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The role of ivermectin on P2X4 receptors in regulating behavior responses in the Parkinson’s disease model

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The role of ivermectin on P2X4 receptors in regulating behavior responses in the Parkinson’s disease model

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Content Copyright 2021 Dongwook Wi

The Role of Ivermectin on P2X4 Receptors in Regulating Behavior
Responses in the Parkinson’s Disease Model

By

Dongwook Wi

A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

AUGUST 2021
ii
Dedication

This thesis is dedicated to
my parents and brother; my mother, Mi Jung Kim, my father, Hee-Soo Wi, and
my brother Dong-Jin Wi.
For their unconditional love, endless support, encouragement, and sacrifices.

iii
Acknowledgments
I want to express the most profound appreciation to my advisor, Professor Daryl Davies,
who has the attitude and the substance of genius: he continually conveyed a suggestion of
adventure regarding research and allowed me to work at the lab. Without his help and persistent
help, this thesis would not have been possible.

Dr. Daryl, Davies, and Ph.D. Alicia Marie Paiva Warnecke supported this work. It allowed
me to evolve as a graduate research assistant, develop my professional lab skills, and learn in their
laboratory. I would also like to thank my lab colleague, Ph.D. candidate Joshua Silva and Lila
Halbers for their lab skills, teamwork, and expertise.

This thesis could not be completed without our group members' effort and cooperation. I
also thank our thesis committee, Dr. Curtis, Okamoto, Dr. Martine, Culty, Dr. Rogan, Duncan for
their guidance and encouragement in finishing this work and even teaching me how to write a
thesis.

Finally, I would like to thank our family: my parents Hee-soo Wi and Mijung Kim, for
giving birth to me in the first place. I would like to thank my girlfriend for her constant source of
inspiration and for supporting me spiritually throughout the unprecedented period COVID-19.

iv
Table of Contents

Dedication ...................................................................................................................................... ii
Acknowledgments ......................................................................................................................... iii
List of Tables ...................................................................................................................................v
List of Figures ................................................................................................................................ vi
List of Abbreviations .................................................................................................................... vii
Abstract ........................................................................................................................................ viii
Introduction ..................................................................................................................................... 1
1.1 Parkinson’s Disease (PD) ..........................................................................................................1
1.2 Parkinson’s Disease Models Mechanism: Compensatory mechanism .....................................4
1.3 Animal models of Parkinson’s disease ......................................................................................5
1.4 Purinergic Signaling Involvement in CNS and P2X4s ..............................................................8
1.5 Ivermectin and the target modulation by Ivermectin ...............................................................13
1.6 Sites of Ivermectin Action on GABAARs and P2X4s .............................................................15
Specific aim ...................................................................................................................................19
Materials and Methods ...................................................................................................................21
Results ............................................................................................................................................27
Discussion ..................................................................................................................................... 39
Conclusion .................................................................................................................................... 42
References ......................................................................................................................................44
v
List of Tables

Table 1: Average AMPH Rotations and Percent lesioned for male (n=8) vs. female (n=12) ...... 27
vi
List of Figures

Figure 1: Site of stereotaxic injection in the medial forebrain bundle (MFB) ................................ 8
Figure 2: Schematic model of fusion activated Ca
2+
entry via P2X4 mediating fusion pore
expansion ........................................................................................................................................ 9
Figure 3: Chemical structure of ivermectin ...................................................................................13
Figure 4: P2X receptors' molecular properties .............................................................................16
Figure 5: Forms of the P2X4R TM1 and ivermectin .....................................................................17
Figure 6: Full-size and zoomed P2X4R rat models and graphic analysis of the ivermectin binding
site .................................................................................................................................................18
Figure 7: 6-hydroxydopamine-lesioned by tyrosine hydroxylase (TH) stains and saline-injected
mice ............................................................................................................................................... 28
Figure 8: Amphetamine-induced (5.0 mg/kg) rotations comparing females with male mice .......29
Figure 9: The difference net AMPH rotation between male and female mice with lesioned and
non-lesioned ...................................................................................................................................29
Figure 10: L-DOPA (5.0 mg/kg) rotations comparing females with male mice ...........................30
Figure 11: The difference net L-DOPA rotation between male and female mice with lesioned and
non-lesioned ...................................................................................................................................31
Figure 12: Contralateral rotations per 10 minutes bin during 90 minutes in male lesioned mice .32
Figure 13: Contralateral rotations per 10 minutes bin during 90 minutes in female lesioned mice
........................................................................................................................................................33
Figure 14: Performances on the Rotarod in five trials of 6-OHDA lesioned male mice treated
with saline, non-lesioned, L-DOPA+IVM, and L-DOPA .............................................................34
Figure 15: Rotarod performance by behavior test across the 5 trials of female 6-OHDA lesioned
treated with saline, L-DOPA+IVM, L-DOPA, and non-lesioned .................................................35
Figure 16: Performances on the Rotarod in 6 trials of male MPTP lesioned mice treated with saline,
L-DOPA+IVM, IVM, L-DOPA, and non-lesioned .......................................................................36
Figure 17: Rotarod performance by behavior test across the 5 trials of female MPTP lesioned
treated with saline, L-DOPA+IVM, L-DOPA, and non-lesioned ................................................ 36
Figure 18: Total time with an object on Novel Object Recognition (NOR) testing by 6-OHDA
males and females treated with the treatment group, and the percentage of time spent with the
object by 6-OHDA lesioned mice treated with the treatment group ............................................ 37
Figure 19: Total time with an object on Novel Object Recognition (NOR) testing by MPTP males
and females treated with the treatment group, and the percentage of time spent with the object by
6-OHDA lesioned mice treated with the treatment group ........................................................... 38

vii
List of Abbreviations
5-BDBD: 5-(3-Bromophenyl)-1, 3-Dihydro-2H-Benzofuro [3,2-e]-1,4-Diazepin-2-one
6-OHDA: 6-hydroxydopamine
AAAD: Aromatic L-amino acid decarboxylase
ATP: Adenosine-5’-triphosphate
BBB: Blood-brain barrier
BDNF: Brain mediated neurotrophic factor
BZ: Benzodiazepine
BzATP: 2',3'-O-(4-benzoyl-benzoyl)-ATP
CNS: Central nervous system
DA: Dopamine
DAT: Dopamine transporter
DAergic: Dopaminergic
ECD: Extracellular Domain
GABA: γ-aminobutyric acid
GABA ARs: GABA type A receptors
GABA A PAMs: GABA A positive allosteric modulator
GluCls: Glutamate-activated chloride channel
GPi: globus pallidus
I.P: Intraperitoneal
IVM: Ivermectin
L-DOPA: Levodopa
LGIC: Ligand-gated ion channels
LID: L-DOPA-induced dyskinesia
MAO-B: monoamine oxidase-B
MFB: Medial forebrain bundle
MOX: Moxidectin
mi RNA: microRNA short interfering RNA (siRNA)
Macrocyclic lactones: (MLs)
MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
NLG: N-linked glycosylation
NOR novel object recognition
P2XRS: P2X receptors
P2X4RS: P2X4 receptors
P2X4 KO: P2X4 knockout
PAM: positive allosteric modulator
PBS: Phosphate buffer saline
PPADS: pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid
PD: Parkinson’s Disease
PFA: Paraformaldehyde
PNS: Peripheral nervous system
SC: Subcutaneous
SCI: Spinal cord injury
siRNA: Short interfering RNA
SN: Substantia nigra
SNPC: Substantia nigra pars compacta
STN: Subthalamic nucleus
TH: Tyrosine hydroxylase
TM: Transmembrane
TNP-ATP: 2'-(or-3')-O-(Trinitrophenyl) Adenosine 5'-Triphosphate
WT: Wild Type
Zfp2x4: Zebrafish P2XR
viii
Abstract
The primary therapy for symptomatic motor benefits linked to patients diagnosed with Parkinson’s
Disease (PD) has been levodopa (L-Dopa). L-DOPA is a precursor of dopamine (DA) and
represents the gold-standard pharmacological treatment for Parkinson's disease. Nevertheless, L-
DOPA has had numerous drawbacks in long-term treatment, leading to motor problems as the
therapy becomes less effective. Presently there is no cure for PD, where the disease has been linked
to a dopaminergic disorder that affects the nigrostriatal pathway triggered by dopamine deficiency.
Purinergic ionotropic P2X4 receptors (P2X4s) have been introduced as a protein candidate that
would regulate dopamine activity. P2X4s are critical dopamine activity modulations that increase
the release of calcium into neurons, leading to an increase in secretion of DA. The goal of my
thesis project is to illustrate the interaction between ivermectin (IVM) modulation of P2X4s using
PD models, 6-OHDA and MPTP to evaluate the effect of P2X4s that are involved in alterations in
motor behavior for PD. This will be accomplished by summarizing drug efficacy via the concept
of IVM's therapeutic targets with the presence of L-DOPA on P2X4s. Overall, this work should
help in the identification of potential therapeutic roles of the purinergic receptor system in the
activation of neurotransmission of DA and the sex differences in the response by IVM in C57BL/6J
mice.
1
Introduction

