a background-subtracted cyclic voltammogram from the maximum current measured for the second peak in Figure

5.5A. By inspection, the anodic peak shape indicates dopamine is oxidized at the electrode surface.

Further characterization of the signal evoked with optogenetic stimulation in

Drosophila is necessary before the measured current can be convincingly attributed to dopamine. Cyclic voltammograms suggest an aspect of the signal is due to dopamine oxidation. Because the working electrode was placed in the MB brain region, signal from oxidation of octopamine, serotonin, and histamine which are electroactive species present mainly in other regions of the Drosophila brain, is unlikely (62). A possible reason for the +200 mV shift in formal potential of the voltammograms shown in this chapter is the difference in placement of the cylindrical electrode in the fly brain. In Chapters 2-4, the microelectrode was inserted into a cluster of dopamine neurons in the PAM area because the uptake of applied dopamine was being quantified. Dopamine neurons in the PAM area project to the MB region of the fly brain, meaning the release of endogenous dopamine occurs here (55). While the PAM and MB regions are just microns apart, the density of the two brain regions is slightly different. The measured signal at the working electrode is low, and the difference in density of the surrounding tissue might cause an observable effect on the measured current.

121

Figure 5.5. Spontaneous release of an electroactive species from a TH- GAL4/UAS:ChR2 mutant fly. (A) Current trace from a mutant fly showing two peaks that do not occur during the blue light stimulation time period. The black arrow corresponds to a 10-s stimulation with blue light beginning at 5 s. (B) Background- subtracted fast-scan cyclic voltammogram (average of 2 scans, 200 V/s) from the maximum measured current of the second peak shown in (A). The wave shape resembles that of dopamine.

122 Conclusions

Chemical, electrical, and optogenetic methods to induce endogenous dopamine release in the Drosophila CNS were studied. FSCV detection with a 5-µm carbon-fiber microelectrode was used to monitor dopamine changes. Methods successfully used in the rat brain for chemical and electrical stimulation of neurotransmitter release were modified for the nanoliter-sized fly CNS. ChR2, an emerging optogenetic tool for controlling neuronal release with blue light, was used to genetically target stimulation of dopaminergic neurons in Drosophila. Results suggest optogenetic stimulation is a useful and noninvasive technique for eliciting dopamine release in the fly CNS. Future investigation of Drosophila mutants genetically altered with the ChR2 ion channel could lead to progression in the novel field of optogenetic neuronal stimulation.

123 References

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128 Chapter 6: Development and Characterization of a Voltammetric

Carbon-Fiber Microelectrode pH Sensor*

*Reproduced with permission from Makos, M. A., Omiatek, D. M., Ewing, A. G., and Heien, M. L. (2010) Development and Characterization of a Voltammetric Carbon-Fiber Microelectrode pH Sensor, Langmuir, accepted. © 2010 American Chemical Society.

129 Introduction

Recently, there has been an interest in developing reagentless sensors to detect small pH changes in non-ideal environments (1). Carbon-based sensing materials are attractive substrates for this application since they are intrinsically biocompatible, conductive, and apt for surface modification. Indeed, ion-selective reporter molecules can be tethered onto a carbon surface through a variety of methods including chemical oxidation of the surface with corrosive acidic solutions and plasma treatment (2, 3), physical adsorption of organic precursors (4, 5), and electrochemically-assisted covalent attachment via the oxidation of amines (6-9) and reduction of diazonium salts (10-16).

Pioneered by Savéant and co-workers in the early 1990s, the reduction of aryl diazonium salts onto carbon surfaces is a well-characterized method for the selective in situ attachment of organic molecules (10). This mechanism involves the electrochemical generation of a solution radical from the diazonium modifier and subsequent covalent linkage to the carbon surface, which possesses marked stability to external stimuli (13).

Electrochemical measurements in the central nervous system (CNS) can quantify redox-active chemical messengers such as catecholamines and indolamines, which are thought to play a fundamental role in the physiological and behavioral aspects of organisms. In vivo voltammetry with carbon-fiber microelectrodes has been used for several decades to monitor neurotransmission of these chemicals in the CNS of various mammalian animal models (17-19). Neurosecretory events are often accompanied by a flux of endogenous species (e.g., H+, ascorbate) which can interfere with the voltammetric signature of the electroactive chemical species of interest (5, 20-26). Of particular interest are pH fluctuations in the surrounding matrix, which are thought to

130 occur as a result of metabolic processes that follow stimulated neurotransmitter release

(23, 27-29). Wightman and co-workers have reported measuring these small acidic pH changes in rat brain slices subjected to electrically stimulated secretion with liquid membrane, ion-selective microelectrodes (ISMs) (23). With the emergence of volume- limited, CNS-containing animal models such as the fruit fly, Drosophila melanogaster, comes the need to develop microanalytical tools capable of measuring the pH fluctuations associated with neurotransmission (30). I have discussed using fast-scan cyclic voltammetry (FSCV) for quantifying in vivo neurotransmitters in the CNS of Drosophila in Chapters 2-5 (31, 32); however, the electrode could not be used to measure fluctuations in the pH of the brain.

