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Evaluating the effects of dihydromyricetin on dopamine mediated behaviors using a Parkinson's disease model

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Evaluating the effects of dihydromyricetin on dopamine mediated behaviors using a Parkinson's disease model

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Content Evaluating the Effects of Dihydromyricetin on Dopamine
Mediated Behaviors Using a Parkinson's Disease Model:

by

Lila Halbers

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
(Clinical and Experimental Therapeutics)

May 2020

Copyright 2020 Lila Halbers

ii

Dedication

I dedicate this thesis to my grandparents, Sandra and Ron Stackler
My mother, Suzanne Myers
and my father, Steven Halbers. May he rest in peace.

iii

Acknowledgements

I would like to thank my advisors, Dr. Daryl Davies, Dr. Michael Jakowec, Dr. Giselle Petzinger,
and Dr. Jing Liang for giving me the opportunity to evolve as a research scientist, develop skills
and learn in their laboratory. I would also like to thank my mentor, PhD candidate Alicia
Warnecke for her patience, teamwork, and expertise.

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Table of Contents:

Dedication…………………………………………………………………………………..….….ii

Acknowledgements…………………………………………………………………………........iii

List of Figures………………………………………………………………………………..........v

List of Abbreviations………………………………………………………………………...…...vi

Abstract…………………………………………………………………………………...…..…viii

Introduction……………………………………………………………………………..…………1

Materials and Methods……………………………………………………………………….……9

Results…………………………………..…………………………………………………..……23

Discussion………………………………………………………………………………………..36

References……………………………………………………………………………………..…38

v
List of Figures:

Figure 1: DHM molecular structure
Figure 2: DA release in rat model
Figure 3: Mice in beaker for rotational behavior
Figure 4: Experimental design study#1
Figure 5: IHC
Figure 6: IHC TH stain (partial lesion)
Figure 7: IHC TH stain (no lesion)
Figure 8: Experimental design study#2
Figure 9: DHM+ L-DOPA induced rotations before and after exercise
Figure 10: DHM+ L-DOPA induced rotations before and after exercise graph (chronic and acute
DHM administration)
Figure 11: DHM+ AMPH induced rotations before and after exercise
Figure 12: DHM versus control rotarod 6-OHDA lesion
Figure 13: DHM versus control rotarod 6-OHDA lesion trial 1 of 3
Figure 14: DHM versus control rotarod 6-OHDA lesion trial 2 of 3
Figure 15: DHM versus control rotarod 6-OHDA lesion trial 3 of 3
Figure 16: DHM versus control rotarod 6-OHDA lesion average of 3 trials
Figure 17: TH-stains of MFB 6-OHDA lesioned mice non lesioned hemisphere
Figure 18: TH-stains of MFB 6-OHDA lesioned mice lesioned hemisphere
Figure 19: DHM, DHM+L-DOPA versus control sucrose preference
Figure 20: DHM, DHM+L-DOPA versus control sucrose preference
Figure 21: Female MPTP lesioned mice rotarod
Figure 22: Male MPTP lesioned mice rotarod
Figure 23: Male MPTP lesioned mice rotarod
Figure 24: Female versus male MPTP lesioned mice rotarod

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List of Abbreviations:

AD Alzheimer’s Disease
ATP Adenosine triphosphate
BBB blood brain barrier
BCA bicinchoninic acid assay
BZ benzodiazepines
CEC cerebral microvascular endothelial cells
CNS central nervous system
DA dopamine
DAT Dopamine transporter
D1R dopamine D1 receptor
D2R dopamine D2 receptor
DHM Dihydromyricetin
EtOH Ethanol
GABAARs gamma-aminobutyric acid receptors
GPi internal globus pallidus
IHC immunohistochemistry
I.P intraperitoneal injection
IVM Ivermectin
MAO-B monoamine oxidase-B
MFB medial fiber bundle

MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

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NOR novel object recognition
NaCl sodium chloride
PD Parkinson’s Disease
PAM positive allosteric modulator
PFA phosphate buffered saline with paraformaldehyde
PBS phosphate buffer saline
PVDF polyvinylidene fluoride
P2X4R P2X purinoreceptor 4
SNPC substantia nigra pars compacta
SNr substantia nigra pars reticulata
SPT sucrose preference test
TH tyrosine hydroxylase
VMAT vesicular monoamine transporter
WT wild type
6-OHDA 6-hydroxydopamine

viii
Abstract:

