Androgen Modulation ofDopamine Transporter Function
in the Corpus Striatum ofMale Rats
by
Samer Mohammed Saleh
Submitted in Partial Fulfillment ofthe Requirements
for the Degree of
Master ofScience
in the
Biological Sciences
Program
YONGSTOWN STATE UNIVERSITY
August, 2003
Androgen Modulation ofDopamine Transporter Function
in the Corpus Striatum ofMale Rats
Samer Mohammed Saleh
I hereby release this thesis to the public. I understand that this thesis will be
made available from the OhioLINK ETD Center and the Maag Library
Circulation Desk for public access. I also authorize the University or other
individuals to make copies ofthis thesis as needed for scholarly research.
Signature:
Approvals:
Robert E. Leipheimer, Ph.D., Thesis Advisor
James R. Toepfer, Ph.D, Committee Member
Mark D. Womble, Ph.D, Committee Member
Peter J. Kasvinsky, Dean ofGra
11'10'3
Date
Date
Date
Date
Date
ABSTRACT
This thesis examined the relationship between the presence or the absence of
testosterone on the function ofthe dopamine transporter system (DAT) in adult male rats.
Eighteen male rats were assigned to control, castrated (GNX), and castrated with
testosterone replacement (GNX+T) groups. Using an in vitro superfusion technique, the
corpus striatum from each rat was divided and treated with dopamine, MPP+, and NMF
infusions. Striatal dopamine and DOPAC levels were measured during baseline
conditions and after drug infusion. Results of our experiments demonstrated that
dopamine and DOPAC recovery were greatest in the GNX rats and were decreased in the
intact or the GNX+T animals. Testosterone appears to be an important steroid that
modulates the function ofthe DAT. Testosterone acts to enhance DAT activity while the
loss oftestosterone reduces DAT function. With respect to activity of neurotoxins (such
as MPP+) to cause symptoms of PD, our results suggest that men may be more prone to
this disease due to the facilitatory actions of testosterone on the DAT activity. Further
experiments are needed to examine in detail the mechanisms ofaction oftestosterone on
DAT function and to explore the relationship DAT function and neurodegenerative
diseases.
111
ACKNOWLEDGMENTS
"In the Name ofAllah, the Benificent, the Most Merciful"
First, I would like to acknowledge the Palestinian people in their struggle for
freedom and I would like to dedicate this thesis to all the heroes who sacrificed their
lives. Second, I would to thank my parents for their continuous support and help and
dedication to improve our lives. I also would like to thank Mala Milkovich and her
mother Ann Milkovich for their help in all possible ways to make this thesis possible, and
for their support in many aspects ofmy life. I also would like to acknowledge my adviser
Dr. Robert Leipheimer for his help on bring the thesis together. Finally, I would like to
acknowledge my friends, Hamid Nawaz, Jeremy Mashburn and Matt Kesic for a
wonderful and fun filled year.
IV
TABLE OF CONTENTS
ABSTRACT .iii
ACKNOWLEDGMENTS .iv
TABLE OF CONTENTS v
LIST OF FIGURES AND ILLUSTRATIONS vi
CHAPTERS
INTRODUCTION 1
Dopamine Circuits in the Brain 3
Dopamine as a Chemical Messenger. .4
Synthesis ofDopamine 5
Treatments for Parkinson's Disease 6
Distribution ofDopamine Receptors and Autoreceptors in the Brain 8
Dopamine Autoreceptor 9
Monoamine Transporters 10
Drugs Acting on the Dopamine Transporter 11
Gender Related Factors Contributing to Neural Protection 14
Specific Aims ofthe Study 19
MATERIALS AND METHODS 20
Animals 20
Surgical Procedure 20
Testosterone Replacement Surgery 21
Solutions 21
Superfusion Chamber. 21
v
Pharmacological Agents 21
Experimental Procedure 22
Tissue Content 25
Data Analysis 25
Criteria ofData Collection '" 25
Baseline Data Collection 26
Release Data Collection .26
Baseline and Release Data Calculation 26
RESULTS 27
EXP 1 27
EXP 2 ;-.28
EXP 3 29
DISCUSSION 55
REFERENCES 58
APPENDIX '" .66
VI
LIST OF FIGURES AND ILLUSTRATIONS
Illustration 1 5
Illustration 2 22
Illustration 3 " 23
Illustration 4 24
Figure 1 32
Figure 2 34
Figure 3 :- 36
Figure 4 38
Figure 5 40
Figure 6 42
Figure 7 44
Figure 8 46
Figure 9 '" 48
Figure 10 50
Figure 11 52
Figure 12 54
Vll
INTRODUCTION
Men and women differ in the risk factors that expose them to Parkinson's disease (PD),
according to a recent analysis. It is not clear why men are more prone to getting the
disease, but it is clear that it is related to a lack of female hormone, estrogen (Dluzen;
McDermott 2000). The general population that is at greater risk for developing PD are
people older than fifty. The disease is related to a motor system disorder that causes the
patient's hands, legs, jaw, face, and arms to tremor. Parkinson's disease may also cause
bradykinesia (slowness ofmovement) as well as a loss ofbalance, and difficulty in doing
ordinary daily activities such as walking, talking, eating, and writing (CaIne and CaIne,
2001). Parkinson's disease occurs when certain nerve cells, or neurons, in an area of the
brain known as the substantia nigra die or become impaired. Normally, these neurons
produce an important brain chemical known as dopamine. Dopamine is a chemical
messenger responsible for transmitting signals between the substantia nigra and the next
"relay station" of the brain, the corpus striatum, to produce smooth, purposeful muscle
activity. Loss of dopamine causes the nerve cells of the striatum to fire out of control,
leaving patients unable to direct or control their movements in a normal manner. Studies
have shown that Parkinson's patients have a loss of 80 percent or more of dopamine 
producing cells in the substantia nigra. The cause ofthis cell death or impairment is not
known but significant findings by research scientists continue to yield fascinating new
clues to the disease. One theory holds that free radicals - unstable and potentially
damaging molecules generated by normal chemical reactions in the body - may
contribute to nerve cell death thereby leading to Parkinson's disease. Free radicals are
unstable because they lack one electron; in an attempt to replace this missing electron,
free radicals react with neighboring molecules (especially metals such as iron), in a
process called oxidation. Oxidation is thought to cause damage to tissues, including
neurons. Normally, free radical damage is kept under control by antioxidants, chemicals
that protect cells from this damage. Evidence that oxidative mechanisms may cause or
contribute to Parkinson's disease includes the finding that patients with the disease have
increased brain levels of iron, especially in the substantia nigra, and decreased levels of
ferritin, which serves as a protective mechanism by chelating or forming a ring around
the iron, and isolating it.
Parkinson's disease (PD) is a progressive neurodegenerative disorder, ofunknown
cause, found mostly in elderly patients. In this disorder, dopamine neurons located in the
substantia nigra are damaged and are gradually destroyed, and therefore, the nigrostriatal
pathway degenerates. The substantia nigra and the striatum are part ofthe basal ganglia,
which is the motor-center ofthe brain. As the cells in the substantia nigra die, the levels
ofdopamine in the striatum also diminish. When these damaged cells are examined under
a microscope one can distinguish pink stained spheres within them. These spheres are
called Lewy bodies and are considered a reliable indicator ofPD. What these bodies are,
or how or why they come about, is still unknown. Individuals with PD have difficulty
initiating and performing complex, sequential movements. PD produces a constellation of
symptoms including rigidity, hypokinesia, and tremor often accompanied by dementia
and depression (Fisher, Hanin et al. 1986)
In the early stages ofPD, the brain can counteract the effects ofmissing dopamine
by increasing the number of dopamine receptors in the striatum, however, once 80% of
the brain's normal level of dopamine is lost, Parkinsonian symptoms begin to appear.
Without the crucial brain messenger dopamine, produced by the neurons in the substantia
nigra, critical brain signals go awry, resulting in symptoms that vary widely from
individual to individual (Koller 1995). Although the most prominent neuropathlogical
abnormality in PD is destruction of the nigrostriatal dopaminergic neurons, other cell
groups, including noradrenergic cells of the locus coeruleus, dopaminergic cells of the
ventral tegmental area, and cholinergic neurons of the nucleus basalis of Meynert, are
also affected in many cases (Koller 1995).
About one in three patients also experience depression and nearly three of every
four ultimately see some decline in their cognitive skills. Stooped posture, shuffling gait,
loss of balance, fatigue, and difficulty speaking and swallowing are common (Koller
1995). Tasks like brushing one's teeth, handwriting, shaving, fastening small buttons, and
stirring coffee gradually become more and more difficult and patients may end up in a
wheelchair or bedridden (CaIne and CaIne 2001).
2
Dopamine Circuits in the Brain
In addition to the dopamine neurons found in the retina of eye, there are three
main divisions of dopamine neurons in the brain. One of these circuits is found in the
hypothalamic-pituitary axis. The neuron cell bodies are found in the hypothalamic region
and they project their axons towards the pituitary gland. This division is considered to be
part ofthe endocrine system.