1.1) Parkinson’s Disease (PD)
Parkinson’s disease (PD), first identified by James Parkinson in 1817, is the second most
common neurodegenerative disorder. It causes cell death as well as the loss of neurotransmitter
activity (Parkinson, J. 1817). Consequently, PD results from a change in the behavior of an adult
brain's normal constituent. The degeneration may be related to the polymerization of an aggregated
α-synuclein in fibrils in the cell bodies of the basal ganglia structure (Burre et al. 2010). About
one million Americans are currently afflicted by PD, with an estimated 60,000 new PD diagnoses
every year (Aminoff, M. J. 1994). PD typically occurs in adults over the age of 50 and is believed
to be the result of a depletion of dopamine pathways of substantia nigra pars compacta (SNPC),
leading to the loss of dopamine and neural circuit disruptions in the basal nuclei (Exner et al. 2012).
Furthermore, the study of the pathophysiology of PD involves the depletion levels of dopamine in
the substantia nigra (SN) with a loss of pigment and the presence of intracellular inclusions. It
indicates that DA gives its coloration to the structure; thus, the brighter the nigra, the lower the
DA loss (Darcy et al., 2012).
Earlier research showing a substantial depletion of dopamine in autopsy PD brains also
recorded a decrease in GABA in certain thalamus regions (Gerlach et al. 1996).
Neurophysiological studies have also demonstrated that the subthalamic nucleus (STN) is
excitable in PD patients because it is not blocked by GABA originating from the external globus
pallidus (GP). These improvements in GABA will potentially have an impact on thalamus
performance and behavioral activities. The study has shown evidence that the pharmacotherapy of
PD is based on a deficit in particular neurotransmitters, including dopamine (DA), which is an
2
essential neurotransmitter of a bilateral dopaminergic pathway, and γ-aminobutyric acid (GABA)
(Umphred et al. 2003).
The majority of PD investigations have focused on dopaminergic nigrostriatal cells’
neurodegeneration as the primary cause of motor impairment. The fundamental motor symptoms
in PD rely on progressive degeneration in the SNPC of dopamine-containing neurons (Blaszczyk,
J. W., 1998). It is possible that the effective functioning of conventional motor and non-motor
functions involves a structurally diverse neuronal circuitry with a significant level of
interconnectedness that uses a combination of various neurotransmitters like dopamine and GABA.
PD's clinical features include both motor and non-motor symptoms, affecting function to a variable
extent. While the motor dysfunction mechanisms are well known, the neuronal and molecular
substrates for non-motor manifestations are far from obvious. The motor symptoms of PD are a
condition in which dopaminergic neurons become impaired or die. As a result, it causes significant
depletion of dopamine input in the corpus striatum. It results in rigidity, akinesia, and motor
disorders that slow down the body (Brooks, D. J., 1998). Non-motor signs, on the other hand,
include fear, stress, fatigue, sluggish thought, and depression (Salawu et al. 2010) that have been
found to predate PD's classical motor features and are positively correlated with GABA deficiency
(Pellicano et al. 2007; Stefanis, L. 2012).
A) Parkinson’s Disease Treatments
Presently there are no drugs that can be used to halt the progression of PD, but rather,
current treatments focus on reducing the symptoms of PD in the patients in an attempt to improve
quality of life. For example, the first drug approved for PD treatment, Levodopa (L-DOPA), acts
locally using the residual DA neurons for DA synthesis (Fahn et al. 2004). However, L-DOPA
itself is not active. It is highly efficient in low doses when administered in the early stages of the
3
disorder, but over time efficacy is limited, and adverse side effects become more apparent
(Parkison's Study group, 2004). These shortcomings illustrate that alternate therapeutic techniques
need to be established since L-DOPA's long-term usage involves dosage escalations, which
eventually contributes to multiple complications, including motor disturbances called L-DOPA-
induced dyskinesia (LID) (Fhan et al. 2004). L-DOPA does not affect various motor PD signs,
along with dysautonomia, sleep disorder, cognitive dysfunction, and apathy (Park & Stacy, 2009).
Many non-dopaminergic therapies have been proposed for PD but have provided few therapeutic
benefits. Also, the oral L-DOPA component that is decarboxylated to dopamine in peripheral
tissues does not reach the brain. It is thus unavailable for the intended treatment purposes of
elevating dopamine in the brain. For this reason, the Levodopa/carbidopa combination is usually
used to inhibit aromatic L-amino acid decarboxylase (AAAD) in peripheral tissue and increase the
impacts of L-DOPA while minimizing dopamine's peripheral adverse effects (Aldred & Nutt,
2010). Therefore, L-DOPA is supposed to be primarily transformed to DA via AAAD and used in
DA neurotransmission.
Furthermore, PD therapy has been used to target DA neurotransmission through
pharmacological strategies such as DA receptor agonists, DA reuptake inhibitor, and DA
enzymatic metabolism inhibitors (Foster & Hoffer, 2004). Since PD patients' striatum's and
SNPC’s have lower DA levels, they can be treated with DA agonists, which mimics dopamine
behavior when levels are low. Unlike levodopa, a dopamine agonist in the brain is not transformed
into DA, and it directly activates dopamine receptors somewhat similar to dopamine, but subtle
differences remain. In mild to moderate PD, DA, a substance that triggers dopamine receptors that
can bypass dopamine synthesis processes and bind to dopamine receptors directly to dopamine
target cells, should be used alone to efficiently reduce the symptoms.
4
There is another active pathway inside the CNS that regulates calcium-mediated processes
and may cause neurodegeneration when disrupted. Neurotransmitter release throughout neural
circuits stimulates adjacent astrocytes that prevent further influx of calcium ions into presynaptic
neurons through GABA (Blaszczyk, J. 2016). The GABA mechanism must regulate the influx of
calcium explicitly through GABAergic receptors and indirectly through the astrocytes and glial
networks of the typical GABAA and GABAB receptors presynaptic receptors to protect the neuron
(Watanabe et al. 2002).
1.2) Parkinson’s Disease Models Mechanism: Compensatory mechanism
Early PD models’ development derives from the need to better understand the
compensatory mechanisms during the initial disease phase in patients. Compensatory processes
were due to improvements and modifications in the nigrostriatal pathway, such as increased
neuronal activity in the SNPC, and increased dopamine synthesis and striatum release. 70% of the
DA nodes are damaged when the typical motor deficits arise, and progressive DAergic neuron loss
has occurred. When preceding the initiation of motor symptoms, during which such neurologic
symptoms occur, mechanisms are triggered to compensate for the DA impairment and sustain the
neuronal metabolic activity (Bezard et al. 2001). However, when these mechanisms are
overwhelmed, motor symptoms such as rigidity and akinesia appear.
Early work by Blesa and colleagues showed that compensatory mechanism occurs at
various locations including synapses and loops, all designed to maintain dopaminergic neuronal
activity. These alterations include increasing dopamine synthesis, DA turnover, and enhancing
tyrosine hydroxylase (TH) activity (Blesa et al., 2012). This results in changes in neurons and the
morphology of synapses. Regarding changes in the behavior, it may be challenging to disentangle
whether alterations in the corpus striatum, prefrontal cortex, brainstem, and prefrontal regions are
5
caused itself or are compensatory mechanisms. While DA turnover is the homeostatic system
capable of preserving the level of physiological DA, this process is postulated to occur only in the
presence of substantial DA degradation (Bezard et al. 2003). Thus, it cannot be treated as an
entirely compensatory mechanism. Previous analysis has provided evidence that DA rates are
lowered, and synthesis and DA turnover in PD patients and animal models are improved (Bezard
et al. 2003). Another factor to recognize is the serotonergic pathway, which is believed to be
overactivated and could be responsible for L-DOPA's pharmacological impact. However,
serotonergic system activation and its role in PD is not straightforward, especially in the early
phase, and literature has recorded discordant results (Matteo et al. 2008). In developing new early
treatment tests and initial treatments for PD, an understanding of the pathways triggered at the
interval between the first neurodegenerative diseases would be of great importance.
1.3) Animal Models of Parkinson’s Disease
To develop new drugs for treatment of PD, an animal model is necessary. In a perfect world,
this PD model would include symptoms and clinical manifestations of PD with DAergic and non-
DAergic systems, movement and non-movement symptoms. Additionally, the PD model should
reflect the age of patients and the age of onset. Unfortunately, none of the existing animal models
of PD fully manifest or mimic all of the cognitive and behavioral changes seen in PD patients.
Given these limitations, animal models have significantly contributed to our overall knowledge of
the disease process and reasonable treatment goals in PD. Present animal PD models may be
classified roughly into two classifications: genetic and neurotoxic models. One limitation of the
genetic models is that they are mainly produced based on defined objectives correlated with
possible pathways believed to trigger PD in individuals (Meredith et al. 2008). However, the
neurotoxic models (MPTP & 6-OHDA) used to damage the bilateral dopaminergic pathway may
6
complement this limitation of genetic models. Currently, using a microRNA (miRNA) or short
interfering RNA (siRNA) cocktail, a very reliable and efficient PD model showing all PD
symptoms has recently been developed (Jagmag et al., 2015). However, neither of the neurotoxic
models reproduce any of the PD characteristics exactly. These animal models have made a
significant contribution to our comprehension of disease mechanisms and future clinical goals in
PD, aside from the limitations. The models are continually developing, but advanced models are
needed.
A) The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Model
The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hypothesis emerged from
findings in the early 1980s that Californian intravenous opioid consumers experienced extreme
PD-like symptoms (Davies et al. 1979). The accumulation of MPP+ follows this conversion in
SNPC DAergic neurons through DAT activity, and MPTP is converted into its active form MPP+
by the enzyme monoamine oxidase-B (MAO-B) (Muralikrishnan & Mohanakumar, 1998). MPTP
produces significant differences in nigral cell depletion, striatal dopamine deficiency, and
behavioral defects, based on the treatment paradigm. However, the motor deficits do not reproduce
entirely those shown in PD. Besides, to gain more new PD findings and identify new therapeutic
methods, MPTP models were used. MPTP mouse models imitate certain disorder features and thus
are valuable ways for studying PD (Meredith & Rademacher, 2011). MPP+ systemic
administration may not harm primary DAergic neurons because it does not reach the BBB quickly
regardless of its charge. But its direct injection into the brain effectively destroys most of the
nigrostriatal DAergic pathway (Yazdani et al. 2006). MPP+ is an exceptional dopamine transporter
substratum (DAT), which describes its specificity of DAergic neurons. While the MPP+ mediated
apoptosis pathways have not been thoroughly described, MPP+ is known to be an essential
7
regulator of complex I respiration in isolated mitochondria. As a result, a significant decrease in
the amount of adenosine triphosphate (ATP) occurs in the striatum and SNPC, which allows us to
know significant disease symptoms by destroying dopamine pathways in the brain's SN (Chan et
al. 1991). Thus, based on multiple findings, MPTP tends to be the most significant animal model
possible to explain the damage to the nigrostriatal pathway in PD and is extremely valuable in the
research of neuroprotective and neurorestorative strategies.
B) 6-hydroxydopamine (6-OHDA) Model
In the early 1960s, 6-hydroxydopamine (6-OHDA), a hydroxylated analog of DA, was first
described (Senoh & Witkop, 1959). Presently, 6-OHDA is one of the most commonly used
neurotoxins used to induce and then study early- or late-stage PD (Blandini & Armentero, 2012).
The neurotoxin is delivered to the brain region of interest using a stereotaxic method allowing for
the toxin to directly impact the brain field of concern to reach individual neurons and circumvent
the blood-brain barrier. Furthermore, 6-OHDA has been the most commonly used drug to stimulate
nigrostriatal pathway neurodegeneration due to an inability to pass the BBB and its efficacy for
damaging SNPC DAergic neurons (Tieu, K., 2011). For this reason, 6-OHDA must be required to
be direct-injected into the brain; common lesioning targets include the medial forebrain (MFB),
striatum, and SNPC. DA deficiency, nigral dopamine cell failure, and neurobehavior abnormalities
have been effectively accomplished utilizing the paradigm (Figure 1). DA deficiency, nigral
dopamine cell failure, and neurobehavior abnormalities have been effectively accomplished using
this paradigm. Also, the previous analysis in Fontoura and Djaldetti’s article showed that the effect
of 6-OHDA intraperitoneal injection (IP) stimulates non-motor as well as motor symptoms, such
as memory loss, depressive disorder, excessive nervousness or sleep disruption, as well as
histology changes (Fontoura et al. 2017 & Djaldetti et al. 2014).
8