Voltammetric pH sensors measure changes in the redox-potential of a surface- bound, electrochemically active species as a function of pH. This methodology for measuring pH has been demonstrated with quinone-based surface modifications of various electrodes (33-36). In a recent study by Tommos and co-workers, the formal potential of a surface-bound quinone on a gold electrode shifted to more negative potentials with increasing solvent basicity (35). While a variety of quinone-modified electrodes have been reported to respond to pH, few have been developed on biocompatible materials that exhibit activity in a physiologically relevant pH range (1,

37). In this chapter, I will describe a procedure for electrochemically grafting Fast Blue

RR (FBRR) salt, a quinone-containing diazonium derivative, to a cylindrical carbon-fiber microelectrode. This results in a microelectrode capable of performing real-time, reagentless pH measurements in biological microenvironments. The redox response of the FBRR-functionalized electrode is characterized using FSCV in biological media set

131 to a physiologically relevant pH range. I demonstrate that modification of a carbon-fiber surface with FBRR is a simple and reproducible method for fabricating a stable pH sensor that is sensitive enough to measure dynamic physiological pH changes in the CNS of Drosophila that are associated with stimulated neurotransmitter release.

Methods

Chemicals. All chemicals were used as received unless otherwise stated. 4-

Benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi(zinc chloride) salt (Fast

Blue RR, FBRR, diazonium salt), tetraethylammonium tetrafluoroborate (TEABF4),

NaCl, KCl, CaCl2, MgCl2, NaHCO3, NaH2PO4, sucrose, Trizma base , and acetonitrile

(ACN, anhydrous, 99.8%) were obtained from Sigma Aldrich (St. Louis, MO). Adult- hemolymph like (AHL) saline (108 mM NaCl, 5 mM KCl, 2 mM CaCl2, 8.2 mM MgCl2,

4 mM NaHCO3, 1 mM NaH2PO4, 5 mM trehalose (Fluka BioChemika, Buchs,

Switzerland), 10 mM sucrose, 20 mM Trizma base , pH 7.5) was made using ultrapure

(18 MΩ cm) water and filtered through a 0.2-μm filter (38). The pH of AHL solutions was adjusted with 0.5 M NaOH and HCl.

Electrode preparation. Cylindrical carbon-fiber microelectrodes were fabricated as described in Chapter 2 (31). Briefly, a 5-μm diameter carbon fiber (T-40 12K, Amoco,

Greenville, SC) was aspirated into a borosilicate glass capillary (1B100-4, World

Precision Instruments, Inc., Sarasota, FL) and sealed using a regular glass capillary puller

(P-97, Sutter Instruments, Novato, CA). The carbon fiber was trimmed to a length of either 50 or 200 μm measured from the glass junction. Electrical contact was made by back-filling the capillary with a silver composition (4922N DuPont, Delta Technologies

132 Ltd., Stillwater, MN), followed by insertion of a tungsten wire, resulting in a 5 µm diameter cylindrical carbon-fiber microelectrode. The 200-µm long cylindrical electrodes were used for all characterization experiments while the 50-µm long electrodes were used for the in vivo Drosophila application.

Chemical modification of the carbon-fiber microelectrode surface. FBRR salt was electrochemically grafted onto the carbon-fiber microelectrode surface using diazonium attachment chemistry. Deposition of the diazonium salt onto the carbon-fiber microelectrodes was carried out using cyclic voltammetry performed with an Ensman

Instruments EI400 microelectrode potentiostat (Bloomington, IN) operated in the two- electrode mode. A 2 mM solution of FBRR salt was prepared in ACN containing 0.1 M

TEABF4. Solutions were purged with Ar (g) for 5 min prior to deposition in order to eliminate signal from the reduction of O2. Electrodes were electrochemically modified via reduction of the diazonium onto the carbon surface by scanning from +0.4 V to -0.8 V vs. Ag QRE (3 mm diameter, Bioanalytical Systems, West Lafayette, IN) at 0.5 V/s.

Data were collected and processed using LabView 8.0 software (National Instruments,

Austin, TX) written in-house. Electrode surface coverage was calculated by subtracting the background current measured for a solution of 0.1 M TEABF4 in ACN from that due to deposition of the diazonium. The typical surface coverage obtained using the experimental conditions listed above for a microelectrode with a 200-µm long carbon fiber was ~20 nmol/cm2.

Electrochemical measurements. Voltammetric responses of the diazonium-modified electrodes as a function of pH were collected using either a Dagan Chem-Clamp potentiostat (Dagan Corporation, Minneapolis, MN) or a flow-injection analysis

133 apparatus with a current amplifier (428, Keithley Instruments, Inc., Cleveland, OH).

Both systems were run by the TH 1.0 CV program (ESA, Chelmsford, MA) (39) coupled with two data acquisition boards (PCI-6221, National Instruments). A Ag/AgCl electrode, which served as the reference in all experiments following the initial deposition of FBRR, was made by chloridizing a silver wire (0.25 mm diameter,

99.999% purity, Alfa Aesar, Ward Hill, MA). Electrodes were positioned using x,y,z- micromanipulators (421 series, Newport, Irvine, CA). All cyclic voltammograms were obtained using a triangular waveform scanned from -0.7 to +0.8 V vs. Ag/AgCl at 20 V/s and repeated every 200 ms unless otherwise noted. Electrochemical responses were plotted and statistical analysis performed using Prism 5.0 (GraphPad Software, La Jolla,

CA). Anodic peak potentials (Epa) were determined using a fifth order polynomial fit from LabView 8.0 software written in-house. Cyclic voltammetry was used to estimate the heterogeneous electron-transfer rate constant, k0, for this system via the method of

Nicholson (40).