The second division, are dopaminergic neurons located next to the substantia
nigra in the ventral tegmental area. They send their axons to the cerebral cortex and the
limbic system. This pathway is involved in one ofthe most devastating mental illnesses
known, schizophrenia.
The third division is the best understood one of all. It is the projection of
dopaminergic neurons from the substantia nigra (dark substance), located in the lower
part of the midbrain, deep below the cerebral cortex in the cerebrum. This circuit
represents about three-quarters of all dopaminergic neurons found in the brain and more
importantly these dopaminergic neurons play a very important role in movement
regulation (Thompson, 1985). The substantia nigra is a brown and black-pigmented
region in the brain. Histological studies done on the substantia nigra and locus coeruleus
in the human brain verified that the pigmentation ofthat region is similar to melanin, so it
was called neuromelanin. As mentioned earlier, an indicative cause ofPD is the selective
death ofthe neurons ofthe substantia nigra. The death ofthese neurons can be linked to
neurotoxic compounds, such as MPTP (l-methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine),
and paraquat an herbicide, which has a similar structure to MPTP. MPTP causes cell
death by its conversion to MPP+ through monoamine oxidase type B, which stops the
respiratory chain at the NADH-CoQl reductase stage (Zecca, 2001).
Following the dopaminergic neurons from the substantia nigra down the axon into
the nerve terminal we arrive at a region called the basal ganglia. The direct correlation
between PD and this region ofthe brain is that the basal ganglia is involved in modifying
minute-to-minute voluntary movements and maintaining posture. The basal ganglia
consists of the corpus striatum, the subthalamic nucleus, and the substantia nigra. The
corpus striatum is the largest structure of all and it is divided into three components, the
caudate nucleus, the putamen, and the globus pallidus. The striatum is considered to be a
3
relay station for the neurons projecting from the substantia nigra. Since dopamine is the
neurotransmitter synthesized in the substantia nigra and released in the corpus striatum, it
is responsible for producing smooth and voluntary muscle activity. The reward pathway
is a neural network in the middle of the brain that prompts good feelings in response to
certain behaviors, such as relieving hunger, quenching thirst or having sex, and it thereby
reinforces these evolutionarily important drives. However, the circuit also responds to
drugs ofabuse, such as heroin, cocaine, amphetamine and nicotine, which seem to hijack
the circuitry, altering the behavior ofits neurons (Creutz and Kritzer, 2002).
Dopamine as a Chemical Messenger
Dopamine is a chemical messenger that tells the body how to mo~e and what
action to take, and it is responsible for how we think and act. Some dopamine present in
the brain activates the frontal lobes, which integrates thoughts, feelings, and sensory
information. The frontal lobes then choose which action the body has to take next.
Therefore, if dopamine is missing, a person becomes unfocused and easily distracted
(Filley 1995). These symptoms can be so faint that they are hardly noticed, but they can
also be very serious. A lack ofdopamine can lead to brain dysfunctions such as PD. It has
also been suggested that an abnormal increase in the amounts of dopamine can lead to
schizophrenia. It is also known that a dysfunction ofthe dopamine system is responsible
for drug addiction (William 1995).
A quarter century ago, in 1975, it was suggested that dopamine was responsible
for depression. This deficiency ofdopamine in our body can be treated with nutrients and
amino acids, which are the raw materials that our body uses to make this neurotransmitter
naturally. If that doesn't work, prescribing a drug called amphetamine, which is also
known as speed, can also treat it. However this treatment may be worse than the
symptoms. Some say that amphetamines are one ofthe most dangerous medications ever
discovered (Fisher 1986). Amphetamines are similar to dopamine, but they can injure
tissue, interfere with growth and development, cause sleep problems or aggressive and
depressed moods. Dopamine is associated with feelings ofpleasure and elation. A hug, a
kiss, a word ofpraise or a winning poker hand can elevate your mood. Scientists say that
4
dopamine is a chemical that transmits pleasure signals. It is also the master molecule of
addiction. Drug addiction may result from malfunctions of the dopamine neuron
neurotransmission system and lead to mental illness (Hadley 1996).
Synthesis ofDopamine
Dopamine synthesis, like that of all catecholamines in the nervous system,
originates from the amino acid tyrosine, which must be transported across the blood 
brain barrier and into the dopamine neuron. Once L-tyrosine gains entry into the neuron it
is converted to L-3,4dihydroxyphenylalanine (L-DOPA) by the enzyme tyrosine
hydroxylase. DOPA is subsequently converted to dopamine by L-aromatic amino acid
decarboxylase. This latter enzyme turns over so rapidly that DOPA levels in the brain are
negligible under normal conditions (Amara, 1998). Because of the high activity of this
enzyme and the low endogenous levels of DOPA normally present in the brain, it is
possible to significantly enhance the formation of dopamine by providing this enzyme
with increased amounts ofthis substrate (Figlewicz, 1999) (see illustration 1).
D_,....
Illustration (1) Internet Source
After dopamine is synthesized, it is stored in vesicles at the active zone of a
presynaptic neuron. Upon the arrival of an action potential it opens voltage gated Ca+2
5
channels located on the neuronal membrane, allowing the influx ofpositive calcium ions.
Extracellular Ca+2 diffuses into the terminals by means of these voltage gated Ca+2
channels (Brunger 2000). Before exocytosis, the vesicle first moves from the cytoplasm
to the plasmalemma. Second, the vesicle becomes attached to the plasma membrane, a
process often referred to as docking. Third, activation involving metabolic energy also
referred to as priming occurs (Skehel, Wiley 1998). Through exocytosis, the vesicles
discharge their contents (dopamine), into the synaptic cleft. The extent to which
dopamine is released depends on the rate and pattern of the neuron's activity (Garris,
Walker and Wightman 1997). Once returned to the sending neuron by the reuptake
system, dopamine is subject to an enzyme named monoamine oxidase (MAO). MAO
usually breaks down dopamine. If no other factors were at work, MAO would keep the
amount of "used" dopamine fairly low. However, dopamine taken back into the nerve
ending can return to the vesicle for storage. Once inside the vesicle, dopamine is
protected from MAO.
A drug named reserpine prevents the reuptake of dopamine and some other
neurotransmitters. Administering reserpine causes dopamine to remain exposed within
the cell and broken down by MAO. This profoundly reduces the available dopamine.
Changing the action of MAO can help us treat diseases that involve dopamine
transmission. For instance, the drug deprenyl inhibits MAO. This increases the stores of
dopamine and slows the progression of Parkinson's disease. In higher doses, deprenyl
enhances the effects ofdopamine on behavior.
Interestingly, one form ofMAO actually protects dopamine. This form ofMAO, found in
dopamine neurons, acts on substances in the neuron other than dopamine. Here MAO
protects the "purity" ofneurotransmission by breaking down other neurotransmitters
Treatments for Parkinson's Disease
Currently available drugs offer temporary relief from the symptoms of the
disorder, but do not stop or reverse the neuronal degeneration caused by this disease.
Available drugs are Levodopa and Carbidopa. Both of these drugs decrease the rigidity,
6
tremors, and other symptoms associated with Parkinson's disease. Levodopa or L-Dopa
(L-3,4-dihydroxyphenylalanine) is the metabolic precursor of dopamine, which crosses
the blood-brain barrier, and be converted to dopamine in the brain. This replaces the
dopamine that has been lost due to Parkinson s disease. Meanwhile, Carbidopa is an
enzyme inhibitor that works to protect L-Dopa in the peripheral circulation so more can
reach the target tissue and be converted into the desired molecule (Sourkes, 1971). Other
drugs are also available such as Bromocriptine, Amantadine, and Deprenyl. These drugs
are dopamine agonistic drugs that target postsynaptic dopamine receptors. They mimic
the effect of dopamine on the postsynaptic neurons and thus are considered in the
treatment ofParkinson's disease (Pearce, 1978 ).
Bromocriptine, pergolide, pramipexole and ropinirole. These four drugs mimic the role of
dopamine in the brain, causing the neurons to react as they would to dopamine. They can
be given alone or with levodopa and may be used in the early stages of the disease or
started later to lengthen the duration of response to levodopa in patients experiencing
wearing off or on-off effects. They are generally less effective than levodopa in
controlling rigidity and bradykinesia. Side effects may include paranoia, hallucinations,
confusion, dyskinesias, nightmares, nausea, and vomiting (Pearce, 1978 ). Treating
Parkinson's disease with surgery was once a common practice. But after the discovery of
levodopa, surgery was restricted to only a few cases. Currently, surgery is reserved for
patients who have failed to respond satisfactorily to drugs. One of the procedures used,
called cryothalamotomy, requires the surgical insertion of a supercooled metal tip of a
probe into the thalamus (a "relay station" deep in the brain) to destroy the brain area that
produces tremors. This and related procedures, such as thalamic stimulation, are coming
back into favor for patients who have severe tremor or have the disease only on one side
of the body. Investigators have also revived interest in a surgical procedure called
pallidotomy in which a portion of the brain called the globus pallidus is lesioned. Some
studies indicate that pallidotomy may improve symptoms of tremor, rigidity, and
bradykinesia, possibly by interrupting the neural pathway between the globus pallidus
and the striatum or thalamus
A great deal of work has been done in the past several years to attempt to find
treatments as an alternative to drug therapy. It has been shown that transplantation of
7
various kinds of catecholamine producing tissues into the striatum of PD patients can be
beneficial as a treatment. The most favorable results have been obtained with the
transplantation of fetal mesencephalic (nigral) tissue into the caudate nuclei of monkeys
rendered Parkinsonian with MPTP. Significant motor behavioral improvement was
observed in these animals (Garris, 1997).