Parkinson’s Disease (PD) is a neurodegenerative disorder distinguished by dopamine
(DA) depletion resulting in motor and cognitive deficits. The current treatment options, DA
replacement therapy and DA agonists, provide symptomatic relief, but do not alter or improve
the underlying pathology. Dihydromyricetin (DHM), a bioactive flavonoid from Hovenia dulcis,
is a positive allosteric modulator of Gamma-amino butyric acid (GABA) receptors that has been
linked to modulation of dopamine activity in the Central Nervous System (CNS). For example, a
downregulation of GABAergic tone is commonly seen in PD patients and is associated with the
loss of nigrostriatal dopaminergic neurons (Magrinelli et al. 2016). DHM potentially regulates
GABAergic tone alleviating excitotoxicity and dopaminergic cell death suggesting that DHM
represents a possible therapy for PD. My thesis tested this hypothesis by investigating the role of
DHM on DA dependent behavior using PD models. Rotational behavior in a 6-hydroxydopamine
(6-OHDA) model and rotarod behavioral model in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) and 6-OHDA models were used to measure the extent of motor impairment in DHM
treated groups versus control groups. DHM versus control group decreased rotational behavior
in 6-OHDA model and DHM versus control group improved rotarod performance in an MPTP
model, suggesting an improvement in motor deficits caused by a dopamine depletion model in
DHM treated mice.

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1. Introduction:

1.1 Background
Parkinson’s Disease (PD) is the second most common age-related neurodegenerative
disease in humans. It is a disorder that is characterized by progressive depletion of dopamine
due to selective loss of dopaminergic neurons in the substantia nigra pars compacta (Mhyre
2012). The pathological features of PD are the loss of 80% striatal dopamine, 40-60% loss of
dopaminergic neurons in the substantia nigra pars compacta, and the presence of Lewy bodies in
affected neurons (Salawu, Danburam and Olokoba 2010). At the present time, the precise
pathogenic mechanism leading to neurodegeneration in PD is unknown, but it is commonly
accepted that neuroinflammation, oxidative stress and mitochondrial dysfunction can contribute
to dopaminergic damage (Qadri et al. 2016). The clinical features of PD include both motor and
non-motor symptoms that can impact the patient’s function, but these changes are variable
depending on the patient. The most prevalent motor symptoms include; tremor, rigidity,
bradykinesia, and postural imbalance, appearing when approximately 50-60% of the nigrostriatal
dopaminergic neurons are lost . Common nonmotor symptoms include; anxiety, depression,
fatigue, delayed thought process, hallucinations, and dysfunctional sleeping patterns (Forbes et
al. 2019, Salawu et al. 2010). However, in PD patients, dementia may be the most debilitating
symptom associated with the disease progression, it is estimated that up to 14% of patients per
year of the age of 65 or older will develop cognitive impairment (Roheger, Kalbe and Liepelt-
Scarfone 2018, Forbes et al. 2019).
Parkinson’s disease is not linked to a singular genetic event or outcome, but rather can
be manifested as familial, secondary, or sporadic diagnoses. Familial Parkinsonism exist within
families in an autosomal dominant manner due to mutation in the alpha-synuclein gene (SNCA)

2
on chromosome 4q2, secondary Parkinsonism cases are a result of toxins, drugs, anoxia, trauma,
or infection, and sporadic/idiopathic Parkinsonism accounts for 90% of all cases and has
unknown exact etiology (Sinha et al. 2005, Jakowec 2019).
Current treatment strategies for patients diagnosed with PD focuses on DA replacement
strategies including administration of compounds such as Sinemet, a combination of L-dopa (the
precursor to DA) and Carbidopa (a peripheral decarboxylase inhibitor). DA replacement
strategies may provide improvement in early stages of PD, but this treatment response is related
to dopaminergic neuronal mass which decreases in advanced stages. PD treatment has been
utilized by targeting DA neurotransmission through pharmacological strategies such as, DA
receptor agonists, enhancement of DA vesicular release, blocking DA uptake through inhibiting
DA transporters, and DA enzymatic metabolism inhibitors (Ghanta, Elango and Bhaskar 2020,
Ambani and Vanwoert 1973, Antonini et al. 2018).
Notably, as society continues to live longer, the incidences of Parkinson’s disease
continues to increase. Currently, there are an estimated seven to ten million people with
Parkinson’s disease worldwide. It is predicted that in 2020 there will be 93,000 people in the
United States living with Parkinson’s disease and by 2030 there will be 1.2 million incidences
(Marras et al. 2018). The prevalence of Parkinson’s disease increases with age, but an estimated
4 percent of people are diagnosed before age 50. The combined direct and indirect cost of
Parkinson’s, including treatment, social security payments and lost income, is estimated to be
nearly $25 billion per year in the United States alone (Marras et al. 2018). These statistics
convey the necessity of therapeutics that can better treat and delay the progression of the disease.