In vivo Drosophila preparation. As in Chapter 5, female flies carrying

Channelrhodopsin-2 (ChR2), a light activated ion channel, were crossed with male flies expressing tyrosine hydroxylase (TH) to produce mutant flies containing dopaminergic neurons that can be controlled through blue light stimulation (TH-GAL4/UAS:ChR2 genotype) (41). Male mutant flies, 3-7 days old, were maintained at 25 °C in the dark and fed yeast containing 10 mM all-trans retinal (light sensitive chemical necessary for

ChR2 function) for 2 days prior to experimentation. Blue light was applied through computer control of a 3-W Luxeon Star LED with a peak intensity of ~470 nm (LXHL-

LB3C, Newark, Chicago, IL). Flies were prepared as described in Chapter 2 for in vivo

134 FSCV measurements (31). Briefly, ice was used to temporarily immobilize flies before they were mounted in a homemade collar (38.1 mm diameter concave plexiglass disk with a 1.0 mm hole in the center) with low melting agarose (Fisher Scientific, Pittsburgh,

PA). Microsurgery was performed on a stereoscope (Olympus SZ60, Melville, NY) to remove the cuticle from the top portion of the head, thus exposing the brain region. The head was covered with 0.1% collagenase solution for 30 min to relax the extracellular matrix in the brain then rinsed and bathed with AHL saline with the preparation maintaining its viability for 1.5 - 2.5 h.

Results and Discussion

Deposition of FBRR diazonium salt onto a carbon-fiber microelectrode surface.

FBRR was electrochemically reduced onto a carbon-fiber surface using cyclic voltammetry by scanning from +0.4 V to -0.8 V vs. Ag QRE at a rate of 0.5 V/s in a 2 M

FBRR/0.1 M TEABF4/ACN solution. The proposed mechanism for this deposition is presented in Scheme 6.1. A representative voltammogram of the diazonium salt reduction onto a cylindrical carbon-fiber microelectrode is shown in Figure 6.1A (blue trace). An irreversible reductive wave is observed around -0.5 V which is attributed to the solution radical formation of the diazonium derivative and its subsequent covalent linkage to the carbon-fiber surface as reported for a similar molecule (12).

The charge (Q) of the diazonium deposited onto the surface is quantified using the current-time integral of the voltammetric trace. The slight charge observed from the solvent background (black trace) has been subtracted from the charge due to diazonium deposition (blue trace). Faraday’s Law (Q = nNF) is used to convert Q to the number of

135

Scheme 6.1. Electrochemical deposition of FBRR salt onto the carbon-fiber microelectrode surface.

136

Figure 6.1. Cyclic voltammograms of a carbon-fiber microelectrode before and after FBRR attachment. (A) Background charge of the carbon-fiber electrode in solvent only (black line). Electrochemical reduction of FBRR on the carbon-fiber surface (blue line). Diazonium concentration = 2 mM in 0.1 M TEABF4/ACN. The potential is scanned +0.4 V to -0.8 V vs. Ag QRE at 0.5 V/s. (B) Cyclic voltammograms (average of 5 scans each) in AHL saline of a bare carbon-fiber microelectrode (dashed black line) and the same carbon-fiber microelectrode after modification with FBRR (solid blue line). The potential is scanned -0.7 V to +0.8 V vs. Ag/AgCl at 20 V/s.

137 moles of diazonium (N) deposited onto the carbon-fiber surface. In this equation the number of electrons exchanged in the reduction reaction, n, is 1, and F is Faraday’s constant (96,485 C/mol). The surface coverage of diazonium on the electrode is calculated by dividing the number of moles of FBRR by the geometric area of the 200

m cylinder (3.2 x 10-5 cm2). This results in a typical coverage of 20 nmol/cm2. As a point of reference, usual monolayer coverage for a small, surface-bound organic molecule has been reported as 300 pmol/cm2 (42). Therefore, this suggests a multilayer deposition of FBRR onto the sensor, a result commonly observed for the reduction of aryl diazonium salts onto carbon surfaces (13). The amount of FBRR deposited onto the carbon-fiber surface (Table 6.1) is dependent on both the scan rate and potential window of the voltammetric sweep. The voltammetric deposition of the diazonium is a time- dependent process; therefore, scanning at slower rates or to an extended negative waveform potential increases the amount of FBRR deposited onto the electrode surface.

There is no significant effect of varying the concentration of diazonium in solution (0.5-

5.0 mM) on the amount of FBRR deposited onto the electrode.