The improvement seems to be due in part to dopamine derived from the fetal
dopaminergic graft. It has been shown that MPTP selectively destroys the dopaminergic
cells in the substantia nigra but spares the mesolimbic system originating from the ventral
tegmental area. Because the effects of MPTP so closely mimic true idiopathic PD, it is
reasonably safe to assume that the same occurs in PD brains (Fisher, 1986). The
implantation of the tissue apparently stimulates these nearby remaining dopaminergic
neurons to develop new dopaminergic fibers into the caudate and a pathway is thus
established to carry host dopamine to the striatum. The transplantation of human fetal
nigral tissue into the striatum ofPD patients has been attempted a few times and has been
relatively successful in combination with immunosuppressive therapy. However,
widespread clinical application of human fetal tissue implantation obviously presents
serious immunological and ethical problems (Kish, 1998).
Distribution ofDopamine Receptors and Autoreceptors in the Brain
The Dopamine Receptors
In the striatum, dopamine interacts with two major subfamilies of receptors, the
D1 subfamily and D2subfamily. Both subfamilies ofreceptors are located on postsynaptic
neurons. The D1 subfamily consists of two different types, the D1 receptor and the Ds
receptor. This subfamily of receptors act via adenylate cyclase. The D2
receptor subfamily contains the D2, D3, and D4 subtype receptors and are found in high
levels in the brain. D2 and D3 receptors are found in the striatum in greater abundance
than the D4 receptor. The D4 receptor is mainly distributed in limbic areas of the brain
(Young at el. 1999). The D2 subfamily receptors have each been shown to inhibit
adenylyl cyclase when expressed in recombinant cells. Although the signal via the D3
receptor has been more difficult to demonstrate and it is generally found at lower levels
than the other receptor subtypes.
8
Overall the Dz, D3, and D4 receptors exhibit pharmacological properties similar to
those ofthe originally defined Dz receptor. They all show high affinities for drugs such as
the butyrophenones, e.g. haloperidol, and the substituted benzamides, e.g. sulpiride, and
these classes ofdrugs provide selective antagonists for the Dzsubfamily receptors (lnase,
Li and Tanji 1997). The Dzlike receptors also show high affinities for phenothiazines and
thioxanthines. Each Dz subfamily receptor does have its own pharmacological profile so
that there are some differences in affinities of drugs for the individual Dz subfamily
receptors. For example, raclopride shows a high affinity for the Dzand D3 receptors, but a
lower affinity for the D4 receptor. Clozapine shows a slight selectivity for the D4 receptor.
The Dz subfamily receptors show moderate affinities for typical dopamine agonists with
the D3 receptor generally showing higher affinities for agonists than the other subtypes.
There are compounds available that are selective agonists for the Dzsubfamily receptors,
e.g. NO 437 and quinpirole (Araki, Tanji, Kato and Itoyama 1998).
Dopamine Autoreceptor
Dopamine autoreceptors are receptors located on the presynaptic nerve terminal
that produce feedback inhibition ofdopamine synthesis and release. There are three kinds
ofautoreceptors classified according to their effects, I) impulse modulating autoreceptor,
II) release modulating autoreceptor; and III) synthesis modulating autoreceptor. All of
these autoreceptors show similar pharmacological profiles, but data suggests a difference
in the second messenger system, which is responsible for the signal transduction cascade
that mediates the autoreceptor response (Perachon, Schwartz and Sokoloff, 1999).
The dopamine autoreceptors are members of the Dz receptor subfamily. They
have a higher binding affinity compared to the effects of dopamine on the postsynaptic
receptors. Several drugs have been synthesized that agonize or antagonize the
autoreceptor. One of these is Pramipexole, a new dopamine receptor agonist with
preference for D3compared to Dz receptors. This drug is considered to be potent against
the dopamine autoreceptor and at the same time it shows no affinity to other receptors of
the Dz subfamily. Given this fact, researchers suggest that such a drug would have limited
side effects when treating PD, due to it's high binding affinity to the D3 receptor and not
to any other receptor. This drug also has been reported to have a neuroprotecting effect
9
for PD and may slow the progressive destruction of dopamine neurons (Grandas, 1999).
Pramipexole produces its neuroprotection by interacting with hydroxyl free radicals in
MPP+ infused rats. It may also protect against oxygen free radicals produced in solutions
and protect red blood cell membranes from lipid peroxidation, which have been reported
in with patients with PD. These patients may refrain from taking L-dopa for several
years as long as they are receiving pramipexole (Grandas, 1999).
On the other hand, antagonist drugs of the dopamine autoreceptor such as
raclopride are known to block the receptor leading to an increase in dopamine neuronal
activity (firing rate, synthesis, and release). These studies showed that raclopride binds to
the dopamine autoreceptor with high affinity in the rat striatum, which in turns provides
an indirect measure of changes in synaptic dopamine (Usidn et aI. 1991; Shimada et aI.
1992).
Monoamine Transporters
Monoamine transporters are plasma membrane transporters, also known as uptake
pumps. They are one of the most efficient means of controlling extracellular
catecholamine concentrations. There are monoamine transporters for dopamine (DAT),
serotonin (SERT), and norepinephrine (NET). These are selectively expressed on the
corresponding neurons and serve as targets of many psychostimulants, antidepressants,
and neurotoxins.
The Dopamine Transporter (DAT)
The dopamine transporter is a plasma membrane protein located on the
presynaptic nerve terminal of dopamine neurons. It is responsible for the termination of
dopaminergic neurotransmission through transmitter reuptake from the synaptic cleft
(Hitri et aI., 1994; Kuhar, 1998). The DAT works through a concentration gradient,
where released dopamine in the synaptic cleft has a higher concentration. This high
concentration of dopamine is sensed by the DAT and dopamine is transported from the
synaptic cleft back into the neuron terminal to be recycled and/or degraded. The
importance of the DAT lies with its regulatory activity as a synaptic modulator of
10
dopamine levels. The re-uptake of dopamine by a presynaptic transporter protein is the
primary mechanism for inactivating dopamine's synaptic effects (Figlewicz, 1999). The
DATs are members of a family of Na+ and cr dependent neurotransmitter transporters
responsible for the rapid clearance of dopamine from synaptic clefts. The primary
sequence of the dopamine carrier contains multiple phosphorylation sites in the putative
intracellular domains for cAMP dependent protein kinase, protein kinase C (PKC), and
Ca+2- calmodulin-dependent protein kinase. Numerous studies have examined the effects
of phosphorylating conditions on DAT activity. The evidence for DAT modulation by
phosphorylation is most compelling in the case of PKC. Activation of PKC inhibits
DAT-mediated uptake through the rapid sequestration/internalization of DAT protein.
Recent studies have demonstrated basal (p32) orthophosphate incorporation into the DAT,
which is increased by PKC activation (Zhu et aI., 1997; Pristupa et aI., 199-8; Melikian
and Buckley, 1999). The DAT undergoes endogenous phosphorylation in striatal
synaptosomes that are regulated by activators of protein kinase C. The activation of
protein kinase C involves sequestering the reaction components into membrane domains.
(Vrindavanam et aI., 1996; Huffet aI., 1997; Vaughan et. aI., 1997), and down-regulation
of transport activity (Kitayama et aI., 1994; Huff et aI., 1997; Vaughan et aI., 1997;
Zhang et aI., 1997).
Studies indicate that the dopamine transporter requires molecules that possess a
phenyl ring with a primary ethylamine side chain for optimal activity, and the B-rotamer
of the extended conformation of catecholamines is transported preferentially (Meiergerd
and Schenk, 1994). Recent transport studies on the cloned human dopamine transporter
suggest that, although B- or phenolic ring hydroxylation ofa substrate results in a change
in the Km over a wide range, the presence ofa phenolic hydroxyl group is not a perquisite
for optimal function of the transporter. Compounds without a phenolic hydroxyl group
such as B-phenethylamine, amphetamines or MPP+, can bind to the carrier and be
transported with same the Vmax (speed) as dopamine (Chen and Justice, 2000).
Therefore, phenethylamine seems to be the most important structural element
accommodated by the dopamine transporter (Giros and Caron, 1993).
Drugs Acting on the Dopamine Transporter
11
The Neurotoxin MPTP (MPP+)
MPTP is a contamination of synthetic heroin and I-methyl-4-phenylpyridinium
(MPP+) is metabolite of MPTP oxidized by the enzyme monoamine oxidase type B
(MOA-B). MPP+-induced dopaminergic neuronal degeneration is similar to that observed
in PD. The amount ofMPP+ produced from MPTP depends on the amount ofthe MOA-B
present. The site of activation of this neurotoxin is thought to be the glial cells found
outside the nerve terminal. This is supported by studies that demonstrated MPTP was
taken up by cultured astrocytes and metabolized into MPP+ (Marin, 1999).