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1.2: The Therapeutic Indication of Dihydromyricetin

Figure 1: Dihydromyricetin molecular structure

Dihydromyricetin [(2R,3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-
dihydrochromen-4- one (DHM)] is a bioactive flavonoid extracted from Hovenia dulcis.
Flavonoids, a subgroup of polyphenols, are broadly present in food and beverage and numerous
studies have suggested that DHM could be useful for preventing or treating neurodegenerative
diseases such as PD in humans (Magalingam, Radhakrishnan and Haleagrahara 2015,
Magalingam, Radhakrishnan and Haleagrahara 2016). DHM has been shown to have a broad
range of therapeutic properties such as the ability to protect cells against inflammatory responses
and oxidative species (Hou et al. 2015, Liang et al. 2015, Silva et al. 2020) DHM acts as a
positive allosteric modulator (PAM) of GABAA receptors, an inhibitory neurotransmitter in the
central nervous system, and in previous studies in our lab has shown to be beneficial in both
Alzheimer’s Disease (AD) models and Alcohol Use Disorders (AUD) (Shen et al. 2012b, Liang
et al. 2014). In the context of AD, transgenic (TG2576) and Swedish transgenic (TG-SwDI)
mice with AD-like pathology were treated with DHM. The results suggested that DHM
improved cognition, anxiety levels and seizure susceptibility. In addition, DHM reversed
progressive neuropathology of mouse models of AD including reducing Aβ peptides, while
restoring gephyrin levels, GABAergic transmission and functional synapses (Shen et al. 2012b,
Liang et al. 2014). In AUD, DHM counteracted acute alcohol (EtOH) intoxication and

4
withdrawal signs in rats including; anxiety, seizure susceptibility, and tolerance. In an
intermittent voluntary ETOH intake paradigm in rats, DHM greatly reduced EtOH. DHM
competitively inhibits benzodiazepines (BZ)-site [ 3 H]flunitrazepam binding (IC50 , 4.36 M),
indicating DHM interaction with EtOH involves the BZ sites on GABAARs. It is suggested that
increased consumption in the rat model is correlated to ethanol-induced plasticity in GABAA
receptors (Shen et al. 2012a).
Notably, previous reports suggest that many patients suffering from PD have
abnormalities in the GABAergic system (Blaszczyk 2016). It is well established that
dopaminergic depletion resulting from neurodegenerative processes affecting dopaminergic
neurons in the substantia nigra pars compacta (SNPC) influence the overactivity of the two main
inhibitory output structures of the basal ganglia, specifically, the internal globus pallidus (GPi)
and the substantia nigra pars reticulata (SNr) (Fearnley and Lees 1991). Major inputs to the
substantia nigra (SN) come from the putamen and caudate nucleus and the pallidum and are
mostly GABAergic (Smith and Bolam 1990). In PD, the loss of nigrostriatal dopaminergic
neurons is associated with a downregulation of the GABAergic tone and a prevalence of the
excitatory system in the SN and basal ganglia (Magrinelli et al. 2016). This change in
GABAergic transmission presumably contributes to excitotoxicity and dopaminergic cell death.
Some of the GABAergic activity of DHM may be related to this selective inhibition of these two
overactive and inhibitory GABAergic structures (the GPi and the SNr). Activity at these
structures can then result in an increased activity of motor cortical areas. This change in activity
may, in part, explain the beneficial effects for these patients as it relates to Parkinsonian motor
symptoms (Daniele 2016). In addition, DHM has also been shown to be a potent neuroprotective
agent for DA neurons by modulating the Akt/GSK-3β pathway (brain insulin signaling pathway)

5
in a MPTP ((1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) prodrug to the neurotoxin MPP+)
dopamine depletion model. MPP+ treatment induced activation of GSK-3β, and DHM treatment
annihilated the MPP+ -induced activation of GSK-3β. In addition, DHM restored MPP+ -
induced suppression of Tyrosine hydroxylase (TH) expression (Ren et al. 2016, Ren et al. 2017,
Jeong and Kang 2018). DHM has been shown to have antioxidant, anti-inflammatory, and
neuroprotective effects, as well as the ability to restore GABA neurotransmission and improve
motor and cognitive behavior (Martinez-Coria et al. 2019, Zhang et al. 2018, Zhao 2005). DHM
appears to be worthy of consideration as a therapeutic modality for Parkinson’s disease treatment
(Li et al. 2017).