The presence of FBRR on the surface has been investigated using FSCV. In

Figure 6.1B, cyclic voltammograms recorded at a bare carbon-fiber microelectrode

(dashed black line) and the same microelectrode following modification with FBRR

(solid blue line) show a clear indication of the presence of the electroactive diazonium salt on the electrode surface. The voltammogram of the FBRR redox system signifies quasireversible behavior with an apparent formal potential of -0.1 V in AHL saline at physiological pH. Integration of the oxidative peak area from the redox-active molecule in Figure 6.1B results in an observed surface coverage of 40 pmol/cm2. This is

138 Table 6.1. Effect of varying voltammetric deposition parameters for FBRR reduction onto a carbon-fiber surface.

Scan rate (V/s) Potential window (V vs. Ag QRE) Γ (nmol/cm2) 0.500 +0.4 → -0.2 2.7 ± 0.9 0.500 +0.4 → -0.4 9.9 ± 3.5 0.500 +0.4 → -0.6 14.5 ± 1.4 0.500 +0.4 → -0.8 21.5 ± 2.7 0.500 +0.4 → -1.0 25.0 ± 2.7 0.050 +0.4 → -0.8 29.4 ± 6.4 0.100 +0.4 → -0.8 24.4 ± 1.2 0.500 +0.4 → -0.8 20.0 ± 2.1 1.0 +0.4 → -0.8 19.7 ± 0.6 5.0 +0.4 → -0.8 9.9 ± 1.4

[FBRR] = 2 mM in 0.1 M TEABF4/ACN. Error is SEM with n = 3 electrodes for each measurement.

139 approximately two orders of magnitude smaller than that calculated from the diazonium deposition in Figure 6.1A. A proposed mechanism for the oxidation-reduction reaction of the surface-bound quinone derivative is illustrated in Scheme 6.2. It is thought that voltammetric cycling of this molecule initially induces a two-electron/two-proton oxidation to convert the p-methoxy moiety on the conjugated ring to its p-quinone analogue. The quinone is then chemically reduced in a two-electron exchange to form the hydroxy derivative of the molecule. Using the method of Nicholson (40), the heterogeneous electron-transfer rate constant, k°, is determined to be 0.13 cm/s. This indicates that the FBRR undergoes outer-sphere electron transfer on the carbon-fiber surface, which is consistent with previous studies that have examined electron transfer kinetics over a wide insulating layer (43).

Electrochemical characterization of the FBRR microelectrode pH sensor. The effect of scan rate on the electrochemistry of a FBRR-modified carbon-fiber microelectrode has been investigated using FSCV. Cyclic voltammograms of a FBRR microelectrode in pH

7.5 AHL saline solution at scan rates of 10, 20, and 50 V/s are plotted in Figure 6.2A.

Because current is directly proportional to scan rate, the current scale on the y-axis has been divided by scan rate to provide a straightforward comparison of the peak positions at the different scan rates. Notably, neither the anodic peak potential (Epa) nor the cathodic peak potential (Epc) significantly shifts in value while varying scan rate in this range. At scan rates higher than 100 V/s (up to 350 V/s), the Epa becomes more difficult to identify due to a decrease in the ratio of the faradaic to the capacitive current. By inspection, the Epa is well resolved from the background current at 20 V/s, a scan rate that should suffice for monitoring rapidly occurring neurosecretory

140

Scheme 6.2. Proposed mechanism for the oxidation-reduction reaction of the surface- bound quinone derivative of FBRR.

141 events during in vivo applications. Therefore, this scan rate has been chosen to monitor pH changes for the remainder of this chapter. Furthermore, the anodic peak current vs. scan rate (Figure 6.2B) is linearly dependent for scan rates 10 – 350 V/s (r2 > 0.99). This confirms that the oxidation and reduction of FBRR is a surface-confined reaction, as expected, and provides evidence that the diazonium compound is sufficiently tethered to the carbon-fiber surface.

The long-term stability of the FBRR microelectrode pH sensor has been studied by continuously cycling modified electrodes in pH 7.5 AHL saline solution for 2.5 h (-0.7 to +0.8 V vs. Ag/AgCl at 5 Hz). This corresponds to 45,000 voltammetric sweeps over the 2.5 h period. An 8% decrease in peak current is observed during the first 10 min of cycling (Figure 6.2C). During the remaining 2.5 h, the peak current remains fairly stable, decreasing by an additional 13%. Therefore, the stability of the FBRR-modified microelectrode provides an ample time window for monitoring the pH in the CNS of

Drosophila during in vivo electrochemical measurements.

The selectivity of the sensor for H+ has been investigated to determine if alternate ionic species present in the biological media could interfere with the voltammetric response. To accomplish this, FBRR microelectrodes (n = 3) have been tested with

FSCV in a series of AHL saline solutions that contained elevated concentrations of various inorganic cations. When the Na+ concentration in the AHL saline solution is

2+ increased by 40%, the Epa remains unaltered. In addition, increasing the Mg concentration by 45%, Ca2+ concentration by 50%, or K+ concentration by 60% does not cause a shift in the Epa. These studies validate that changes in the concentration of these four cations do not contribute to the measured shift in the Epa, which suggests charged

142

Figure 6.2. Electrochemical characterization of the FBRR microelectrode pH sensor in pH 7.5 AHL saline solution. (A) Cyclic voltammograms (average of 5 scans each) of the FBRR redox couple at 3 scan rates. The current scale on the y-axis has been divided by scan rate so the peak positions can be easily compared between the different scan rates. In this scan rate range, neither the anodic peak potential (Epa) nor the cathodic peak potential (Epc) significantly shifts in value. (B) Anodic peak current (ipa) as a function of scan rate (SEM bars are too small to see). (C) The effect of continuous cycling of the electrode on ipa. SEM bars are too small to see (n = 3).