MPP+ is considered to be a major neurotoxin that can mImIC the
neurodegeneration ofneurons seen in PD. The effects ofthis neurotoxin that mimics PD
are related to the chronic presence of MPP+ in the neuron's powe~house, the
mitochondria. MPP+ selectively accumulates inside dopaminergic neurons and causes the
death ofthese neurons (Irwin and Langston, 1985). The mechanism of action ofMPP+ is
thought to be through the generation of superoxide. When MPP+ is found in sufficient
amounts, it can overcome the powerhouse by inhibiting the mitochondrial complex I of
the electron transport system leading to the generation of reactive oxygen species and
eventually cell death (Marin, 1999).
MPP+ also has an acute effect; once it reaches the inside of the neuron it causes
the displacement of dopamine from their synaptic vesicles into the intracellular
cytoplasm. Since the DAT moves dopamine down its concentration gradient, the
displaced dopamine in the active zone of the neuron activates the DAT and causes the
dopamine to be transported out ofthe nerve terminal and into the synaptic cleft leading to
dopamine depletion (Qu, 1988).
In a study by Mandavilli and Van Houten (2000), the levels of dopamine and its
metabolites, DOPAC and HVA, were measured by HPLC equipped with electrochemical
detection. DNA damage was also measured by quantitative PCR in both mitochondrial
and nuclear (beta-polymerase) targets from the caudate-putamen, substantia nigra, and
cerebellum regions ofcontrol and MPTP-treated mice. They found MPP+ treatment led to
damage in both mitochondrial and nuclear DNA ofthe substantia nigra, while there was
no damage in either the cerebellum or caudate putamen (Mandavilli, 2000). These
12
findings indicate that MPP+ enters dopaminergic neurons of the nigrostriatal system via
the dopamine reuptake system, where it is concentrated within mitochondria by active
transport. MPP+ inhibits NADH-ubiquinone reductase, which is the first enzyme-protein
complex of the mitochondrial respiratory chain. By inhibiting this complex, MPP+
probably induces nigrostriatal cell death by depleting cellular ATP levels (Dluzen 1996).
As mentioned before, MPP+ is selective for the nigrostriatal system but it is not
well understood why. The degeneration of dopamine neurons are detected in the nigro 
striatum in brains with PD (Iwata, Keikilni and Gengy 1997). Also during aging, the
number of dopamine neurons in the substantia nigra is known to decrease at a rate of
about 5-10% neurons per decade, suggesting that dopamine neurons in this region are
more vulnerable than in other regions and other types ofneurons (Goulet, et. aI., 1999). It
has been thought that aging might account for the death ofselective neuronal-populations
in certain progressive neurodegenerative disorders, including Alzheimer's disease and PD
(Goulet, et. aI., 1999).
Cocaine
Cocaine is a popular drug of abuse that possesses the properties of a local
anesthetic and psychomotor stimulant. Cocaine blocks the DAT resulting in an increase
in the extracellular concentration of dopamine and the accompanying physiological
effects associated with cocaine (Ritz et aI., 1987). This includes to the enhancement of
sympathetic activity and potentiation of the action of dopamine and other
neurotransmitters (Amara and Sonders, 1998). Chronic cocaine users eventually face
depletion of their striatal dopamine, which also leads to some of the neurological and
psychiatric complications which increase the risk for narcoleptic movement disorders
(Wilson, 1996). The depletion ofdopamine triggers the vicious cycle ofcraving for more
cocaine. The behavioral effects of cocaine result from a powerful stimulation of the
cortex and the brain stem. Cocaine acutely increases mental awareness and produces a
feeling of well-being and euphoria, but it can also cause hallucinations, delusions and
paranoia. It increases motor activity and at high doses causes tremors and convulsions
followed by respiratory and vasomotor depression. Addiction to cocaine, once fully
developed, may represent a true biological dependency on the drug. Cocaine can also
13
temporarily relieve the neurological defects seen with PD. This occurs because cocaine
blocks the DAT and thus more dopamine is present in the synaptic cleft, which relieves
some ofthe symptoms associated with PD (Amara and Sonders, 1998).
The DAT and the serotonin reuptake transporter (SERT) are the only monoamine
transporters that can facilitate cocaine reward in chronic users. Neurotransmitter
rearrangements in single and double knockout mice demonstrate compensations for
transporter deletion. Similar changes might also follow long-term DAT or SERT
blockade by drugs. Researchers have investigated the influence ofcocaine in single DAT
or SERT knockout mice that can retain some of the cocaine reward and in double
knockout mice that do not experience a cocaine reward. The data obtained by the
investigators indicates that the presence ofdopaminergic neurons and the DAT are more
important for cocaine reward than are serotonergic neurons (Sora, 2001).
Nomifensine
Nomifensine (NMF) is an antidepressant drug that possesses the ability to block
the DAT. In contrast to cocaine, nomifensine does not have an addictive side effect to it.
Thus, this drug potently inhibits the reuptake of dopamine, by inhibiting presynaptic
DAT as well as the autoreceptor activities. Blockade of the DAT causes increased levels
of synaptic dopamine, which results in prolonged postsynaptic receptor activation and
ultimately motor activation (Garris, 2003 ).
Research done by Disshon and Dluzen (1999), demonstrated that after infusion of
NMF into superfused striatal tissue the outcome showed an increase in dopamine
recovery. Chronic infusion of d-amphetamine (AMPH) causes dopamine depletion,
degeneration of nigrostriatal dopaminergic neurons, decreased activity of tyrosine
hydroxylase and reduces in the number of DAT of rats. Treatment with NMF protects
against AMPH-induced long-term dopamine depletion (Dluzen, 2000).
Gender Related Factors Contributing to Neural Protection
Monoamine transporters are known to be sensitive to changes in sex steroids and
any variation in the levels of these hormones may alter the expression of these
14
transporters, which is known to be related to the pathophysiology ofmovement disorder,
depression, schizophrenia, and obsessive compulsive disorder (Rehavi, 2000).
The impact ofthe female gonadal hormone estrogen upon the DAT has been well
documented. Estrogen acts in the striatum to rapidly inhibit dopamine clearance from the
synaptic cleft by inhibiting the DAT (Dluzen, 2000). Estrogen also decreased
amphetamine (AMPH)-induced dopamine release from striatal tissue (McDermott and
Liu 1994), enhanced dopamine turnover (Kelly and Lagrange 1999), and decreased
dopamine receptor density in the striatum (Garris and Walker 1997). The Estrogen
metabolites, estrone, estriol, and the non-steroid analog diethylstilbestrol do not produce
these effects (Ekue and Boulanger 2002). Thus the steroidal pattern and hydroxylation on
the A-ring of estrogenic compounds may be important determinates of ligand binding to
the putative estrogen-binding site in the striatum (Lindford and Wade 2000). Estradial
conjugated to bovine serum albumin mimics the effect ofestradial to enhance stimulated
straital release. These findings suggest that the modulatory effects of estrogen are
mediated by a specific membrane-bounded receptor mechanism (Kelly et. aI., 1999).
Numerous studies have demonstrated that estrogen provides neuroprotectivity
within the dopaminergic system. One ofthe ways that estrogen exerts its neuroprotection
is by acting as an inhibitor of the DAT. Since the DAT does not discriminate between
dopamine and neurotoxins such as MPP+, transport of MPP+ with dopamine into the
presynaptic neuron leads to destruction (neurodegeneration) ofthe neuron. Estrogen acts
as a neuroprotectant by inhibiting the dopamine transporter function. This influence leads
to the blockade of the transporter and therefore, the DAT is unable to recover the
neurotoxin and the dopamine, which are found in the cleft (Dluzen, 2000).
Evidence that estrogen blocks the DAT comes from experiments examining the
effects of methamphetamine on the nigrostriatal dopaminergic system and therefore the
DAT. Methamphetamine, like MPP+, induces dopamine displacement from synaptic
vesicles and is transported out of the presynaptic vesicle via the DAT. Infusion of
methamphetamine into striatal tissue significantly increases dopamine release, an effect
that is prevented in the presence of estrogen, indicating that estrogen is acting to inhibit
DAT functioning (Myers, 2003). The DAT is therefore unable to recover the neurotoxin
and dopamine, which remain in the synaptic cleft (Dluzen, 2000).