Figure 1: Graphical illustration of the stereotaxic injection site in the medial forebrain bundle
(MFB) shows dopaminergic fibers connecting the striatum and the SNPC. (De et al. 2015)

Since α-synuclein overexpression or toxic synuclein protofibril injections that make the
quantification of an automatic motor or sensory-motor activity take a relatively time-consuming
duration and require preparations, rotational amphetamine testing is becoming an ever more
common method for proving the functional restoration of the transplant. D-amphetamine reduced
the ipsilateral rotation rate or changed the rotation direction caused by amphetamine (Bjorklund &
Dunnett, 2019). Like MPTP, the 6-OHDA model offers a method for early diagnosis of PD and
evaluates antiparkinsonian therapies’ efficacy (Gerlach et al., 2012).
1.4) Purinergic Signaling Involvement in CNS and P2X4s
The definition of purinergic signaling was introduced in the early 1970s, as ATP as an
extracellular signaling agent with neurotransmitter properties (Burnstock et al., 1970). P2X
receptors are classified into a family of ATP-gated ion channels ranging from P2X1 to P2X7 for
human beings, mice, or rodents (North, R. A., 2002). Purinergic receptors activate the binding of
signals to their corresponding receptors, primarily to ATP nucleotides. Under physiological
9
conditions, the extracellular ATP is released from cells and extracellular ATP levels are regulated
by ectonucleotidases, which catalyze their degradation (Zimmermann, 2001). P2X receptor
subtypes each have phenotype-dependent structures, ionic characteristics, and responsiveness to
various allosteric modulators, agonists, and antagonists (Li et al. 2009). For example, the majority
of P2X receptors are calcium-permeable and receptor activation results in membrane
depolarization and increased ionized calcium’s intracellular concentration (Eagan & Khakh, 2004).
These ionotropic receptors are essential for intracellular signaling pathways induced by ionized
calcium (Khakh et al. 2001). Besides, stimulation of P2X receptors by ATP results in an increase
in Ca
2+
current based on the subtype. Upon P2X4 channel activation in the ATP cell membrane,
the substantial cation influx, Ca
2+
, contributes to membrane depolarization (Figure 2). Ca
2+
is
essential, for instance, for neurons for neurotransmitter release regulation (Neher & Sakaba, 2008)
and different microglial functions (Farber & Kettenmann, 2006).

Figure 2: Schematic model of fusion activated Ca
2+
entry via P2X4 mediating fusion pore
expansion. (Miklavc et al., 2011)
In most neurons and glial cells in the CNS, P2X4 overwhelmingly expresses functional
ATP-gated purinergic receptors among P2X receptors. For example, P2X4 is almost one thousand
times more sensitive to ATP than other purinergic receptors (Burnstock & Kennedy. 2011), which
10
has the highest Ca
2+
permeability. Besides, P2X4 plays an essential part in modulating synaptic
transmission. The interaction between synapses and adjacent glial, which is the most prevalent cell
type, contributes to 70 % of total cells in the CNS (Leanne et al., 2017). P2X4 activation of spinal
microglia contributes to the release of brain-derived neurotrophic factor (BDNF), which transmits
pain hypersensitivity to glial cell and spinal interneurons by disinhibiting GABAergic information
(Ulmann et al. 2008; Trang et al. 2009). Also, active neuroglia plays a crucial function in the
pathogenesis of certain disorders, like spinal cord injury (SCI) and neurodegenerative (Parkinson's
disease), and P2X4 may also lead to the pathogenesis of such conditions.
A) The structure of the P2X4 receptor
In plasma membrane trimeric structures, the P2X substituents are arranged in homomeric
or heteromeric configurations (Saul et al., 2013). In the extracellular P2X domain, three inter-
subunit sites were confirmed for ATP. For example, Hattori et al. showed in 2012 that three
molecules of ATP should bind the ECD (Extracellular Domain) for receptor activation, triggering
a non-selective cation channel created from the transmembrane domains (Hattori et al. 2012). For
its feature, the P2X4 receptor's conformational structure is important for its function. According
to the early work by Kawate, P2X4's trimeric organization was verified by the resolution of
zebrafish P2XR (zfP2X4) in a closed state at a resolution of 3.1A (Kawate et al. 2009). The
homomeric zfP2X4 has an ECD spanning approximately 70 Å above the membrane and the other
transmembrane region protruding 28 Å across the membrane (Kawate et al. 2009). Each receptor's
extracellular portion can be represented as a broad disulfide-rich domain (Hattori & Gouaux, 2012),
loaded with expressing N-linked glycosylation (NLG) moieties (Kawate et al., 2009). Six helices,
two from each subunit, form the receptor complex’s TM region (Kawate et al., 2009). The TM
11
helices are aligned nearly antiparallel to each other within a subunit, and to date, P2X4s are
identified as the first P2X receptor crystal structure in 2009 (Kawate et al., 2009).
B) A novel P2X4 antagonists
Rat's subunit P2X4 is comparatively resistant to traditional antagonists of the P2X receptor.
This receptor subunit's mouse and human sequence homology indicate some decreased
susceptibility to pharmacological agents, including pyridoxal phosphate-6-azophenyl-2', 4'-
disulphonic acid (PPADS) and 2'-(or-3')-O-(Trinitrophenyl) Adenosine 5'-Triphosphate (TNP-
ATP) (Khakh et al., 2001; Fever et al., 2006), were confirmed to be an antagonist for P2X4s. Due
to the current lack of P2X4 receptor antagonists and an ineffective antagonist for P2X4s is
troublesome, novel P2X4 receptor-selective antagonists, including 5-(3-Bromophenyl)-1, 3-
Dihydro-2H-Benzofuro [3,2-e]-1,4-Diazepin-2-one (5-BDBDs), were introduced. BZ
(benzodiazepine) derivative binds to P2X4 receptors at submicromolar concentrations, thus
disrupting the activity of the receptor.
C) A novel P2X4 receptor-selective agonists
ATP is still the most active agonist for P2X4s. The ATP dose-response relationship
between P2X4 in rats and humans were slightly different, with EC50 values of 6.9 ±0.5 and 7.2
±0.5 μM, respectively (Soto et al., 1996; Garcia-Guzman et al., 1997). Besides, P2X4s is also
stimulated by 2', 3'-O-(4-benzoyl-benzoyl)-ATP (BzATP), alpha,beta-Methylene-ATP (αβ-
meATP), and 2-methylthioadenosine 5'-triphosphate (2-meSATP) (Khakh et al., 2001; Jarvis et
al., 2009). However, initially, αβ-meATP was confirmed to be inactive, but a later study found that
its activities were species-dependent (Jones, Chessell, et al., 2000). αβ-meATP was found to be a
partial agonist for the mouse isoforms and humans, but instead an antagonist of the rat's P2X4
receptor. Specifically, several P2X4R ectodomain residues have been classified as essential for
12
receptor fuction, including Lys 67, Lys 313, and Arg 295, for ATP binding. Previous studies
showed that the ectodomain sequence of Lys67-Lys313 includes an ATP binding region and partly
accounts for the sensitivity of these areas for the receptor agonist (Stojilkovic et al., 2010). And as
the basic framework for producing receptor-specific agonists in further research and therapeutics,
P2X4R will serve as the clinical pharmacology receptor model.
D) Positive modulators
Given the potential of IVM to modulate both GABA and P2X4s, animal behavior
experiments may potentially expose results mediated by each receptor. Knockout (KO) mouse
models have been used to evaluate further which effects are related to P2X4 activity (Khoja et al.,
2019; Khoja et al., 2016). Even though there have been several analyses on all the other molecules,
IVM is the P2X4 modulator, most widely used in livestock for parasite control. Nevertheless,
because of its possible neurotoxicity, there have been some issues with IVM usage in humans. A
serious IVM’s adverse effects have been observed in individual humans with high filarial
nematode burden. IVM may cause side effects or PNS induced by natural exposure in animals like
that of deficient Pgp activity (Menez et al., 2012). As a P2X4 modulator, other anthelmintics that
are macrocyclic lactones (MLs) such as moxidectin (MOX) have been in progress due to IVM's
limitation. Even if the blood-brain barrier structure is damaged, MOX does have a more
considerable safety of effectiveness than IVM. The in vivo neurotoxicity is attributed to the
multiple effects of the medications on GABA-gated channels. These findings supported
understanding of ML toxicity and opened up new opportunities for potential human usage of MOX.