143 species, other than H+ ions, in the AHL saline solution are not affecting the pH response of the FBRR microelectrode. pH response of the FBRR microelectrode sensor. To calibrate the voltammetric response of the sensor, the FBRR-modified carbon-fiber microelectrode has been investigated in AHL saline solutions of varying pH. The peak characteristics of cyclic voltammograms recorded over a pH range of 5.0 – 9.0 with a scan rate of 20 V/s have been examined. Figure 6.3 depicts representative voltammograms of the FBRR microelectrode in three different pH solutions. The Epa noticeably shifts to more negative potentials as pH is increased. The Epc follows the same trend with pH as the Epa; however, the peak becomes difficult to distinguish from the background current in higher pH solutions ( pH 8), as reported previously for chemically modified electrodes in physiological media (5). Therefore, the Epa was chosen as the identifier for the sensor calibration and subsequent in vivo studies instead of the formal potential.

FSCV has been used to determine the response of the sensor to pH changes. In a pH range of physiological relevance (6.5 – 8.0), a sigmoidal fit best describes the relationship between Epa and pH (Figure 6.4, n = 9 electrodes). Linear regression of these data yields a slope of 38 mV/pH unit which is less than the theoretical value of 59 mV/pH unit for a reversible, two-electron/two-proton redox reaction at room temperature

(44). This deviation from the predicted Nernstian value suggests the attachment of FBRR to the carbon-fiber surface alters the electrochemistry of the quinone couple. pH- sensitive, glassy carbon electrodes tethered with alternative reporter molecules have been previously fabricated that exhibit expected Nernstian behavior, but practical limitations, such as high capacitive currents, larger diameters (millimeter), and lengthy time scales to

144

Figure 6.3. Cyclic voltammograms of a microelectrode modified with FBRR in AHL saline solutions of different pH. Asterisk (*) corresponds to the Epa for each voltammogram (20 V/s, average of 5 scans) with the dashed vertical line included for comparison purposes. As the pH increases, the Epa visibly shifts to more negative potentials. (A) pH 6.5 (B) pH 7.5 (C) pH 8.0.

145

Figure 6.4. The anodic peak potential, Epa, as a function of AHL saline solution pH for FBRR-modified electrodes. The Epa has a sigmoidal relationship with changing pH in a physiological relevant pH range (6.5-8.0). Error bars are SEM (n = 9 electrodes).

146 obtain stable readings, limit their biological usefulness (34, 36). For example, Shiu et. al. have reported the development of a glassy carbon electrode (3 mm diameter) modified by adsorption of an anthraquinonesulfonate film that possessed a near Nernstian slope of

56.4 mV/pH unit in aqueous pH buffers (34). However, it would not be feasible to use an electrode of this size to measure dynamic events associated with in vivo neurosecretion in volume-limited model systems such as Drosophila.

Microelectrode response time to a pH change. Flow-injection analysis has been used to study the dynamic response of the sensor by introducing plugs of AHL saline solution of varying pH to a FBRR-modified microelectrode. Ideally, a fast electrode response time to a minor change in pH of the surrounding solution would produce a square-shaped

Epa vs. time trace. Figure 6.5 shows the Epa response of the modified electrode sensor to

0.2 pH unit changes. After initial immersion in an AHL saline solution of pH 7.4, the electrode is exposed to a bolus of AHL saline solution of pH 7.2 (Figure 6.5A).

Likewise, AHL solution of pH 7.6 is introduced to the electrode in pH 7.4 solution in

Figure 6.5B. By inspection, the modified electrode response to a 0.2 change in pH is square-like and consistent for measurement of either an acidic or a basic pH change.

Indeed, flow-injection calibration of the sensor revealed marked sensitivity for H+ with the modified electrode capable of detecting pH changes as small as 0.005 (based on S/N

3) with a time response equal to 1.6 s (τ determined from exponential decay).

Measuring dynamic in vivo pH changes in the Drosophila CNS. I utilized the pH electrode to monitor a dynamic pH change associated with neurotransmitter release in the fly brain. Optogenetic stimulation using blue light with the mutant fly TH-

GAL4/UAS:ChR2 has been demonstrated to evoke dopamine release in Drosophila

147

Figure 6.5. Plot of anodic peak potential vs. time during flow injection changes of 0.2 pH units in AHL saline. The electrode is able to consistently measure either an acidic or a basic pH change. (A) Initial AHL saline solution of pH 7.4 is decreased to pH 7.2. (B) Initial AHL saline solution of pH 7.4 is increased to pH 7.6.

148 larvae by Venton and co-workers (45). In addition, a procedure for using optogenetic stimulation with adult Drosophila was described in Chapter 5. The TH-

GAL4/UAS:ChR2 mutant fly expresses blue light sensitive cation channels which are specific to dopaminergic neurons, allowing dopamine release to be elicited through timed blue light stimulations. Following microsurgery, a micromanipulator is used to insert a cylindrical FBRR-modified electrode into the CNS region of an adult mutant fly. A 5-s stimulation with blue light is used to induce neurotransmitter release which causes a change in Epa corresponding to an ~0.034 acidic pH change in the fly (Figure 6.6, solid red line). This value is in agreement with pH fluctuations observed as a result of stimulus-induced neurosecretion in rat brain slices (0.047 unit pH change in the cortex) reported by the Wightman lab using ISMs (23). To ensure the response is due to a biological change in the fly, the experiment has been repeated with the electrode in the surrounding solution outside of the fly brain (solid black line). This experiment demonstrates the high temporal sensitivity of the FBRR sensor and highlights its utility for real-time analyses of pH fluctuations associated with neurotransmitter release in volume-limited biological microsystems.