15
Additional support for the hypothesis that estrogen acts as a dopamine uptake
inhibitor comes from experiments examining the effect ofestrogen in male animals. After
castrating male rats and treating them with estrogen, it has been observed that estrogen
exerts neuroprotective effects upon the nigrostriatal dopaminergic system (Gao and
Dluzen 2001). Estradiol effect on the DAT has been suggested to be mediated by a
membrane bound receptor, rather than having a genomic action. This suggestion is
supported by in vivo electrochemistry experiments, where researchers explored the effect
ofadministering MPP+ in the presence or absence ofestrogen into the corpus striatum of
rats. The amplitude of the dopamine release was decreased by ten fold when MPP+ was
administered with estrogen, which implied an inhibitory effect of estrogen on the DAT
activity. The researchers also measured the clearance rate of dopamine after MPP+ alone
and MPP+ with estrogen treatment. They found that there was a significant reduction in
the clearance rate of dopamine after treatment with estrogen. These results suggest that
estrogen exerts its actions very rapidly, which is most likely due to a membrane action
rather than by classical genomic effects in protein synthesis (Arvin, 1998).
As mentioned before, PD is a neurodegenerative disease that is more prevalent in
older populations versus younger ones. Another study done by Leipheimer and Arvin
(2000) examined the effects ofinfusing MPP+ alone or in combination with estrogen on
the release and clearance ofdopamine in the corpus striatum ofaged female versus young
female rats. Their in vivo electrochemistry experiments demonstrated that estrogen was
effective in aged animals and significantly inhibited the MPP+ induced release of
dopamine and significantly inhibited the clearance rate of dopamine from the
extracellular fluid. This study provides evidence that estrogen retains its inhibitory action
on DAT activity in aged animals.
The action of estrogen of appears to be inhibitory to DAT activity, however the
site ofestrogen acting is still a matter of debate. To address this issue a study was done
by Leipheimer and Arvin (2001) to further understand the specificity of estrogen action.
This electrochemistry study was done with female rats infused with dopamine alone,
dopamine plus estrogen, or the combination ofdopamine plus estrogen plus tamoxifen (a
dopamine antagonist) in the corpus striatum. In this study, tamoxifen completely reversed
the inhibitory action ofestrogen on the clearance rate ofdopamine. These results suggest
16
that estrogen is acting via a specific receptor mechanism to inhibit the activity of the
DAT.
Studies done at Harvard University explored the effects of low levels of
testosterone on depression in man. They found that the low levels of testosterone are
directly related to depression and to the expression ofthe serotonergic and dopaminergic
transporters. Furthermore, it was found that depressed men receiving regular injections of
testosterone had significant mood improvement which suggests the importance of
testosterone in regulating the expression of dopaminergic and serotonergic transporters
on psychological conditions (Treatment up date, 2000).
Although much evidence is accumulating concerning the role if estrogen in the
modulating neurotransmitter function, there is little information concerning the role of
testosterone.
A study done by Leipheimer, Fedrokova, and Arvin (1999) explored the effects of
castration on the K+ or MPP+-stimulated dopamine release from the nucleus accumbens
and the corpus striatum of male rats. Using real time recording of both the release and
reuptake ofdopamine, it was found that castration changed the dynamics ofthe dopamine
transporter especially in the nucleus accumbens after MPP+ infusion. These results
suggests that the effects ofandrogens on the DAT activity vary with specific brain areas,
and that testosterone may have greater effects on DAT activity in the nucleus accumbens
than in the corpus striatum.
In a study done by Dluzen and Ramirez (1989) Testosterone and DHT was
investigate and compared to estrogen action on MPP+-induced dopamine depletion.
Studies have shown that the male gonadal steroid hormone testosterone did not weaken
the effects of MPP+-induced dopamine release nor did DHT. Meanwhile, estrogen
showed a reduction in striatal dopamine concentrations (Dluzen and Ramirez 1989).
Furthermore, striatal specific binding to the DAT was measured using [1251] RTI-121
and [3H] dihydrotetrabenazine autoradiography. With MPP+ treatment the DAT
concentration was significantly reduced in control rats, meanwhile estrogen prevented
this reduction in the concentration ofthe DAT but testosterone failed to do the same. The
role of testosterone was also explored in the substantia nigra with MPP+ treatments and
the observed results suggested that androgen did not prevent MPP-induced decrease of
17
the DAT mRNA, while estrogen significantly prevented MPP+-induced depletion of
DATmRNA.
Furthermore, a recent study revealed that testosterone did not have the same
modulatory effects as estrogen upon amphetamine evoked dopamine release from the
superfused corpus striatum. This study emphasized the effects of estrogen in producing
neuroprotectency upon the striatal tissue, meanwhile it suggested that infusing
testosterone had no protective effects against the depletion of dopamine from the striatal
tissue (Myers, 2003). In another study, investigators found that estradiol treatment
reduced MPTP induced neurotoxicity and lowered dopamine depletion in mice. However,
treatment with testosterone or DHT did not reduce MPTP-induced neurotoxicity. These
androgen treated mice had dopamine depletion similar to control mice treated with MPTP
alone (Ekue, 2002 ).
In conclusion, steroid hormones, estrogen and testosterone, seems to exert
differential modulatory effects upon dopamine and DOPAC output from striatal tissue,
and this could be the basis for the difference in occurrence ofthis disease between males
and females.
18
Specific Aims ofthe Study
The main focus of this thesis was to investigate the role of testosterone (T) on
dopamine transporter function. Using in vitro superfusion techniques, the dopamine
transporter was studied under three conditions. These included intact male animals
(control), castrated or gonadectomized male animals (GNX), and castrated males with
testosterone replacement (GNX+T).
Striatal tissue isolated from rats in each group was treated with the neurotoxin MPP+,
with nomifensine a DAT inhibitor, or with dopamine. The amount of dopamine released
from the tissue and recovered in the superfusion solution served as an indicator of DAT
activity. Increased dopamine release and recovery indicated a decrease in DAT function.
We hypothesized that testosterone has a facilitatory effect on the DAT. Therefore it was
expected that castrated male rats (GNX) would show increased dopamine recovery when
compared to control rats or castrated male rats with testosterone replacement.
19
METHOD
Animals
Eighteen male Sprague Dawley rats about 3-5 months old, with an approximate
weight of 125g, were housed in groups of two or three per cage. The animals were kept
on a twelve-hour light/dark cycle, with lights on at 0600. Food and water was available
ad lib. Six animals remained intact and were used as the control group. Twelve animals
were castrated and six of these received testosterone replacement therapy. These
experiments were approved by the Institutional Animal Care and Use Committee
(lACUC) at Northeastern Ohio Universities College ofMedicine (NEOUCOM.).
Surgical Procedures
Castration
Animals were weighed and gIven an intramuscular injection of a mixture of
Ketamine (60mg/kg) and Xylazine (13 mg/kg). Ketamine or ketamine hydrochloride, is a
non-barbiturate, rapid-acting disassociative anesthetic used on both animals and humans.
Xylazine is a clonidine analoque, an agonist that acts on the (xz adrenergic receptors on
the presynaptic and postsynaptic neurons in the central and peripheral nervous system,
which causes the muscles to relax. One disadvantage of Xylazine that it causes
cardiopulmonary depression, so it was found that a combination or a mixture of both
Ketamine, which is, considered a good cardiovascular stimulant and Xylazine, which
provides long periods ofanesthesia, can be good and suitable for surgery (Zandieh, 2000)
Following anesthesia, the animals were checked for reflex withdrawal reactions to
ensure complete anesthesia. An incision was made in the lower abdomen and the testes
were pushed up toward the incision. After locating the testes, the ductus deferens was
clamped using a hemostat at a point above the epididymis and the testes. The ductus
deferens and the arteries and veins supplying the testes were tied off with a silk thread.
Using scissors, the testes were removed and the tissue was checked for bleeding before
20
unclamping the hemostat. The remainder of the ductus deferens was put back carefully
and the incision sutured. The animals were allowed to recover ten to fourteen days and
until all the testosterone was absent (Dluzen, 2000).
Testosterone replacement
Testosterone was replaced in some GNX rats by implanting capsules containing
testosterone. The capsules were made with silastic medical grade tubing (1.6 mm I.D.,
3.2 mm O.D.), Dow Corning (Midland, MI). They were filled with testosterone to an
active length of 40 mm. The tubing was sealed on the ends with 5 mm. wooden plugs
and silastic medical adhesive silicone type-A, Dow Corning (Midland, MI). These
capsules were made with modifications according to methods described by Smith et al.
(1977) and are similar to capsules reported to maintain sexual reflexes in castrated male
rats (Leipheimer and Sachs, 1993). The capsule was inserted subcutaneously in the mid
dorsal thoracic region under light Halothane anesthesia. The animals were given a week
to allow testosterone levels to return to normal levels (Dluzen, 2000).
Solutions
Kreb Ringer Phosphate Buffer (KRP)
The superfusion medium (KRP) consisted of: 120 mM NaCl, 4.8 mM KCL, 0.8
mM ofCaCb, 1.2 mM ofMgS04, 10.2 mM Na2HP04, 1.8 mM NaH2P04 and 3.25xl04
mM glucose adjusted to a pH of7.4 (Dluzen, 2000).
Pharmacological Agents
We used three different drugs for the purpose of this thesis: First, MPP+ (lO/lM,
Research Biochemical International) to cause dopamine release, second, Dopamine
(l/lM, Sigma Chemical Company), and third, Nomifensine (lmM, Sigma Chemical
Company) was used to block the DAT.