13
1.5) Ivermectin and the target modulation by Ivermectin
In the late 1970s, Satoshi Ōmura and William Campbell identified ivermectin (Burg et al.
1979) (Figure 3). IVM kills pathogens by stimulating glutamate-activated chloride channels
(GluClR) and targeting multiple ligand-gated ion channels and receptors, namely, GABAAR
(Adelsberger et al. 2000), P2X4 receptors (Chen & Kubo, 2018), and human P2X7 receptors
(Nörenberg et al., 2012), but no other P2X receptors can be potentiated by IVM and it may be
delivered orally or through injection (Klement et al. 1996). It works by linking GABA-gated
chloride to PNS, blocking nerve impulse conduction (Ricart et al. 2010). Besides, IVM is one of
the primary therapeutic agents used in anthelmintic and insecticides. It is the preferred medicine
for the treatment and prevention of onchocerciasis, known as Robles’ disease and lymphatic
filariasis (Remme et al. 2006).

Figure 3: Chemical structure of ivermectin: Ivermectin is a 16-member macrocyclic lactone
derivative with spiroketal benzofuran and disaccharide moieties. The molar mass is around 875 g
/mol. IVM is a complex of elements of 80% B1a and 20% B1b. (Burg et al., 1979)

14
However, IVM is a class of lipophilic molecules which can pass the blood-brain barrier
(BBB) but it also quickly fluxed out (efflux) via P-glycoprotein efflux transporter. Nonetheless, to
get sufficient bioavailability, it was thus deemed free of the potential to stimulate neural harmful
drug effects, even in overdose cases (Chung et al. 1999). Classification of the avermectin-binding
protein in invertebrates has shown that it binds to the membrane with a high affinity (Rohrer et al.
1992). However, avermectin-binding protein in a vertebrate is lower by about 100 compared to
nematode or insect membranes (Rohrer et al. 1992). It also accounts for the comparatively low
toxicity of this drug to mammals.
A) Modulation of P2X receptors by Ivermectin
Similar to GABA receptors, IVM can positively modulate P2X4s by partitioning into the
lipid membrane and transmitting the transmembrane regions of P2X4s (Silberberg et al. 2007).
Previous studies report that IVM attaches to the lipid-protein interface, operating on locations
inside the transmembrane section and the P2X4R ectodomain-transmembrane interface (Jelinkova
et al. 2006, 2008 & Silberberg et al. 2007). IVM functions as a positive allosteric modulator (PAM)
of the ATP-gated receptors of P2X, P2X4 in particular, but not the receptors P2X2 and P2X3
(Khakh et al. 1999). In other words, the most noticeable difference between P2XR species is their
sensitivity to IVM. For example, the P2X4 receptor displays a strong response to IVM, while other
P2XR subgroups are IVM-insensitive (Khakh et al. 1999). Entire-cell current studies have shown
that IVM enhances the susceptibility of P2X4R to ATP and raises current amplitude in response
to supramaximal doses of agonist about twice, and substantially exacerbates the deactivation of
current after agonist wash (Jelinkova et al. 2006). Single-channel research made it clear that IVM
stabilizes the channel’s open state (Priel & Silberg, 2004). The IVM-dependent enhancement of
15
P2X4R specificity for ATP has been used to classify the extracellular sequences engaged in ATP
binding/gating.
According to Krusek et al., “IVM by itself does not activate the P2X4R but preincubation
of 0.1–10 μM IVM oocytes had significant impacts mostly on ATP-activated current” (Krusek, et
al. 1994). Since P2X receptor-mediated receptor activation is unknown, postsynaptic P2X4
receptors could be involved in GABAergic epileptic transmission. The role of P2X receptors in
the CNS is strongly correlated with GABAergic inhibition (Robertson et al. 2001). In contrast,
P2X2 immunohistochemistry was decreased in the pathway and the Schaffer collaterals. It states
that levels of GABA could be involved in altering the expression of the receptor P2X. The GABA-
mediated activity can influence P2X receptor expression rates in the small mammal’s hippocampus
(Kang et al. 2003). But these alterations do not give enough explanation about the influence of
GABA on the expression of the P2X receptors. It is because GABA’s inhibitory roles are regulated
by two different receptor groups, such as the GABAARs and the GABABRs (Benke et al. 1999).
Additionally, GABAAR antagonism by bicuculline decreased P2X expression levels in seizure-
resistant gerbil’s hippocampus even though P2X4 immunohistochemistry was significantly
increased in the mossy route. Those same results indicate that the GABAAR in the gerbil cerebral
cortex facilitates the regulation of expression of P2X receptors (Kang et al. 2003).
1.6) Sites of IVM action on GABA
A
Rs and P2X4s
As described above, GABAARs are the family of the Cys-loop ligand-gated ion channel
(LGIC), including glycine receptors (GlyRs). These Cys-loop receptors can be triggered by
different neurotransmitters like glycine and glutamate while controlling exiting or inhibiting
synaptic transmission of the CNS (Lynagh & Lynch, 2012). Such receptors are all organized as
heteropentamers of subunits with four TMs, an intracellular loop between TM3 and TM4, a large
16
ECD, and a short C terminal tail. Although its binding site is not identified, the IVM stimulates
the GABAAR. Understanding the molecular associations between IVM and its GABAAR binding
sites will identify a potential therapeutic drug that can help achieve effective neurological care.
There are still many concerns about the fundamental foundation for modulation (not activation) of
such GABAAR by IVM. The structural blueprint given by the GABAAR crystal structure will help
in the attempts to address these questions.

Figure 4: P2X receptors' molecular properties. A figure reveals that a subunit of the mammalian
P2X receptor comprises two transmembrane domains, including TM1 and TM2 bound by an
extracellular domain and N- and C-termini intracellular domains. (Khakh et al., 2001)

Contrary, the newly identified family of receptors includes ATP and proton-gated-channels.
While these are not sequentially related, the two-channel families consist of the trimeric or
heteromeric assembly of subunits with two TMs, a large ectodomain, and intracellular COOH- and
NH2-termini (Khakh et al. 2001) (Figure 4). Previous tests indicated that IVM affects the role of
P2X4R when added extracellularly but not intracellularly (Priel & Silberg, 2004), meaning it can
bind to the ECD. However, in contrast to the ATP-binding properties (He et al. 2003), the
transmission of P2X4R ECD sequences to the P2X2R backbone did not transfer the sensitivity for
17
IVM (Jelínkova et al. 2006). These findings have pushed us to look at the possible importance of
TM helices to IVM binding. It has been shown that the transition of P2X4R TM regions is adequate
to express IVM's susceptibility, indicating that IVM directly interacts with the two TM helices at
the protein-lipid interface (Silberberg et al. 2007). The areas of helices associated with IVM
interaction must be reasonably large, as demonstrated by comparing the appropriate location of
the IVM molecule and P2X4R region TM1 in (Figure 5). There are no specific selective agonists
for P2X4Rs to date, but Jelnkova's early experiments showed that IVM directly improves P2X4
receptor-channel function and acts as P2X4Rs PAM (Jelnkova et al. 2008).

Figure 5: Forms of the P2X4R TM1 helix (left figure) and Ivermectin (right figure). Graphic
analysis of the sizes of the P2X4R TM1 and IVM molecule segments modeled as the standard
alpha-helix. (Jelínkova et al., 2008)
The molecular dynamics simulation method was used to visualize residues that were found
to be relevant for IVM activity and ethanol (Trp46 and Met336) and to validate potential
associations between these and other residues (Franklin et al., 2014). Gouaux’s group introduced
the homology model of rat P2X4 using the resolution of zebrafish P2XR (zfP2X4) in a closed state
at a resolution of 3.1A (Kawate et al., 2009). As shown in figure 5, this model showed a pocket
18
made up of Trp50, Trp46, Met336, and Asp331, which was proposed to play an essential role in
ethanol and IVM activities. As shown in (Figure 6), the Trp50 and side chains of the first α-helix
face Asp331 and Met336 and Asp331 in the final α-helix of the adjacent subunit. Manual rotations
of the C-alpha formed a location for the C-beta bonds of Trp46 and Trp50 to C-beta bonds of the
two different Trp side chains and the Met336 sulfur interaction. This first P2X4s model showed
that these amino acid residues considered essential for ethanol's actions on P2X4 receptors were
localized around a small pocket in the three-dimensional structure, although relatively broadly
distributed in the primary P2X4R sequence (Franklin et al. 2014).