Conclusions

A carbon-fiber microelectrode pH sensor was developed via the voltammetric reduction of the FBRR diazonium salt. The stability and sensitivity of the sensor for H+ was characterized in biological media set to a physiologically relevant pH range. FSCV was used to probe the surface-bound diazonium derivative as a function of pH. The peak corresponding to Epa for the FBRR-modified electrode was correlated to small changes in

149

Figure 6.6. Physiological pH measurements in an adult Drosophila CNS. A representative trace of a dynamic, acidic pH change associated with neurotransmitter release being measured with a FBRR-modified electrode in a mutant fly CNS (red line). A control stimulation of the same electrode in AHL saline solution only is plotted for comparison (black line). The black arrow corresponds to a 5-s stimulation with blue light.

150 pH. Flow-injection analysis was used to characterize the temporal response of the sensor in solutions of varying pH, resulting in a limit of detection to 0.005 pH units.

Furthermore, direct in vivo measurements of pH were made in the Drosophila CNS after stimulated neurotransmitter release, revealing an acidic change in a brain region dominated by dopaminergic neuron innervations. These data demonstrate the utility of this easily fabricated sensor for measuring dynamic changes in extracellular pH in the fly and other microanalytical animal models.

151 References

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154 Chapter 7: Future Directions for Quantifying Neurochemicals in

Drosophila Using Electrochemical Detection

155 Drosophila is a useful model system for studying several human physiological processes including addiction. Many central nervous system (CNS) pathways in flies and mammals are evolutionarily conserved because of the genetic similarity between the two species. Research has demonstrated fruit flies exhibit behavioral responses to psychostimulants that are amazingly comparable to human behaviors. The overall goal of my thesis was to develop methods capable of quantifying neurochemicals in

Drosophila. Chapters 2-4 were focused on measuring changes in uptake of exogenously applied dopamine in the fly CNS in the presence of drugs of abuse. Approaches for stimulating release of endogenous dopamine in Drosophila were investigated in Chapter

5. Chapter 6 described the development of a microelectrode pH sensor for monitoring in vivo pH fluctuations associated with neurotransmitter release. These methods could lead to a more analytical view of the basis behind addiction. In this chapter, I will discuss the future directions of this project with respect to three aspects: biological application, kinetics of dopamine uptake, and stimulating dopamine release.

Investigating Alcohol Addiction with Drosophila

The majority of the applications discussed in my thesis involved cocaine and amphetamine addiction. One future application of this project is to utilize the tools I developed in conjunction with recently identified Drosophila mutants to investigate the mechanisms underlying alcohol tolerance and abuse.

Addiction is defined as compulsive drug use that has escalated to a level the user can no longer control with drug use persisting despite significant negative consequences.

One mechanism of addiction is activation of dopaminergic fibers in the brain (1).

156 Additional psychomotor actions occur which vary depending on the particular addictive substance in question. Ethanol affects γ-aminobutyric acid (GABA) receptors in the CNS by increasing their usual function (2-4). Because GABA is an inhibitory neurotransmitter, this leads to a depression in CNS activity. In addition, ethanol interacts with N-methyl-D-aspartate (NMDA) receptors. This prevents the action of glutamate, an excitatory neurotransmitter, on NMDA receptors which causes a further decrease in CNS function (5, 6). In the brain, extracellular dopamine increases with alcohol consumption because ethanol is thought to stimulate dopamine release in certain regions of the CNS

(7, 8).

While the main actions of ethanol in the brain are known, less is understood regarding the changes in neuronal activity following short-term ethanol exposure and their contribution towards alcohol tolerance and sensitivity (9). Alcohol addiction has a strong genetic correlation, and there remains much to be discovered about the specific genes that increase the genetic risk of a person developing an addiction to alcohol as well

(10, 11). Because the Drosophila genome possesses little genetic redundancy, it is an attractive model system for identifying individual genes that influence particular behaviors (12, 13). Additionally, the behavioral response of flies to ethanol has been shown to model that of mammals (14-16).

Recently, several mutant fly types have been developed by Heberlein and coworkers that exhibit unique behavioral responses toward alcohol consumption (17-19).

Table 7.1 is a summary of the modified behaviors the genetically altered flies cheapdate, tipsy, barfly, and hangover display following exposure to ethanol. The effects of ethanol on fly behavior were measured using a fly inebriometer (Figure 7.1). This home built

157

Table 7.1. Drosophila mutants that display altered behavioral responses to ethanol.

Fly mutant Modified behavior References

increased sensitivity to alcohol (17) cheapdate

tipsy increased sensitivity to alcohol (18)

barfly reduced sensitivity to alcohol (18)

reduced development of hangover (19) tolerance to alcohol

158

Figure 7.1. The fly inebriometer. This device is used to measure changes in fly postural control upon ethanol exposure. (Reprinted from (18), with permission from John Wiley and Sons).

159 apparatus allows the ethanol-induced loss of postural control of flies to be quantified (18,

20). Flies are introduced into the top of a 4-ft glass column where a controlled concentration of ethanol is circulated. Over time, flies become intoxicated and lose their postural control. They tumble down the column where they elute out the bottom and are counted. Fly research labs can analyze the effects of ethanol on the motor control of hundreds of flies simultaneously with this device.