Superfusion Chamber
The superfusion chamber consists of a 1cc syringe with a 21-gauge needle at the
end. The chamber must house the tissue for the whole experiment and must prevent the
tissue from leaving the chamber. This was accomplished using filter paper and corks with
21
the corks and the filter papers positioned in the chamber as follows. The filter is first
inserted into the syringe and pushed down until it reaches the end of the syringe
(chamber). The cork is then inserted and also pushed all the way in. Striatal tissue is put
into each chamber, followed by a second filter and cork. Caps with an attached air hose
were placed into each chamber to guarantee adequate delivery of oxygen to the tissue at
all times. Prior reaching the tissue chamber air was passed through distilled water to
moisten the air and collect unwanted particles. The tissue chambers were submerged in a
water bath at a temperature of37?C. Samples were collected in microcentrifuge tubes on
Superfusion Chamber
Collection tube
Drug
KRP infusions l'lllKRF
the time except
4 and 5
Drug infuion only
at min 40 8: 50
ice every 10 minutes at a flow rate of25J.lllmin [see illustration (2)].
Illustration (2)
Experimental Procedure
On the day of the experiment, three rats from the assigned groups were obtained
(control, castrated plus testosterone treated, and castrated with no treatment) and
decapitated using a sharp guiotine. Decapitation was done with no prior anesthetesia,
because anesthesia interferes with catecholamine concentrations in the brain. The brain
was harvested and the striatal tissue was removed from the brain by making small
22
incisions on the posterior and anterior sides of the lateral ventricles. Once the incisions
were made, the lateral ventricles were pushed aside exposing the corpus striatum. The
striatum is characterized by having striations from side to side that can be seen with the
naked eye.
The striatal tissue is then collected in small beakers containing KRP buffer and
put on ice to preserve the tissue. The corpus striatum from each animal was cut into
approximately three equal parts and then minced prior to treatment with the three drugs
(see illustration 3).
? =Striatum
MPP+ Dopamine Nomofensin
A B C D E IF IIG II H ~ I I
11\
I"
?IntactGNX+T GNXIntactGNX+T GNXIntact
I I
~ ~ ~
? ?GNX+T GNX
Animal 1 Animal 2
Illustration (3)
Tissue was placed into a superfusion chamber with KRP buffer being infused at
25ul/min. The tissue was placed allowed to equilibrate for one hour before samples were
23
collected. KRP saline buffer from the tissue chamber was collected for 10min and pooled
as a single sample. Sample 1-3 were collected during the infusion ofcontrol KRP buffer.
The experimental drugs were infused during the collection of sample 4-5. Control buffer
was again infused during collection of the remaining samples (6-10). Ten samples were
collected from each chamber every ten minutes for a total of ninety samples for each
experiment (see illustration 4).
KRP infusion /1 0 min
QJ00 0~~ [2] ~~~
'0
Drug infusion
Illustration (4)
Prior to their use, the centrifuge tubes for sample collections were weighed and
recorded. After the samples were collected, the tubes were weighed again, which allowed
the determination ofthe final volume in the tubes. The samples were analyzed by a high 
pressure liquid chromatography (HPLC) for dopamine and DOPAC concentrations
(Dluzen, 2000).
HPLC separates complex mixtures using high pressure to force a sample that has
been dissolved in a solvent (the mobile phase), through a narrowly packed column (the
stationary phase). As the solute is being infiltrated through the column, the molecules
become separated from each other due to different chemical and physical interactions
with the packing material. Because ofthese interactions, the components are retained by
the stationary phase and move at different rates through the column. As the separated
24
components elute from the column, they pass through a electrochemical detector which
detects the amounts ofneurotransmitters present (Pokrasen et. aI, 1997 ).
Tissue Contents
Following sample collection, the striatal tissue was recovered from the tissue
chambers, placed on a paper towel to dry, weighed, and put into 500 1-11 of 0.1 N
perchloric acid (HCI04) to preserve it. The tissue was then sonicated for twenty seconds,
centrifuged for fifteen minutes, and the supernatant removed and stored in 500 1-11 of0.1 N
ofHCI04 ? Tissue concentrations ofdopamine and DOPAC were analyzed from the HPLC
instrument in pg and then expressed as pg/mg of tissue. The tissues were analyzed for
dopamine and DOPAC content at the end of the superfusion experiments to verify that
these experiments did not result in the complete depletion ofthese catecholamines in the
tissue. There were no significant differences in the tissue content between treatment
groups in this study and therefore the data is not presented in this thesis.
Data Analysis
Data was analyzed using the SPSS software program (SPSS Inc.). Dopamine and
DOPAC releases from all three groups (control, castrated with testosterone replacement
treatment, and castrated with no treatment) were analyzed for variation between the
groups. Oneway ANOVA was utilized, followed by LSD Post Hoc tests and significant
differences between groups were noted when p:s 0.05.
Percent change for each group was determined by taking the stimulated release
value minus the baseline value, divided by the baseline value, multiplied by 100. The
percent change numbers were then converted to arcsine numbers for statistical analysis.
Criteria ofData Collection
This thesis is based on six experiments, where we collected 540 released samples
and 54 tissue samples. Each released sample was plotted in Excel and visually assessed.
We considered data to be valid ifthe baseline equilibrated between sample one and four
(where the baseline concentration ofdopamine and DOPAC was very low), followed by a
25
response to drug stimulation (an increase from baseline) followed by a decline from the
peak response to baseline after wash out with KRP buffer. Tissue responses that did not
fit these criteria were eliminated.
Baseline Collection
The lowest concentration in pg/mg/min of dopamine or DOPAC between sample
one and four was taken to be the baseline reading for that particular release. Baseline data
were grouped together based on the animal model and what kind of treatment the group
received. For example, data from GNX animals treated with dopamine infusions were
grouped together and the average ofthe six runs (experiments) was collected for analysis
with SPSS.
Release Data Collection
For the release study, the data were examined and the average values were
determined for the release ofdopamine and DOPAC. Release data was grouped together
based on the animal model the treatment received. For example, data from GNX animals
treated with dopamine infusions were grouped together and the average of the six runs
(experiments) was collected and analyzed with SPSS.
Baseline and Release Data Calculation
Baseline and release values were determined usmg the following formulas:
Baseline Concentration = [(l/tissue weight) x 0.005] x volume in the centrifuge tube x
concentration in pg collected from HPLC machine. The final concentration is given as
pg/mg oftissue/min.
Release Concentration = [(l/tissue weight) x 0.005] x volume in the centrifuge tube x
concentration in pg collected from HPLC machine. The final concentration is given as
pg/mg oftissue/min. Our hypothesis is that testosterone acts to increase DAT activity. By
removing testosterone, we predict that castration will reduce DAT activity. To test this
hypothesis, striatal tissue was obtained from intact (control), castrated (GNX), and
castrated plus testosterone (GNX+T) male rats. The striatal tissues were infused with
KRP buffer plus either dopamine (l/lM), NMF (lmM), and MPP+ (lO/lm). The amount
ofdopamine recovered from the tissue was then determined.
26
Results
Experiment (1). The effects of infused dopamine on the release of dopamine and
nopAC in corpus striatum tissue.
Dopamine Recovery
Infusion of control KRP buffer provided baseline data for dopamine release from
the striatal tissue. After 30 min ofcontrol infusion, the saline was changed to KRP buffer
plus dopamine (111M) for 20 min. Control buffer was then infused for the reminder ofthe
experiment.
There were no significant differences in the levels ofbaseline dopamine recovery
from striatal tissue obtained from the three animal groups (Fig. 1). Baseline values were
0.06 ? 0.16 pg/mg/min for control animals (n = 6), 0.22 ? 0.20 pg/mg/min for GNX
animals (n = 6), and 0.25 ? 0.10 pg/mg/min for GNX+T animals (n = 6).
As expected, infusion of dopamine increased the amount of dopamine recovered
for all groups (Fig.1 infusion section ofthe graph). There were no significant differences
between the control group (1.11 ? 0.55 pg/mg/min; n = 6) and the GNX+T group (0.60 ?
0.14 pg/mg/min; n =6). However, the amount of dopamine recovered from the GNX
group was greatly elevated to 9.08 ? 3.21 pg/mg/min (n = 6), which was significantly
greater than the control group (p=0.0009) and the GNX+T group (p=0.006).
Figure 2 shows the percent change in dopamine recovery between the baseline
and stimulated response for the three groups. Dopamine infusion increased dopamine
recovery by 23.9 ? 8.4% for the control group, 38.8 ? 8.3% for the GNX group and 13.9
? 4.9% for the GNX+T group. The observed increase for the GNX group was
significantly greater than GNX+T group (p = 0.031). Due to variability in control tissue
responses, there were no significant differences between the GNX and intact groups.
DOPAC Recovery
Examination of DOPAC, a metabolite of dopamine, released from the striatal
tissue showed a similar response to the infusion of dopamine (Fig. 3). Baseline values
were 0.83 ? 0.27 pg/mg/min for control animals (n = 6), 0.90 ? 0.47 pg/mg/min for the
27
GNX group (n = 6), and 0.067 ? 0.19 pg/mg/min for the GNX+T (n = 6) group. There
were no significant differences in the DOPAC recovery level between the control and the
GNX+T groups. However, the GNX group was significantly higher than GNX+T group
(p = 0.050), but not significantly different from the intact group.