Figure 6: (A) Full-size and zoomed P2X4R rat models, (B) Graphic analysis of the IVM binding
site. (Franklin et al., 2014)

19
Specific Aim
My thesis's main objective was to understand the role of IVM on P2X4 receptors in
behavior change in PD models. I tried to figure out the role of IVM on DA neurotransmission by
investigating the influence of IVM on DA-dependent behavior change, rotation rate in the
unilateral striatal dopamine depletion (6-OHDA) lesioned rodent model. It results in a high content
of dopamine released in intact striatum of mice occurring on the same side of the lesion, initially
leading us to hypothesize that IVM will improve rotations.
I evaluated the hypothesis that P2X4s represent a new candidate for DA regulation in
controlling DA neurotransmission over the brain's dopaminergic pathway (Khoja et al., 2018;
Franklin, Asatryan, Jakowec, et al., 2014). These results indicate that purinergic neurotransmission,
and DA neurotransmission, including consuming alcohol, are strongly linked (Khoja et al., 2016;
Khoja et al., 2018). However, accurate descriptions of the fundamental processes, though, remain
elusive.
In the case of P2X4s, based on our laboratory, the manipulation of P2X4s by IVM will
change the PD model's rotational activity when present in combination with L-DOPA. Improved
cycling with L-DOPA and IVM is an example of increased dopaminergic activity. For our
secondary aims, based on my laboratory's evidence, I hypothesized that P2X4s are the primary
target agent for dopamine modulation activity.
The specific aim 1 & aim 2 of these steps were:
Aim 1
[1-1] Performing a minimally invasive method of surgical procedure (stereotaxic surgery) that
uses a three-dimensional coordination device to find and perform activities include lesion and
injection on small targets within the C57BL/6J mice (female/male)
20
[1-2] Assessing the degree of 6-OHDA lesion after undertaking the behavioral research by testing
immunohistochemical staining for tyrosine hydroxylase (TH), which reflects a percentage
decrease in immunostaining by analyzing contralateral and dorsal striatum ipsilateral lesions
[1-3] Evaluating the influence of macrocyclic lactones, ivermectin on DA-dependent behavior,
primarily by examining the rotational frequency (AMPH rotation vs. L-DOPA rotation) after
dosage in the DA-depletion unilateral 6-OHDA lesioned mouse model
[1-4] Clarifying the basis of differences between male vs. female in the 6-OHDA mice in terms of
each mouse's pharmacological differences in the model and if these variations are consideration
factors to analyze in the novel's clinical benefits P2X4R-targeted adjunct therapy or not.
Aim 2
[2-1] Administering an MPTP injection in a sequence of four i.p (intraperitoneal) injections at 2-
hour intersections with a cumulative administration of 80 mg/kg that produces a reproducible
lesion of the dopamine pathway after its systemic administration
[2-2] Conducting a rotarod performance test based on a spinning rod with forced motor movement
to evaluate parameters, including riding time of endurance for mice
[2-3] Performing a Novel Object Recognition (NOR), useful for studying cognitive impairments
in mice's transgenic strains in the MPTP model

21
Materials and Methods
Aim #1
Animals
C57BL/6 J male (6-OHDA & MPTP) and female mice (6-OHDA & MPTP) (Jackson
Laboratories; Bar Harbor, Maine, USA) were grouped in 5 to a cage with 12-hour light and 12-
hour dark period cycle with ad libitum food and water. The mice used in this experiment were
supervised by the Institutional Animal Care and Use Committee (IACUC) of the University of
Southern California. Training and qualification review of mice was conducted during the
protocol's administrative pre-examination to ensure they are eligible to perform specific
procedures described in the protocol and are aware of the risks involved with the analysis.
Stereotaxic surgery
C57BL/6 J mice (Jackson Laboratories; Bar Harbor, Maine, USA) were treated with
desipramine hydrochloride (25 mg/kg in 0.9 percent saline, i.p) to prevent damage to the
noradrenergic pathways (Sigma-Aldrich) 30 min before surgery. (Khoja et al., 2016). After halving
the isoflurane concentration to 2% until the mice were anesthetized entirely and so that steady
anesthesia could be developed without the respiratory system's risky side-effects.
6-OHDA lesioning in mice
While using a 10 μL Hamilton syringe and a microliter syringe pump, 2 μL of prepared 6-
OHDA (4 mg / mL in 0.2 % ascorbic acid and 0.9 % saline; Sigma-Aldrich) was unilaterally micro
infused through the left medial forebrain bundle (MFB) with a rate (0.5 μL/min) of infusion. To
ensure complete absorption while following 6-OHDA delivery, a thin tube inserted into mice
allowed it to remain in place for 5 minutes, and the cannula was then increased at a rate of 0.1 mm
per 15 s.
22
IVM behavioral testing: Rotation Behavior
6-OHDA lesioned mice were conducted by enabling them to remain for four weeks, and
D-amphetamine was used to evaluate the degree of lesioning of 6-OHDA mice. Each mouse was
tested to an established baseline rotation for 10 minutes before each compound was tested. AMPH
was then dosed, recording the number of ipsilateral rotations beginning following infusion and
over 120 minutes. Mice that showed more than 180 rotations for 2 hours were regarded as lesioned
and recorded in the analysis, while the mice falling onto the threshold were excluded in the report.
Statistical information of rotation has been reported to figure out the degree of 6-OHDA lesioning
and screen the net rotations for every 10-min cycle and overall net rotations to test mice's activity
collected after the 120 min.
Rotation behavior was tested with L-DOPA, IVM, and moxidectin (MOX) after following
a week washout period. The total rotations of ‘contralateral’ and ‘ipsilateral’ were measured every
10 minutes over 90 minutes. Besides, 5 mg/kg of L-DOPA with 1.25 mg/kg benserazide in 0.9%
saline was administered via S.C. injection, and 5 mg/kg of L-DOPA+IVM in 0.9% saline via I.P.
injection, and 5 mg/kg of L-DOPA+MOX in 0.9% saline, I.P., was given to evaluate rotational
behavior. Considering that IVM was shown to reach maximum concentration in the blood plasma
after 8 h (Yardley et al. 2012), IVM was given 8 h after the first L-DOPA injections to determine
the rotational behavior which was recorded by videotaped. 6-OHDA lesioned mice were observed
during behavioral testing, and the level of 6-OHDA lesioned was identified at the end of the trial.
Immunohistochemistry staining for tyrosine hydroxylase
Mice were narcotized with 25 mg/kg of avertin via I.P. and 4 % phosphate-buffered saline
(PBS), a non-toxic solution supplemented by 4 % paraformaldehyde (PFA) in PBS was used for
tissue fixation. Tissues were isolated from the brain and washed overnight at 4 % PFA in PBS,
23
supplemented by 20 % sucrose solution in PB, and were rapidly frozen on dry ice in methyl butane.
In the coronal plane, a freezing microtome of Leica CM1900 was used for cutting the striatal slice
of brain tissues at a thickness of 25 μm, and cut portions were soaked in a PBS solution. After that,
sliced tissues were immersed into TBS (TRIS-buffered solution) and blocked in 4 % normal goat
serum with TBS with 0.2% Triton® X-100. Subsequently, tissues were stained with TH primary
antibody at dilution of 1:5000 (cat. no. MAB318, Millipore) in 2% normal goat serum and TBS
with 0.2 % Triton® X-100 in 48 h at 3 to 5 °C.
After 48hrs, slices were washed in TBS for 3 times for 5 minutes. Slices were then
incubated in a solution containing IRDye 800CW secondary antibody (Dilution range: 1:20000),
2 % natural goat serum, and TBS with 0.05% Triton® X-100 for 1 h at room temperature. Portions
were washed with TBS with 0.5% Triton® X-100, then TBS and images collected using an
Odyssey
®
Fc Imaging System. The degree of immunostaining for each portion of the dorsal
striatum was investigated utilizing the Image J1 (Image processing program), which is a Java-
based image processing program, and it was used to figure out the level of 6-OHDA lesioning. Its
degree was evaluated as a percent decline in immunostaining by analyzing the difference in
fluorescence of the ipsilateral and contralateral dorsal striatum.
Statistical Analysis
Statistical analysis was conducted using R, a programming language and software
environment for statistical computation and graphics supported by Statistical Computation's R
base (Morandat et al. 2012). An inter-subject design was implemented to test significant
differences between the treated 6-OHDA animals versus untreated 6-OHDA animals following the
6-OHDA injected versus saline-injected animals. A two-way ANOVA with replication was
conducted to assess the effect of P2X4R modulation on behavioral outcomes. Treatment with 6-
24
OHDA lesioned vs. 6-OHDA non-lesioned) by to different sexes and treatment (6-OHDA
lesioned vs. 6-OHDA non-lesioned) by period (every 10-min cycle over 120 min) was carried out
for AMPH study. In addition, treatment (6-OHDA lesioned vs. 6-OHDA non-lesioned) by sex
difference and treatment (6-OHDA lesioned vs. 6-OHDA non-lesioned) by duration (every 10-
min cycle over 90 min) were also assessed for L-DOPA evaluation.
Aim #2
Animals
C57BL/6 J male (6-OHDA & MPTP) and female mice (6-OHDA & MPTP) were grouped
of 5 to a cage with 12-hour light and 12-hour dark period cycle with ad libitum food and water.
The mice used in this experiment were supervised by the Institutional Animal Care and Use
Committee (IACUC) of the University of Southern California. Training and qualification review
of mice was conducted during the protocol's administrative pre-examination to ensure they are
eligible to perform specific procedures described in the protocol and are aware of the risks involved
with the analysis.
MPTP injection
The overall amount of MPTP solution, which can be derived from adding all mice's weights
to be injected with MPTP, was measured. The mice were categorized into treatment groups (saline
or MPTP). To prevent overloading fluid animals (which could lead to fatal heart failure), 10 mL
of MPTP injection per 1g body weight was administered (i.e., about 0.25 ml for a 25 g mouse).
The cumulative amount was then determined by applying multiple injections in the schemes to
MPTP-treated mice's total weight. Four i.p injections (the best way of systemic administration of
MPTP mice) in duration of 2-hour for a total of 80 mg/kg (20 mg/kg per injection) were injected
into the MPTP mice.
25
Behavior testing
Compounds including L-DOPA, serazide, ivermectin, and moxidectin were prepared to
investigate the testing of behavior. Behavior activity was conducted after at least a week after
MPTP injection due to the more precise estimates of DAergic neuronal loss in all MPTP mouse
models (Meredith et al. 2008). Each compound was diluted to perform the behavior test: (Using
1% IVM stock, create a working solution using dilution factor of 1:20 (IVM: 0.9% sterile saline,
ex. 50 uL IVM + 950 uL saline), (5 mg/kg of L-DOPA with 1.25 mg/kg serazide in 0.9% saline),
and (L-DOPA with IVM, and saline (0.9%)). A rotational station was prepared, and every behavior
testing was monitored by visualizing and our group members counted the rotations.
Rotarod Performance Test
The rotarod performance test was performed by following the protocol that was previously
handled in our lab. Mice were trained for three days during the experiment by setting different
seconds at a different speed. On day 1, mice were taken into the behavior room and allowed to
acclimate for 30 minutes, then returned to their usual housing cage in the morning. The mice were
acclimatized during the afternoon, and mice were familiarized with the rotarod for 90 seconds at
a speed of 5 rpm, and if the mice fell before the end of the 90 seconds, they were placed back on
the rotarod. On day 2, mice were handled in the same way as day 1, but mice were introduced to
the rotarod for 120 seconds at a speed of 10 rpm, 15 rpm, respectively, in the morning and
afternoon. On the last day, mice were treated in the same way as day 2, but mice were introduced
to the rotarod for 200 seconds. The machine was designed to switch the direction to forward and
reverse every 25 to 30 seconds, and the speed was set to increase and decrease from 0 to 30 rpm
in this time range, and this procedure was repeated 3 to 5 times with 90 seconds rest between each
trial.
26
NOR (Novel Object Recognition)
To assess alterations in learning and memory for mice, NOR research was used. On the
first day, mice were put for 5 minutes in a white box with bedding. After day one, with two types
of the same item, including a big blue Lego and a glass beaker, mice were put back into the box
and allow to roam around the box for 10 minutes while each time a mouse interacts with an object,
the time was recorded using a stopwatch to monitor the recorded video. If the mice did not hang
around the object more than 20 seconds, it was excluded from the NOR analysis. The mice were
put in a box 24 hours later, with a beaker and a Lego. The sides of the novel objects were switched
up and randomized, and mice were investigated by allowing them to explore each object and the
discrimination index percentage was recorded.
Statistical analysis
Statistical analysis was tested using a statistical computing and graphics programming
language and software environment assisted by Statistical Computation's R base (Morandat et al.,
2012). All those other behavioral statistical analyses were performed using a two-way ANOVA
with replication to assess the effects of P2X4 modulation and sex variations. An inter-subject
approach was used to determine the significant variations between the treated 6-OHDA / MPTP
animals vs. untreated 6-OHDA / MPTP models and 6-OHDA / MPTP injection vs. saline-injected
animals.