1.3 DHM the Blood Brain Barrier and the Neurotransmitter Dopamine
The blood-brain barrier (BBB), an interface between the peripheral circulation and
central nervous system (CNS), represents a major obstacle to the delivery of drugs to the CNS.
The two main mechanisms dominant in drug delivery are protein transporters in brain endothelial
cells and transmembrane diffusion (Jorgensen, Ulmschneider and Searson 2020). Most drugs in
clinical use are lipid soluble molecules that cross the BBB by transmembrane diffusion. The
BBB consists of several barriers in parallel, with the two most prominent being the vascular BBB
composed of cerebral microvascular endothelial cells (CEC) (O'Brown, Pfau and Gu 2018).
Drug delivery tends to focus on the vascular BBB, but in some instances the blood-cerebrospinal
fluid barrier is used (Banks 2009). Studies from the Liang Laboratory suggests that DHM, a
lipid soluble molecule, can readily cross the blood brain barrier – a critical requirement for any
therapy linked to neurodegenerative diseases (J. Liang, personal communication).

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Preliminary data in our lab suggests that extracellular dopamine is increased when DHM
is injected successively. Using dialysis (dialysis probe in the nucleus accumbens) with naive
Wistar rats (about 300g), DHM or vehicle (NaCl) was systemically injected and 20 minutes later
ethanol or vehicle was injected. DA was continuously monitored every 20 minutes for a total of
180 minutes following the final injection. This data suggests that DHM can affect ethanol
induced DA release in the nucleus accumbens (Liang J et al. 2007). The goal of my current work
is to investigate DA release in the striatum.

Figure 2: Liang, J, et al., 2007

1.4 Exercise and Parkinson’s Disease
Previous studies have supported the use of exercise for improving motor performance in
PD patients and in models of PD to facilitate neuroplasticity (Petzinger et al. 2013). The MPTP
animal model can be used to investigate molecular mechanisms of exercise-induced
improvement in motor behavior. The dopamine D1 and D2 receptors (D1R and D2R) modulate
physiological properties and cell communications and are the main targets of striatal medium

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spiny neurons. The D2R specifically plays a role in synaptic plasticity through glutamatergic and
dopaminergic neurotransmission that leads to changes in motor function in the dorsolateral
striatum. Exercise, specifically intensive treadmill running promotes neuroplasticity through
increased expression of striatal D2R. In addition, exercise enables a down regulation of the
dopamine transporter (DAT) (clears dopamine from extracellular space) protein within the
striatum, resulting in increased dopamine in the synaptic cleft. Exercise can be beneficial for
neuroprotection, neurogenesis, and neurorestoration and should be regarded as an essential
treatment for Parkinson's disease (Fisher et al. 2008, Fisher et al. 2013, Petzinger et al. 2007,
Petzinger et al. 2010, Petzinger et al. 2013, Petzinger et al. 2015, Bergen et al. 2002).

1.5 Justification for Animal Dopamine Depletion Models:
Animal models of PD have proved effective in the discovery of novel treatments for
motor and non-motor symptoms. These animal models of PD mimic, at least partially, a
Parkinson-like pathology and thus are believed to reproduce specific features of the human
disease. Early models were developed using dopaminergic neurotoxins, agents that destroy
catecholaminergic systems, such as 6-hydroxydopamine (6-OHDA) and MPTP. A common
feature of these neurotoxin induced models is that they affect mitochondria specifically; they
inhibit mitochondrial complex I or complex II (Mari and Bodis-Wollner 1997, Schober et al.
2004). 6-OHDA is systemically administered stereotactically into the brain, because 6-OHDA
fails to cross the blood–brain barrier. The preferred injection sites are the substantia nigra,
medial forebrain bundle, and striatum. Induced toxicity is rather selective for catecholaminergic
neurons, resulting from a preferential uptake of 6-OHDA by dopamine and noradrenergic
transporter molecules. Inside neurons, 6-OHDA accumulates in the cytosol and induces cell