The percent change in DOPAC recovery was also determined. Dopamine infusion
increased recovery by 12.4 ? 4.30% for the control group, 11.8 ? 5.76% for the GNX
group, and 5.83 ? 1.99 for the GNX+T group. There were no significant differences
between groups.
Experiment (2). The effects of infused MPP+ on the release of dopamine and
nopAC from the corpus striatum tissue.
Dopamine Recovery
There was no significant difference in the levels of baseline dopamine recovery
from striatal tissue obtained from the three animal groups (Fig. 5). Baseline values were
0.95 ? 0.16 pg/mg/min for control animals (n = 6), 1.19 ? 0.60 pg/mg/min for GNX
animals (n = 6), and 2.23 ? 1.08 pg/mg/min for GNX+T animals (n = 6).
Following MPP+ infusion, there also were no significant differences in dopamine
recovery between groups. Average dopamine recovery for the intact group was 4.45 ?
1.75 pg/mg/min (n=6), 10.1 ? 7.06 pg/mg/min (n=6) for the GNX animals, and 10.5 ?
6.63 pg/mg/min (n=6) for the GNX+T group.
Figure 6 shows the percent change in dopamine recovery for each group between
the baseline and stimulated responses. MPP+ infusions increased dopamine recovery by
11.37 ? 1.29% for the control group, 14.75 ? 2.13% for the GNX group, and 10.16 ?
2.06% for the GNX+T group. The observed increases were not significantly different in
between groups.
DOPAC Recovery
Baseline analysis for the DOPAC release showed some variation. Baseline values
were 0.84 ? 0.15 pg/mg/min for the intact group (n = 6), 1.33 ? 0.41 pg/mg/min for GNX
group (n = 6), and 3.38 ? 1.25 pg/mg/min for the GNX+T (n = 6) animals. There was a
28
significant difference between the GNX+T and intact groups (p = 0.03), however, there
were no significant differences between other groups (Fig. 7).
After MPP+ infusion, there were no significant differences in DOPAC recovery
between groups (Fig. 7). Average DOPAC recovery for the intact group was 1.64 ? 0.38
pg/mg/min (n = 6), 0.96 ? 1.28 pg/mg/min (n = 6) for the GNX group, and 3.16 ? 1.76
pg/mg/min for the GNX+T (n = 6).
Figure 8 shows the percent change in DOPAC recovery between the baseline and
stimulated response for the three groups. MPP+ infusions increased DOPAC recovery by
6.52 ? 1.29% for the control group, 8.78 ? 1.75% for the GNX group, and 5.94 ? 2.95%
for the GNX+T group. The observed increases were not significantly different in between
any group.
Experiment (3). The effects ofinfused NMF on the release ofdopamine and DOPAC
from the corpus striatum tissue.
Dopamine Recovery
Infusion of control KRP buffer provided baseline data for dopamine release from
the striatal tissue. After 30 min ofcontrol infusion, the saline was changed to KRP buffer
plus NMF (lmM) for 20 min. Control buffer was then infused for the reminder of the
experiment.
There were no significant differences in the levels ofbaseline dopamine recovery
from the striatal tissue obtained from the three animal groups (Fig. 9). Baseline values
were 0.34 pg/mg/min ? 0.17 for control group (n = 6), 0.10 ? 0.04 pg/mg/min for GNX
group (n = 6), and 0.03 ? 0.04 pg/mg/min GNX+T (n = 6) group.
There were also no significant differences in the levels of stimulated dopamine
recovery from striatal tissue obtained from the three animal groups (Fig. 9). The mean
values were 1.18 ? 0.81 pg/mg/min for the control group (n = 6),2.52 ? 0.62 pg/mg/min
for the GNX group, and 0.90 ? 0.52 pg/mg/min for the GNX+T group (n = 6).
In contrast, evaluation of the percent change data for dopamine recovery
demonstrated significant effects following NMF infusion (Fig. 10). Gonadectomy resulted
in a significant increase in average recovery following NMF infusion when compared to
29
the control group (p=0.0032). There were no significant difference between the other
groups 6.78 ? 3.97% for the control group, 30.99 ? 5.24% for the GNX group and 20.13
? 13.45% for the GNX+T group.
DOPAC Recovery
Baseline analysis for DOPAC release showed some variation, however there were
no significant differences between groups. Baseline values were 0.93 ? 0.32 pg/mg/min
for intact group (n = 6), 0.53 ? 0.17 pg/mg/min for GNX group (n = 6), and 0.60 ? 0.24
for the GNX+T pg/mg/min (n = 6) (Fig. 11).
The NMF DOPAC stimulated recovery levels also showed no significant
differences between groups. The mean values were 3.50 ? 1.37 pg/mg/min for the control
group (n = 6), 2.73 ? 0.75 pg/mg/min for the GNX group (n = 6) and 1.28 ? 0.79
pg/mg/min for the GNX+T group (n = 6) (Fig. 11).
Figure 12 illustrates percent change in DOPAC recovery after NMF infusion.
Average DOPAC recovery for the groups was 7.26 ? 1.17% for the control group (n = 6)
animals, 14.65 ? 2.85% for the GNX group (n = 6), and 12.47 ? 3.24% for the GNX+T (n
= 6). There was a significant increase in DOPAC recovery between GNX and intact
groups (p=0.005). There were no significant differences found between the other groups.
Experiment 4. Content Study.
Dopamine and DOPAC Tissue Content
Statistical analysis ofthe dopamine and DOPAC tissue content did not show any
significant differences between groups; GNX, GNX+T or intact. This statement applies
to all ofthe superfusion experiments done in this thesis (data not shown).
30
Figure
infusions.
no
recovery
when
for
31
14.00 1 -------------,----------------,
Dopamine tnfU$ion
*12.00 t--------------+------+------~
10.00 t-------......-..,.------+-------I----------I
4.00 t---------------l-----
2.00 t---------------+--=l=--
0.00 -I-_...,';II;,...--.J_" ...L.
c:E
8.00 BlntactCJ
E -GNXCJ
-GNX+TQ.
Gl Blntactc:
E -GNX!
6.000 -GNX+T
c
Baseline Infused
32
-
Figure
recovery.
GNX
each
33
6000.00 ------------------------l
*
5000.00 l-------------I-------------~
0.00-1----
1000.00 -1----
4000.00
CD
? Intact
IIIIII
CD..
.GNX
u.E
3000.00
.GNX+T
CD
Cl.s
c:CD
u..
CDD.
2000.00
34
Figure
dopamine
baseline
the
each
35
14.00 -.----------------,.----------------,
Dopamine Infusion
*
12.00 +--------------+-------1--------1
10.00 +--------------t-------II----------j
~ 8.00 +---..........----------+------..
"61i
o:
8 6.00 +--------------+----
4.00 +---..........-------.----+------'
2.00 +--------------+-........---..............
0.00
Blntact
BGNX
BGNX+T
Blntact
BGNX
BGNX+T
Baseline Infused
36
Figure
recovery
significant
37
9000 r--------------------.,
8000 r-----------r~---------J
7000 r--------------+----------J
6000
II)
IIIIII
II) 5000...
u.= -Intact
II)
tllS -GNX
c
II) 4000 -GNX+Tu...
II)
Q.
3000
2000 r-----------1I-------------J
1000 ,---------=--.....__+-----------J
0-1---.1---1-
38
Figure
MPP+
in
group).
39
25,--------------r----------------,MPP+ Infusion
20 +--------------+---------------1
c: 15E Blntact
"al -GNXE
~ -GNX+T
CD Blntactc:
E -GNXII
0 -GNX+T0
10
5+--------------t-----'F---
o
Baseline Infused
40
Figure
recovery
significant
41
1000.00 ,------------------------------,
900.00 -1---------------__------------------1
0.00+----
800.00 +---------------+-----------------j
700.00 -1------------
300.00 +----
100.00 +--~-
200.00 +----
600.00
GIIII
IIIl!!
Blntactu
.E 500.00 BGNX...
c:: BGNX+TGI
~:.
400.00
42
Figure
MPP+
significantly
[p=O.03]
between
43
7.00 .----------------r------------------,
MPP+ Infusion
6.00 +----------------t------------t------"1
5.00 +----------------+-------------11---------1
*
t:'E 4.00
ClE
~
g: 3.00 +-------------
2.00 +------------
1.00 +------------;1----
0.00
-Intact
-GNX
-GNX+T
-Intact
-GNX
-GNX+T
Baseline Infused
44
Figure
recovery
significant
45
350
300
250
50+----
o
100
200
-Intact
GI
-GNX
fIl
~
-GNX+T
u.5
CGI
U...
GIa..
150
46
Figure
NMF
differences
periods.
