in vivo electrochemical measurements in
TRANSCRIPT
The Pennsylvania State University
The Graduate School
Department of Chemistry
IN VIVO ELECTROCHEMICAL MEASUREMENTS IN
DROSOPHILA MELANOGASTER
A Dissertation in
Chemistry
by
Monique Adrianne Makos
© 2010 Monique Adrianne Makos
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
May 2010
ii
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
iii
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
iv
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.
v
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
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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.
vii
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
viii
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
ix
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
x
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
xi
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
xii
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
xiii
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
xiv
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
xv
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
xvi
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.
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, 2
nd, and 3
rd 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.
2
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
8
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
12
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,
17
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.
20
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.
23
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.
25
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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
38
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
40
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
42
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
44
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,
46
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
49
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
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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
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 composition (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. 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
61
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
63
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
65
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).
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. 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).
68
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
70
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
73
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
Table 3.1. Change in [DA]max for four drugs of abusea.
cocaine amphetamine methamphetamine methylphenidate
IC50 (µM)
Drosophila
dopamine
transporter
6.0b
2.7c
4.9b
(+) 6.6; (-) 34.0c
4.5b 6.8
b
wild type:
[DA]max
normalized (20
min treatment)
223 ± 40 % 117 ± 8 % 129 ± 22 % 174 ± 31 %
fmn mutant:
[DA]max
normalized (20
min treatment)
91 ± 8 % 102 ± 8 % 99 ± 4 % 102 ± 11 %
aMaximum changes for dopamine ([DA]max) values are for (+) amphetamine and (+)
methamphetamine. [DA]max values are mean ± SEM for 1.0 mM drug concentrations (n
= 5-6). Literature IC50 values are included for comparison. bReference (56).
cReference
(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
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Lundberg, L. M. F., Carvelli, L., Javitch, J. A., and Galli, A. (2000)
Amphetamine-induced loss of human dopamine transporter activity: An
internalization-dependent and cocaine-sensitive mechanism, Proc. Natl. Acad.
Sci. U.S.A. 97, 6850-6855.
51. Melikian, H. E., and Buckley, K. M. (1999) Membrane trafficking regulates the
activity of the human dopamine transporter, J. Neurosci. 19, 7699-7710.
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(2001) Methamphetamine-induced rapid and reversible changes in dopamine
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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
90
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
92
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
95
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.
97
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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
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Table 5.1. Eliciting dopamine release via chemical stimulation.
Stimulant Action References
potassium depolarizes cell membrane
in vivo rat and mouse CNS (21, 22)
in vitro rat brain tissue (23)
veratridine depolarizing agent that acts as a
sodium channel agonist
in vivo rat CNS (24)
in vitro rat brain tissue (25)
in vitro guinea pig cochleae (26)
caffeine adenosine receptor antagonist
in vivo rat CNS (27, 28)
nicotine nicotinic acetylcholine receptor
agonist
in vivo rat CNS (29-31)
barium
thought to induce exocytotic
release by a mechanism similar to
calcium
bovine cell culture (32)
rat neuronal preparation (33)
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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
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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/cm
2. 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.
[FBRR] = 2 mM in 0.1 M TEABF4/ACN. Error is SEM with n = 3 electrodes for each
measurement.
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
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
increased by 40%, the Epa remains unaltered. In addition, increasing the Mg2+
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
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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
cheapdate increased sensitivity to alcohol
(17)
tipsy increased sensitivity to alcohol
(18)
barfly reduced sensitivity to alcohol
(18)
hangover reduced development of
tolerance to alcohol
(19)
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
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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:
slope = 0.69 ± 0.04 nA/μM
r2 = 0.98
n = 3
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
0 10 20 30 40 500
10
20
30
40
[DA] M
i ma
x (
nA
)
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.