A future proposal might be to use in vivo electrochemical detection to quantify uptake of exogenously applied dopamine in the mutant flies cheapdate, tipsy, barfly, and hangover. The mutants could also be studied following short-term exposure to ethanol in a fly inebriometer. Comparison of these data to wild-type flies exposed to identical ethanol concentrations will provide information about the neurochemical changes behind the altered behavioral response of the mutant flies toward ethanol. As evidence suggests that neurotransmitter systems affected by ethanol are conserved between flies and humans (21), this could potentially lead to a better understanding of the role genes affecting dopamine neurotransmission play in alcohol addiction. While no animal model is a perfect model for alcoholism, utilizing the genetic advantages of the fruit fly will allow aspects of this complex disease to be studied and will give insight into the effects of ethanol on the CNS.

Quantifying the Kinetics of Dopamine Uptake in Drosophila

In vivo uptake has been characterized in the rat brain using experimental data and has also been modeled by simulations (22-26). Following the work reported in this

160 thesis, a future investigation might be to model dopamine uptake in the Drosophila CNS, and to compare it with experimentally obtained results.

Modeling neurotransmitter uptake involves understanding the relative importance of diffusion vs. uptake processes which can be mathematically examined with the classic

Michaelis-Menten equation (27). Figure 7.2A is a depiction of a typical Michaelis-

Menten plot that can be used to determine kinetic parameters, such as Vmax and Km, for simple kinetic behavior. Michaelis-Menten kinetics involves assumptions based on

Fickian diffusion, which must be kept in mind when examining physiological processes.

It has been demonstrated that ion diffusion through medium as complex as the extracellular space of the brain cannot necessarily be assumed to obey Fick’s Laws (28,

29). Tortuosity, or the extent to which diffusing particles are hindered by obstructions in their path, has been shown to affect small cations moving through extracellular space. In addition, volume averaging takes into account the variations in extracellular vs. intracellular space. It has been suggested that equations originally developed to describe macroscopic problems are valid to describe uptake in the CNS when tortuosity and volume averaging are taken into account (28, 30, 31).

A suggestion for a future direction is to model dopamine uptake and quantify the kinetic parameters Vmax and Km for Drosophila to provide a better understanding of in vivo measurements in the fly CNS. Quantification of the measured dopamine signal can be divided into two phases: the rising phase and the falling phase (Figure 7.2B). The rising phase consists of the amount of dopamine that is transported into the tissue and is oxidized at the electrode surface. The falling phase of the signal is due to the clearance of dopamine from the tissue. This is a combination of the uptake, metabolism, and

161

Figure 7.2. Modeling dopamine uptake. (A) The classic Michaelis-Menten plot for determining kinetic parameters Vmax and Km. (B) Representative signal measured during dopamine uptake.

162 diffusion of dopamine out of the tissue, making the falling phase of the signal a nontrivial component to accurately model.

Improving the Detection of Stimulated Dopamine Release in Drosophila

The electrochemical measurements of dopamine reported in my thesis were made with a 5 µm diameter carbon-fiber electrode. This ~50 µm long cylindrical microelectrode adequately detected the changes in uptake of exogenously applied dopamine described in Chapters 2-4; however, the measured signals from dopamine released via optical stimulation in Chapter 5 were less robust. It is likely that when the endogenous dopamine released from the fly CNS reaches the microelectrode surface it is of a lower concentration than the dopamine measured in exogenously applied experiments. A future step in the analytical development of measuring optically stimulated dopamine release in Drosophila is to increase the sensitivity of the working electrode to improve in vivo detection limits for dopamine.

The sensitivity of carbon-fiber microelectrodes is dependent on several properties of the electrode including the size and the surface roughness or chemistry. Increasing electrode sensitivity by using a methane/oxygen flame to etch carbon-fiber electrodes down to < 1 µm diameters has been demonstrated previously by our laboratory (32).

Decreasing the overall size of the working electrode results in lower signal due to background current since double layer charging current is proportional to surface area, as well as lower signal arising from the analyte of interest (33). By increasing surface roughness of a carbon-fiber electrode of a given size, the sensitivity for measuring dopamine can be improved. Electrochemical over-oxidation of carbon increases the

163 surface roughness which allows the addition of more oxide groups (34, 35). These oxide groups act as adsorption sites for cationic species, such as dopamine, thus leading to increased sensitivity of the electrode. Typically, high positive potentials are avoided when using carbon electrodes in biological applications to prevent the interference of water oxidation which has been observed to cause instability and inactivation of pyrolytic graphite electrodes (36). Several groups have reported nanomolar detection limits for dopamine using carbon-fiber microelectrodes scanned to a positive potential of 1.4 V instead of the more traditional 1.0 V (37-39). In addition, carbon-fiber microelectrodes that were both flame-etched and electrochemically overoxidized have been successfully used to measure in vivo dopamine concentrations of < 25 nM in the rat CNS (39).

A future direction here would be to use a methane/oxygen flame to etch a 5 µm diameter working electrode down to ~1 µm diameter. This electrode would then be used as the working electrode to measure dopamine release in vivo the Drosophila CNS following optical stimulation. In addition, the anodic scanning potential of the applied waveform would be increased to 1.4 V. Preliminary work measuring dopamine using fast-scan cyclic voltammetry with a 5-µm carbon-fiber microelectrode scanned to 1.4 V has demonstrated a significant increase in signal over dopamine measured with a waveform scanned to 1.0 V (Figure 7.3A). An improvement in voltammogram shape has been observed for exogenously applied dopamine measured in the Drosophila mushroom bodies when the applied waveform is extended to 1.4 V as well (Figure 7.3B).