27
Results
Aim #1
6-OHDA lesioning quantified by tyrosine-hydroxylase staining
In order to evaluate the predictive quantity of TH positive cell loss in SNPC concerning
doses of 6-OHDA, optical TH density normalized was measured by using an injection of 6-OHDA
into the MFB with saline-injected mice. I also defined situational parameters based on this
knowledge that can be used to estimate the degree of lesioning in the 6-OHDA model.
Table 1. Average AMPH Rotations and Percent lesioned for male (n=8) vs. female (n=12). The
overall number of net ipsilateral rotations was conducted for 120 min and the SEM-image of dorsal
striatal tyrosine hydroxylase immunoreactivity (TH-ir) depletion with the lesion.

Number Treated Percent Lesioned
(%)
AMPH Net Rotation (120 min)
Male 8 100.42±1.24 281.38±62.27
Female 12 94.94±2.73 337.67±62.27

For TH staining, saline-injected mice were not analyzed as they exhibited no rotational
activity. However, compared with the non-lesioned hemisphere, TH's optical density in the
lesioned hemisphere reduced to 98.5 percent (Figure 7). However, in view of analyzing the level
of dorsal striatal TH-ipsilateral deficiency, there was no noticeable difference between 8 male mice
(percent lesioned: 100.4±1.2) and 12 female mice (percent lesioned: 94.9±2.7), which were carried
out in 6-OHDA lesioning quantified by TH staining (Table 1).
28

Figure 7: IHC TH stain Lesion Percentage= 98.5%: median forebrain bundle (MFB) 6-OHDA
lesioned by tyrosine hydroxylase (TH) stains and saline-treated mice A) Saline treated, non-
lesioned, male mouse B) Male mouse with 6-OHDA MFB lesions.
AMPH-induced rotation
The evaluation of the amphetamine-induced rotation test showed an improvement in
ipsilateral net turns as cell loss increased. In this analysis, ipsilateral rotational activity induced by
AMPH was investigated in 8 male and 12 female mice over the 120-min period. 5.0 mg/kg AMPH
rotation for both mice was performed to assess the level of motor dysfunction caused by the lesion
in this study. As illustrated in Figures 8 and 9, throughout the overall rotations in the range to 20-
min period, female mice displayed a higher peak height than male. In view of analyzing the number
of total rotations during the 10 to 20-min cycle period, female mice (52.25±6.82 net rotations)
were higher than male mice (20.25±5.95 net rotations) (Figure 8) (Warnecke et al. 2020). On the
other hand, as shown in Figure 9, no significant relevant differences in the total amount of net
rotations between two different sex mice (male=281.38±62.27 and female=338.08±131.58)
performed over the entire 120-min duration. Also, lesioned mice had more significant net AMPH
rotations compared to non-lesioned mice.
29

Figure 8: The number of rotations was measured every 10-minute cycle over the 120 minutes after
an administration of amphetamine (5.0 mg/kg) by comparing females with male 6-OHDA lesioned
mice obtaining six daily rotations. A graphic showing the number of rotations done every 10
minutes for 120 minutes during AMPH (5.0 mg/kg) treatment with the male (n = 8) vs. female (n
= 12), two-way ANOVA, p < 0.01, Tukey p < 0.01. Males are less active than females when testing
mice in the 6-OHDA MFB model and undergo varying amounts of exercise (*p<0.05), with female
mice performing more rotations throughout 10-20 min bin.

Figure 9: The difference net AMPH (5.0 mg/kg) rotation between male and female mice with
lesioned and non-lesioned: Diagram showing the difference in net ipsilateral rotations per 10 min
bin per 120 min after AMPH (5.0 mg/kg) treatment in 6-OHDA of 12 female mice and 8 male
mice and non-lesioned treated with saline in 10 female mice and 10 male mice.
30

L-DOPA-induced rotation
In this analysis, L-DOPA-induced rotational behavior in 8 male and 12 female mice was
monitored throughout the 90 minutes. Consequently, the L-DOPA-induced rotational number
returned to the baseline at the end. In this study, 5.0 mg/kg of L-DOPA-induced contralateral
rotation between male and female mice was performed to analyze the number of total rotations
during the 10 to 20-min cycle period. As seen in Figure 10, male mice displayed a higher peak
height than female. However, the gap differences of both (male/female) mice in rotations per every
10-minute were decreased after time went. In contrast to AMPH-induced rotation, the result of
relevant differences in the total number of L-DOPA-induced rotations between male mice
(309.75±62.15 net rotations) and female mice (132.58±40.19 net rotations) varied greatly
throughout the 90-min period (Figure 11).

Figure 10: The number of rotations was measured every 10-minute cycle over the 90 minutes after
an administration of L-DOPA (5.0 mg/kg) by comparing females with male 6-OHDA lesioned
mice obtaining six daily rotations: Diagram showing the number of rotations per every 10 min
cycle over the 90 min after treated with L-DOPA (5.0 mg/kg) in 8 males vs. 12 females, (p < 0.05).
31
6-OHDA lesioned in male mice has a higher number of rotations over the 90 minutes than female
mice, indicating a sex differences in the 6-OHDA MFB model.

Figure 11: The difference net L-DOPA (5.0 mg/kg) rotation between mice with lesioned and non-
lesioned in female and male: Figure shows the difference in net contralateral rotations per every
10 min bin over the 90 min after L-DOPA (5.0 mg/kg) treatment in 6-OHDA lesioned mice (female
mice n = 12, male mice n = 8) and injected with saline in 8 female mice and 10 male mice (two-
way ANOVA, p < 0.05, Tukey p < 0.01).