47
4.000
NMF Infusion
3.500
3.000
0.500
1.000
0.000
2.500
-IntactI:
-GNX~
[]GNX+Ti
-Intact2.000
-GNX
QlI:
-GNX+T
"e1lI
Co8
1.500
Baseline Infused
48
Figure
recovery
greater
(n=4
49
9000.000 *
8000.000 L----------+---------l
0.000
2000.000 .!----------
7000.000
3000.000 L----------
6000.000 -1----------
1000.000
CD
II) 5000.000
-Intact
IIICD
IIGNX
..u
.5
-GNX+T
..c
CDu..
4000.000CD0.
50
Figure
NMF
differences
51
6.000 NMF Infusion
5.000
4.000
c'Er
~ 3.000 L----------,
~8
2.000
1.000
0.000
Baseline
II Intact
IIGNX
IIGNX+T
II Intact
IIGNX
IIGNX+T
52
Figure
recovery
greater
(n=4
53
8OO~-----------------1
700 L---------~*~---------1
600L---------+--------i
500
QI
-Intact
II)
IIIl!
-GNX
u.E
400
-GNX+T
..c::
QIU...
~
300
200+----
100+----
0+----
54
Discussion
An exploration of the literature suggests a greater occurrence of Parkinson's
disease in males versus females. Clinical observations in geriatric medicine revealed the
first gender differences in relation to Parkinson's disease. This literature supports the
notion that men are more at risk of acquiring the disease than women (Dluzen and
McDermott, 2000).
Substantial amounts of data from experiments in animal models suggest a reason
for this gender difference. Estrogen has been reported to act as a neuroprotectant of the
nigrostriatal dopaminergic system by inhibiting DAT function and thereby reducing the
uptake of specific neurotoxins that in turn promote the degeneration of neurons within
substantia nigra neurons (Arvin et. al. 2000, Dluzen 2000, Disshon and Dluzeii 1997).
The results for the dopamine infusion experiment show higher concentrations of
dopamine and DOPAC recovery in the GNX animals versus the intact and the GNX+T
rats. As indicated by the results of experiment one, the loss of testosterone appears to
slow the DAT activity and lead to an increase in the recovery in the superfusion fluid.
During dopamine infusion, less dopamine and DOPAC were recovered from tissue taken
from animals having testosterone (either intact or GNX+T). In contrast, data acquired
from experiments infusing estrogen into ovariectomized rats have demonstrated that
estrogen blocks the DAT (Arvin et. al. 2000). When methamphetamine was infused into
striatal tissue, it significantly increased dopamine release, but this was prevented in the
presence of estrogen. This indicates that estrogen acts to inhibit DAT function (Myers,
2003), and therefore the striatal neurons were unable to recover the neurotoxin, which
remained in the synaptic cleft (Dluzen, 2000).
The neurotoxin MPP+ causes a selective destruction of dopaminergic neurons of
the nigrostriatal pathway in humans, rats and mice and therefore has been used as a
model for investigating PD. It is taken into the neuron terminal via the DAT and acts by
displacing dopamine from its storage sites in synaptic vesicles, causing an increase in
dopamine concentration gradient in the cyotsol. This reverses the direction of the DAT
and eventually causes the release of dopamine into the extracellular space (Dluzen,
2000). Examination of figures 6 and 8 indicates a similar trend in the data following
55
MPP+ infusions. We consistently found that the greatest percent increase in dopamine or
DOPAC recovery was from the gonadectomized rats. The dopamine and DOPAC
recovery from intact or GNX+T rats was reduced. These results support the hypothesis
that DAT activity is reduced in the absence oftestosterone. A study done by Dluzen and
Disshon (1998) indicated that when estrogen was present and mice were treated with
MPP+, the outcome was increased dopamine released into the superfusion fluid.
However, when they blocked the effect ofestrogen with Tamoxifen (an estrogen receptor
antagonist), the dopamine concentration was significantly less. This finding suggests that
estrogen acts to inhibit DAT activity. In contrast, the results ofthe present study suggest
that the presence oftestosterone serves to enhance dopamine transporter function within
the nigrostriatal dopaminergic system in the male brain. The increased dopamine released
from corpus striatal tissue in castrated male animals in this study indicate~ that DAT
function is decreased in the chronic absence of testosterone. These findings then suggest
that in the presence of testosterone, DAT activity is increased. This elevated activity
would enhance the ability ofthe dopaminergic neurons to take up neurotoxins, resulting
in the death ofthese neurons and the eventual onset ofPD.
This study confirms that nomifensine is working as a DAT blocker, as
increased concentrations of dopamine and DOPAC were recovered during
nomifensine treatment (figure 9 and 11). Analysis ofthe percent change data indicates
that GNX animals had eight times more dopamine recovered in the superfusion fluid
than either the GNX+T or intact groups (figure 10). These results for nomifensine
treatment support the results obtained for dopamine infusion. Both studies confirmed
that dopamine and DOPAC recovery rates were greatest from GNX animals when
compared to GNX+T and intact rats. These results suggest that DAT activity is
significantly inhibited in the absence of testosterone. In contrast, DAT activity is
increased in the presence of testosterone suggesting a role for testosterone in the
physiological modulation of the efficiency and efficacy of the DAT. In contrast, a
study done by Disshon and Dluzen (1999) shows results of an in vitro superfusion
study using estradiol, which like nomifensine increased the recovery of infused
dopamine from the striatal tissue ofovariectomized female rats.
56
The experiments presented in this thesis suggest that androgens may function to
increase the activity or efficiency of the DAT. In contrast, many experiments have
reported that estrogen inhibits the activity of the DAT (Arvin, 1998; Dluzen, 2000;
Disshon and Dluzen 1999; McDermott and Anderson 1999). Therefore, with respect to
neurotoxins such as MPP+ that are known to gain entry to dopamine neurons via the
DAT and lead to symptoms of classic PD it appears that men (higher levels of
testosterone) maybe at greater risk of accruing the disease than women (higher levels of
estrogen). The mechanism(s) by which the presence or absence of testosterone (or
estrogen) facilitates or inhibits the DAT remain unknown. However, it has been reported
that the activation ofprotein Kinase C (PKC) results in the phosphorylation ofthe DAT.
This phosphorylation is linked to the internalization and subsequent down regulation of
the DAT, which inhibits the reuptake of dopamine (Drew and Werling, 2001) Thus it is
interesting to speculate that steroid hormone may alter DAT activity through mechanisms
that involve the regulation ofPKC.
In conclusion, testosterone is an important steroid that appears to modulate the
function of the DAT. Results of our experiments demonstrated that dopamine and
DOPAC recovery were greatest in the GNX rats and were decreased in the intact or the
GNX+T animals. These findings suggest that testosterone acts to enhance DAT activity
while the loss of testosterone reduces DAT function. With respect to activity of
neurotoxins (such as MPP+) to cause symptoms ofPD, our results suggest that men may
be more prone to this disease due to the facilitatory actions of testosterone on DAT
activity. Further experiments are needed to examine in detail the mechanisms ofaction of
testosterone and on DAT function and to explore the relationship between DAT activity
and neurodegenerative diseases.
57
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65
Northeastern Ohio Universities College of Medicine",
TO:
FROL\I:
Dean E. Dluzen. Ph.D.
Associate Professor, AnJ.lOmy
Gary D. Niehaus. Ph.D.
IACUC ChJ.irperson
SUBJECT
DATE:
Protocol :\pproval by the Northe:lstem Ohio Universities College of Medicine
(0iEOlJCO~l) Institution:ll AnimJ.1 C:lre :lod? Use Committee (L-\CUC)
December 10,2002
The following j'.,cOUCO~l protocol was revie"ved J.ild approved by this Institution's Animal Clfe ~Jnd
Use Committee (L-\CUC) on December 10,2002. Protocols involving the use of hum:m ti"ssues rc::quire
InstitutionJ.1 Review Board (ffiB) appron!.
NEOUCOl\I Protocol No.:
Ti tie of Protocol:
Type of Vertebrate:
Funding Agency:
02-033
Gonadal Steroid Hormonal Modulation of Dopamine
Tr:.1nsporter FUllction \Vithin Male Rats
Rats
Internal Funds
This institution has an AnimJ.1 WelLue Assur:lnce on file with the Office of LJ.bor:ltory Anim;ll Welbre
(OLA\y') The AssurJ.nce number is .A3-1-74-0 1. This institution is J.lso registered with the United Sutes
Department of Agriculture (USDA). The USDA registcltion number is 31-R-0092.
The Comp;lrJ.tive Medicine Unit (GvlU) J.t the Northeastem Ohio Universities College of ~redicine
(l'-fEOUCOM) hJ.s been J.ccredited "'lith the Association for Assessment for Accreditation of LaborJ.tory
AnimJ.l CJ.re (A.A..j),lAC) IntemationJ.1 since June 8, 1982. Full J.ccreditJ.tion was lJ.st renewed on July 8.
2002.
Thank you.
GDN:lkn
Cc: Gary B. Schneider, Ph.D.
Associate Dean of BJ.sic Medical Sciences
Associate Dean for Research
N"EOUCOM Institutional Official
Shannon Russell
Research & Sponsored Programs
File