Decreasing the size of the electrode and extending the waveform to a more positive potential will improve detection limits for measuring optically stimulated dopamine release in Drosophila.

164

Figure 7.3. Voltammetric measurements of dopamine using an applied waveform of 1.0 V vs. a waveform extended to 1.4 V. (A) Comparison of an identical dopamine concentration measured by a 5-µm carbon-fiber microelectrode scanned -0.6 V to 1.0 V vs. -0.6 V to 1.4 V (mean ± SEM; Student’s t-test, p = 0.0004 (***), n = 4 measurements for each potential). (B) Cyclic voltammograms (200 V/s, average of 5 scans each) of exogenously applied dopamine measured in vivo the Drosophila CNS with an applied waveform of 1.0 V (black line) and a waveform extended to 1.4 V (red line).

165 References

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168 Appendix

1) Calculations for exogenously applied dopamine: The radius was determined by ejecting the dopamine solution into mineral oil and measuring the diameter of the bubble that formed. This was tested for both the single-barrel and three-barrel glass micropipets. Micropipets were manually cut so each opening would be ~ 5 μm. volume = V = 4/3πr3 = 4/3π(0.065 cm / 2)3 V = 1.44x10-4 cm3 ~ 150 nL

150 nL of 1.0 mM DA = 150 pmol dopamine applied

2) Electrode calibration plot:

(nA) slope = 0.69 ± 0.04 nA/μM

m ax 20 i r2 = 0.98 n = 3 10

0 0 10 20 30 40 50 [DA] M

3) FSCV volume sampled: Diffusion layer radius (δ) = (2Dt)1/2 = (2* 5x10-6 cm2/s * 0.007 s)1/2 = 2.6x10-4 cm δ + radius carbon fiber = r = 2.6x10-4 cm + 2.5x10-4 cm = 5.1x10-4 cm volume δ and carbon fiber = V = πr2h = π(5.1x10-4 cm)2 * 5.0x10-3 cm = 4.1 pL volume carbon fiber = V = πr2h = π(2.5x10-4 cm)2 * 5.0x10-3 cm = 0.98 pL volume sampled = 4.1 pL – 0.98 pL ~ 3 pL

Amperometry volume sampled: Diffusion layer radius = δ ~ 6r (where r = radius of carbon fiber) = 6 * 2.5x10-4 cm = 1.5x10-3 cm δ + radius carbon fiber = r = 1.5x10-3 cm + 2.5x10-4 cm = 1.8x10-3 cm volume δ and carbon fiber = V = πr2h = π(1.8x10-3 cm)2 * 5.0x10-3 cm = 50.1 pL volume carbon fiber = V = πr2h = π(2.5x10-4 cm)2 * 5.0x10-3 cm = 0.98 pL volume sampled = 50.1 pL – 0.98 pL ~ 50 pL

169 Vita: Monique Adrianne Makos

EDUCATION: Ph.D. in Chemistry, May 2010, The Pennsylvania State University B.S. in Chemistry, May 2005, The University of Texas at Austin

AWARDS: Society for Electroanalytical Chemistry Graduate Student Award, SEAC (2010) Norma Robinson Award for outstanding graduate research, PSU (2009) Travel Award for oral presentation, PSU (2007) The Roberts Award for select incoming graduate students, PSU (2005)

PUBLICATIONS: Makos MA, Heien ML, Ewing AG “Oral Administration of Methylphenidate Blocks the Effect of Cocaine on Uptake at the Drosophila Dopamine Transporter” ACS Chemical Neuroscience, in preparation.

Makos MA, Omiatek DM, Ewing AG, Heien ML “Development and Characterization of a Voltammetric Carbon-Fiber Microelectrode pH Sensor” Langmuir, accepted.

Makos MA, Han KA, Heien ML, Ewing AG “Using In Vivo Electrochemistry to Study the Physiological Effects of Cocaine and Other Stimulants on the Drosophila melanogaster Dopamine Transporter” ACS Chemical Neuroscience 2010, 1, 74-83.

Makos MA, Kuklinski NJ, Berglund EC, Heien ML, Ewing AG “Chemical Measurements in Drosophila” TrAC Trends in Analytical Chemistry 2009, 28, 1223-1234.

Makos MA, Kim YC, Han KA, Heien ML, Ewing AG “In Vivo Electrochemical Measurements of Exogenously Applied Dopamine in Drosophila melanogaster” Analytical Chemistry 2009, 81, 1848-1854.

ORAL PRESENTATIONS: Makos MA, Heien ML, Han KA, Ewing AG “Quantifying Real-Time Neurotransmitter Changes in the Central Nervous System of Drosophila melanogaster Using Fast-Scan Cyclic Voltammetry” ACS invited session at Pittcon, Orlando, FL. March 2010.

Makos MA, Kim YC, Han KA, Heien ML, Ewing AG “In Vivo Electrochemical Monitoring of Dopamine Uptake in Drosophila melanogaster” Pittcon, Chicago, IL. March 2009.

Makos MA, Kim YC, Han KA, Ewing AG “In Vivo Electrochemistry in the 8-nL Brain of the Fruit Fly” Pittcon, Chicago, IL. February 2007.