IVM Behavioral testing rotational results
An influence of IVM on L-DOPA-induced rotations was tested in 6-OHDA mice. Using a
mouse line without P2X4R expression (P2X4RKO) did not exhibit the same increased rotation
numbers. P2X4s co-localization in the SNPC with dopaminergic neurons associated with proof
that P2X4RKO mice may not display the same rotational activity as wild-type C57 / BL6, when
provided L-DOPA and IVM, indicate that P2X4R mediates the influence of IVM on DA. In areas
of DA depletion, L-DOPA's impact on rotational activity arises from enhanced dopaminergic
release. Increased rotation found in the presence of L-DOPA with IVM is an indication that
dopaminergic activity has increased. It is hypothesized that positive modulation of P2X4 with IVM
causes increased rotational behavior in a PD mouse model. In support of the hypothesis, the impact
32
of IVM's treatment effect on L-DOPA-induced rotations was studied in male and female 6-OHDA
mice.
The number of net rotations in L-DOPA treated alone groups versus L-DOPA with IVM
treated groups in 8 male mice was performed to determine the IVM's impact on behavior testing
rotational results. Treatment effect (F (2,210) = 14,450, p<0.01) with the Tukey Test was
performed to evaluate a different behavior change between L-DOPA and L-DOPA combination
group with IVM by comparing rotations per every 10-minute and total rotations along with using
a two-way ANOVA for treatment vs. time (Warnecke et al. 2020) (Figure 12). The Tukey test
showed a significant difference in L-DOPA rotations with IVM (498.1±54.54) vs. L-DOPA treated
alone (309.8±62.15), suggesting an increase of rotational behavior when using IVM with L-DOPA
treated groups in the male study.

Figure 12: The diagram shows the rotations per every 10 min cycle during 90 min after ad-
mistering with 5 mg/kg of L-DOPA alone, (5 mg/kg of L-DOPA with 5 mg/kg of IVM), and 5
mg/kg of L-DOPA with 5 mg/kg of moxidectin in 8 male 6-OHDA lesioned mice with SEM
representing bars. Treatment (p<0.01) with a Tukey Test evaluating a substantial different
behavior change (rotation number) between treated with L-DOPA vs. L-DOPA + IVM and L-
DOPA + IVM vs. L-DOPA + MOX (p<0.01) were measured by comparing rotations per every 10
min along with using a two-way ANOVA for treatment group versus time (p<0.01).
33

On the other hand, the number of net rotations in L-DOPA treated alone groups versus L-
DOPA with IVM treated groups in 12 female mice was also performed to evaluate the IVM's
impact on behavior testing rotational results. Treatment effect (F (2,210) = 14,450, p<0.01) with a
Tukey's Honest Significant Difference test was performed to evaluate a different behavior change
between L-DOPA and L-DOPA combination group with IVM by comparing rotations per every
10-minute and total rotations along with using a two-way ANOVA for treatment vs. time
(Warnecke et al. 2020) (Figure 13). The Tukey test showed a significant difference in L-DOPA
rotations treated with IVM (973.17±166.79) vs. L-DOPA treated alone (132.58±40.19), suggesting
an increase of rotational behavior when using IVM with L-DOPA treated groups in the female
study. Based on the findings, IVM impacted the behavior changes in male and female mice, but
with having a different degree of influence on the variable number of total rotations.

Figure 13: The diagram shows the rotations per every 10 min cycle during 90 min after ad-
mistering with 5 mg/kg of L-DOPA alone, (5 mg/kg of L-DOPA with 5 mg/kg of IVM), and 5
mg/kg of L-DOPA with 5 mg/kg of moxidectin in 12 female 6-OHDA lesioned mice with SEM
representing bars. Treatment (p<0.01) with a Tukey Test evaluating a substantial different
behavior change (rotation number) between treated with L-DOPA vs. L-DOPA + IVM and L-
DOPA + IVM vs. L-DOPA + MOX (p<0.01) were measured by comparing rotations per every 10
min along with using a two-way ANOVA for treatment group versus time (p<0.01).
34
Aim #2
Rotarod test
6-OHDA mice
On average, the pattern of latency to fall(s) for 6-OHDA male mice with L-DOPA treated
showed a value of (68.350 ± 15.610), but the value of latency to fall(s) was relatively short
compared to the L-DOPA+IVM treated group (Figure 14). On the other hand, mice in saline-
treated produced results that did not fall off the rotarod in less than 90 seconds. The pattern of all
treatments in 6-OHDA males was relatively irregular compared to the trend of female mice (Figure
14).

Figure 14. Performances on the Rotarod in five trials of 6-OHDA lesioned male mice treated
with saline, non-lesioned, L-DOPA+IVM, and L-DOPA.

In 6-OHDA female mice, the latency to fall(s) from the rotarod between (saline-treated
mice) and (treated mice) was substantially different. On average, the saline-treated mice did not
have irregular latency to fall(s) cycles during five trials, and they were remaining on the rotarod
for less than 50 seconds. The latency to fall(s) of control mice was shorter than the latency to falls
of non-lesioned females (182.267 ± 13.385), and L-DOPA + IVM treated mice (110.320 ± 15.038)
(Figure 15).
35

Figure 15. Rotarod performance by behavior test across the 5 trials of female 6-OHDA lesioned
treated with saline, L-DOPA+IVM, L-DOPA, and non-lesioned
MPTP-mice

In MPTP male mice, the latency pattern to fall(s) in a mouse with IVM treated could not
predict the result as with latency to fall(s) increases and decreases as the trial number increases.
Besides, compared to 6-OHDA male mice, MPTP male mice had a very irregular latency pattern
to fall(s) in mice with all treatment (Figure 16). On the other hand, the latency pattern of mice
treated with IVM in MPTP female mice increased with an increasing number of trials since the
second attempt. The latency pattern to fall(s) of mice treated with L-DOPA+IVM in an MPTP
female mouse increased the number of trials until the third attempt, but the pattern of latency to
fall(s) was irregular from subsequent attempts. Compared to 6-OHDA female mice, MPTP female
mice showed very distinctive latency patterns in all treated mice (Figure 17).
36

Figure 16. Performances on the Rotarod in 5 trials of male MPTP lesioned mice treated with saline,
L-DOPA+IVM, IVM, L-DOPA, and non-lesioned.

Figure 17. Rotarod performance by behavior test across the 5 trials of female MPTP lesioned
treated with saline, L-DOPA+IVM, L-DOPA, and non-lesioned.
Novel Object Recognition
6 OHDA-mice
The gap difference between total time with the object(s) treated with L-DOPA was higher
than in other treatment groups (non-lesioned, saline, and L DOPA+IVM) in 6-OHDA mice (Figure
37
18). Besides, all 6-OHDA female mice stepped more around on the novel object than males except
in the 6-OHDA mice treated with IVM+L-DOPA (p<0.06). The proportion of time spent with the
object was also assessed to determine the 6-OHDA treatment groups' performance by dividing the
time with a novel object by the total time with objects. In 6-OHDA mice with L-DOPA treatment
groups, there was no evidence related to the treatment group's effect on the performance. Also, no
significant difference between females' and males' percentage of time spent with the object was
found in all treatment groups (saline, non-lesioned, and L-DOPA+IVM) (p>0.05) (Figure 18).

Figure 18. Total time with an object on Novel Object Recognition (NOR) testing by 6-OHDA
males and females treated with the treatment group: ((n=5): saline males and females; (n=5): L-
DOPA+IVM females; (n=3): L-DOPA and non-lesioned females and males; (n=3): L-
DOPA+IVM males). The percentage of time spent with the object by 6-OHDA lesioned male and
female mice treated with the treatment group ((n=5): saline males and females; (n=5): L-DOPA +
IVM females; (n=3): L-DOPA and non-lesioned females and males; (n=3): L-DOPA+IVM males).

MPTP-mice
There was no gap between total time with the object(s) in MPTP mice treated with saline
between males and females. In contrast to 6-OHDA mice, all MPTP male mice stepped around
more times with the object than females (Figure 19). The percentage of time spent with the object
38
was also measured to determine the MPTP treatment groups' performance by dividing the time
with a novel object by the total time with objects. In MPTP mice treated with saline, non-lesioned,
L-DOPA+IVM, IVM, and L-DOPA, there was no clue about the effectiveness of their
performance after being treated with treatment groups. A two-way ANOVA for the sex/treatment
group showed no significant difference between females' and males' percentage of time spent with
the object was found in all treatment groups (p>0.05) (Figure 19, Right). However, A two-way
ANOVA for females’ and males’ effect on total time with objects, females spent less time with
objects than males in all treatment group, including saline, non-lesioned, and L-DOPA+IVM, L-
DOPA, and IVM) (p>0.05) (Figure 19, Left).

Figure 19. Total time with an object on Novel Object Recognition (NOR) testing by MPTP males
and females treated with the treatment group: ((n=6): saline females and males; (n=5): L-DOPA,
IVM, and L- DOPA+IVM for male mice; (n=4): non lesioned males, L-DOPA, IVM, L-
DOPA+IVM and non-lesioned for female mice. The percentage of time stepped around on the
object by 6-OHDA lesioned male and female mice treated with the treatment group ((n=6): saline
females and males; (n=5) L-DOPA, IVM, and L-DOPA+IVM for male mice; (n=4) L-DOPA,
IVM, L- DOPA+IVM and non-lesioned for female mice, and non-lesioned males).

39
Discussion
In recent years, as reviewed by a previous study (Burnstock, G. 2017), the purinergic
signaling mechanism has been introduced as an essential research topic for identifying and
managing many pathologies. Purinergic receptors regulate DA neurotransmission as a critical
modulator of various signaling pathways correlated with neurodegeneration (Ribeiro et al., 2016
& Burnstock, 2016). Since the sex-dependent differences in DA signaling within a group of the
subcortical nuclei responsible for motor control have been established as a possible parameter in
testing the experimental therapeutics approach to treatment for PD, I used both male and female
mice to investigate P2X4R positive allosteric modulation pharmacology. Since the information on
P2X4 receptor KO mice for antagonists has not been specified to date, I analyzed the influence of
P2X4, determining the particular contributions of a goal to behavioral effects caused by IVM.
Besides, the 6-OHDA and MPTP models were assessed to investigate the IVM’s function to
modulate DA-mediated behaviors.
The purpose of this study is to test the function of ivermectin on P2X4Rs in regulating
behavior responses in the PD model. This study was based on the earlier reviews that support the
research's focus that IVM might enhance contralateral rotation numbers in male C57BL/6J mice
with 6-OHDA lesioned mice in the MFB (Khoja et al., 2016). In this experiment, I assessed the
response with L-DOPA treatment to identify the therapeutic effects of utilizing a supportive
P2X4R modulator as a combination treatment for PD (Asatryan et al. 2010). Furthermore, the
unilateral 6-OHDA model was used to examine ivermectin’s ability to modulate DA-mediated
behaviors. IVM enhanced ATP-mediated calcium currents by around 20% from P2X4Rs at lower
dosages (0.5 M), according to early work by Khoja. At higher dosages of 1.0 M, other macrocyclic
lactones (MLs), like MOX, did not increase ATP-mediated calcium currents (Khoja et al.,
40
2016). It seems to show that the influence of IVM on behavior changes can result in the binding
to the higher affinity site from P2X4Rs than other MLs.
The P2X4R positive modulation pharmacology was studied for female and male mice in
this experiment to analyze more biological sex differences. Many PD characteristics have to do
with the distinctions between males and females. However, few studies have looked into the
rotational behavior in the 6-OHDA MFB model that PD or other hormone-related diseases can
cause and how they are affected by biological sex, precisely and during the disease's earlier stages
(Betancourt, Wachtel, Mchaelos, et al., 2017). The significant difference in dopamine depletion
was performed to support the further study by using the 6-OHDA model. 6-OHDA lesions
performed in this research indicated that the reduction in tyrosine hydroxylase was not
significantly influenced by sex. However, it showed that changed sex-based motor behavior may
be related to neurochemical changes between various sexes in the dopaminergic system (Tamas,
et al., 2005). Given the MFB 6-OHDA model, this experiment was not enough to explain the result
of rotational activity in female versus male mice. Besides, variations in the target of the 6-OHDA
lesion, the different dosages of neurotoxin administered, and the usage of varying rat species used
in other experiments may explain the lack of a substantial difference in tyrosine hydroxylase (TH)-
immunoreactive (ir) observed in this research after dopamine depletion.
Rotational reaction with AMPH administration is commonly used as a behavioral indicator
to test dopamine neuron loss in the unilateral 6-OHDA lesion model. (Ungerstedt U., 1968;
Munhall et al., 2007). A 6 turns/min rate of rotation following AMPH administration was shown
to correspond with a DA deficiency level adequate to cause rare Parkinsonian disorder symptoms
characterized by unilateral body atrophy in 6-OHDA-lesioned rats (Putterman et al., 2007; Winkler
et al., 2002). Following the previous study, L-DOPA-mediated rotational activity was substantially
41
improved by IVM. I found that IVM treatment had a potential effect on the rotational action of L-
DOPA. Likewise, the induced rotation activity of AMPH and L-DOPA appeared in female and
male mice at different degrees. IVM treated along with L-DOPA changed the more significant
DA-associated activity in female than in male mice, based on rotation numbers.
IVM is more significantly toxic than other MLs, it has also been well known that individual
invertebrate animals, such as dung beetles (Lumaret & Errouissi, 2002) and Anopheles mosquitoes
(Butters et al., 2012) are identified to be susceptible to IVM. While this knowledge is essential for
optimizing the use of MLs in humans and the treatment of animals, the mechanisms for these
variations remain unclear. However, IVM may act by many pathways, including increasing the
secretion of DA and reducing the half-lives. There might also be variations in calcium uptake level,
where males and females have different abilities (Cerri et al., 2019). Since IVM is known as a
positive allosteric modulator (PAM) of the P2X4Rs, capable of fluxing calcium at levels close to
glutamate and ion channel protein receptors, allowing calcium to travel across the plasma
membrane in females may result in a more significant function in buffering the intracellular
calcium excess that occurs under pathological conditions (Costa et al., 2020). This rise could be a
possible process by which IVM in female and male mice can improve rotational activity rate.
However, there would be sex differences in rotational behavior due to the calcium flux level in
both mice with dopamine (DA)-depleting brain lesions of various sizes. Further investigations are
required to be performed to monitor the DA release of different sex hormones and DA receptor
dynamics to establish post-synaptic or presynaptic variations. In general, the gender-dependent
variations observed in the current research point out the need for trials of both male and female
mice. Based on the limitation, our research could support the possibility of recognizing gender
differences in terms of the treatment of PD patients.
42
Furthermore, the influence of IVM on the level of net rotation was decreased in P2X4RKO
mice (Khoja et al., 2016). It indicates that the impact of IVM on rotation behavior is possibly due
to the association of IVM with P2X4Rs. These numerous pharmacological characteristics can
explain the function of IVM on P2X4s in our experiments. However, there were no differences in
the performance of the rotarod in mice treated with MPTP lesions. The result may be attributed to
a lack of test accuracy, with MPTP-lesioned mice requiring a more demanding motor test to enable
motor deficits to be identified. Previous studies have also shown that despite DA depletion, MPTP
treatment regimens, including subacute and acute MPTP lesions, were not sensitive to motor
deficits. For example, in mice that did exercise after four weeks of MPTP treatment, even if the
53 % loss of tyrosine hydroxylase (TH) inside substantia nigra (SN) and 73% loss of TH inside
the dorsal lateral, it had the same behavioral improvement compared to the mice that did not
exercise after four weeks of MPTP treatment with the same loss of TH. (Sconce et al., 2015).
Following the study, our findings reveal that MFB 6-OHDA lesions were useful for analyzed
rotarod analysis, but they were not reliable to MPTP lesions. Besides, both models were not
susceptible to test performance on the NOR test. Identifying lesion models that affect the cerebral
cortex covering the frontal lobe or other regions involved in learning is required in a future study.

Conclusion
The possible application of IVM in conjunction with L-DOPA for PD patients was
introduced as a new treatment therapy in this study. By showing the efficacy of IVM on two PD
animal models, including MPTP and 6-OHDA, IVM’s function to modulate DA mediated
behaviors in PD would suggest as a new therapeutic for neurodegenerative disease in the future.
43
The result stated a substantial difference in net contralateral rotation between female and
male mice when the L-DOPA-induced rotation test was conducted. I found that all mice with L-
DOPA-induced rotation could be more beneficial on rotational behavior than an AMPH
administration of 5.0mg/kg. When designing research based on impairment of voluntary
movement in the mice 6-OHDA model, this study had brought consequences that had been taken
into account for the hypothesis. Our results show that the AMPH-induced rotation test indicated a
less ineffective indicator than the L-DOPA-induced rotation test of dyskinesia development in
male mice, at least at the dose of AMPH (5.0mg/kg). On the other hand, the L-DOPA-induced
rotation test showed a less low indicator than the AMPH-induced rotation test of dyskinesia
development in female mice, at least at the dose of L-DOPA (5.0mg/kg).
Future research is expected to establish the processes by which IVM as a new therapy in
PD for the treatment of behavior indications regulates mesolimbic dopamine neurotransmission,
which would be shown to impact its behavioral responses depending on different dose levels for
males and females in several species.

44
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Abstract (if available)

Abstract The primary therapy for symptomatic motor benefits linked to patients diagnosed with Parkinson’s Disease (PD) has been levodopa (L-Dopa). L-DOPA is a precursor of dopamine (DA) and represents the gold-standard pharmacological treatment for Parkinson's disease. Nevertheless, L-DOPA has had numerous drawbacks in long-term treatment, leading to motor problems as the therapy becomes less effective. Presently there is no cure for PD, where the disease has been linked to a dopaminergic disorder that affects the nigrostriatal pathway triggered by dopamine deficiency. Purinergic ionotropic P2X4 receptors (P2X4s) have been introduced as a protein candidate that would regulate dopamine activity. P2X4s are critical dopamine activity modulations that increase the release of calcium into neurons, leading to an increase in secretion of DA. The goal of my thesis project is to illustrate the interaction between ivermectin (IVM) modulation of P2X4s using PD models, 6-OHDA and MPTP to evaluate the effect of P2X4s that are involved in alterations in motor behavior for PD. This will be accomplished by summarizing drug efficacy via the concept of IVM's therapeutic targets with the presence of L-DOPA on P2X4s. Overall, this work should help in the identification of potential therapeutic roles of the purinergic receptor system in the activation of neurotransmission of DA and the sex differences in the response by IVM in C57BL/6J mice.

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Creator Wi, Dongwook (author)

Core Title The role of ivermectin on P2X4 receptors in regulating behavior responses in the Parkinson’s disease model

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2021-08

Publication Date 07/29/2021

Defense Date 07/27/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,blood-brain barrier,Dopamine,ivermectin,Levodopa,novel object recognition,OAI-PMH Harvest,P2X4 receptors,Parkinson's disease,positive allosteric modulator

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Advisor Culty, Martine (committee member), Davies, Daryl (committee member), Okamoto, Curtis (committee member)

Creator Email DWi@mednet.ucla.edu,wid@usc.edu

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