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death without apoptotic aspects (Deumens, Blokland and Prickaerts et al. 2002). Comparatively,
MPTP enters the brain by crossing the blood-brain barrier after systemic injection. MPTP is
taken up by astrocytes and converted by the enzyme monoamine oxidase-B (MAO-B) into its
active form MPP+. MPP+ is then released from astrocytes into extracellular space and is
transported by DAT into dopaminergic neurons. Inside the dopaminergic neuron MPP+ can be
allocated into mitochondria or can be sequestered by a vesicular monoamine transporter
(VMAT) into synaptic vesicles (Duty and Jenner 2011, Luthman et al. 1989, Mari and Bodis-
Wollner 1997, Schober 2004).
The animal models of PD substantially alter dopaminergic drug treatment of the motor
signs and successfully capture the prevention and reversal of drug induced side effects that
develop with disease progression. However, so far, there is little evidence of established models
that reflect the progressive nature of the disorder or mimic the pathological and biological
complexity (Stvolinsky, Fedorova and Boldyrev 2007, Luthman et al. 1989).
1.6 Ivermectin Modulation of P2X4Rs in a 6-OHDA Unilateral Model:
Ivermectin (IVM), an anthelmintic medication, positively modulates P2X (subtype
receptor 4) (P2X4R) (Asatryan et al. 2010). P2X4Rs are ligand-gated ion channels activated by
Adenosine triphosphate (ATP) and distributed within the peripheral and central nervous system.
P2X4RS are co-localized to dopaminergic neurons in the SNpc and with GABAergic projection
neurons in the striatum. It is suggested that P2X4Rs regulate post synaptic NMDA receptors,
AMPA receptors and GABAA receptors currents as well as presynaptic release of glutamate and
GABA (Asatryan et al. 2014, Khoja et al. 2016, Xu et al. 2016).
The effects of IVM on DA-neurotransmission has been investigated using a unilateral 6-
OHDA mouse model of dopamine-depletion that targets the medial fiber bundle (MFB) (Khoja

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et al. 2016). Lesioning the MFB results in total denervation of the dopaminergic nigrostriatal
pathway (Boix et al. 2015). A rotational behavior model can successfully be used to explore
changes in DA including its presynaptic release and its postsynaptic activation of DA receptors.
Rotational behavior induced by compounds affecting presynaptic release overactiving the intake
hemisphere leading to rotations toward the lesioned side (ipsilateral rotations). In contrast,
rotational behavior induced by compounds (DA-agonists) affecting supersensitive postsynaptic
DA receptors on lesioned striatum leads to rotations away from the lesioned side (contralateral
rotations) (Konieczny et. al 2017). Ivermectin enhanced DA-agonist induced rotational motor
behavior and suggests close interactions between purinergic neurotransmission and DA
neurotransmission, however the underlying mechanism still remains unclear (Asatryan et al.
2010, Khoja et al. 2016).
Considering P2X4RS are expressed on DA neurons and GABAergic medium spiny
neurons of the basal ganglia, it was predicted that DHM through modulation of GABAergic
activity would increase DA agonist induced rotations in a similar manner to IVM.
1. Materials and Methods
Study #1
2.1 Animals:
All mice utilized in the experiments were eight-week-old male C67BL/6J mice purchased from
Jackson Laboratories (Bar Harbor, Me). In this study I used 31 mice for the stereotaxic surgery.
Mice were kept on a 12h light/dark cycle food and water available ad libitum. Experiments were
carried out under the accordance with the Institutional Animal Care and Use Committee of the
University of Southern California.
2.2 Stereotaxic Surgery:

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31 wild type C57BL/J6 mice from Jackson Laboratory mice underwent stereotaxic surgery
carried out as previously reported (Khoja et al. 2016). 30 minutes prior to surgery all mice were
injected with desipramine hydrochloride (HCl) (25 mg/kg in 0.9% saline, I.P; Sigma-Aldrich
(I.P. 25mg/kg) to prevent damage to the noradrenergic pathways. Mice were anesthetized with
4% isoflurane plus O2 (400 mL/min). After mice were anesthetized. The isoflurane was reduced
to 2%, this allows steady anesthetization without adverse respiratory effects. Mice are placed
into the small animal stereotaxic apparatus (Stoelting model 51730) and their nose cones (4%
isoflurane plus O2 (400 mL/min) and ear bars are put in place. Ophthalmic ointment was applied
to both eyes following induction of anesthesia to prevent corneal drying. An incision using a
feather surgical blade (stainless steel No. 10) and forceps (stainless steel 10cm curved) was used
to make an incision from the base of the neck to the interfrontal bone between the eyes. During
surgery 6-OHDA( (2 µL of 4 mg/mL in 0.2% ascorbic acid and 0.9% saline; cat. no. H4381,
Sigma-Aldrich) was loaded into the stereotaxic syringe and was positioned over the following
coordinates (relative to bregma): anterior–posterior (A/P) = −1.3mm; medio-lateral (M/L) =
+1.1mm and dorso-ventral (D/V) = -5.0mm, the left medial forebrain bundle (MFB). A bore hole
was then made above the left MFB using a stereotaxic micro drill (Kopf model 1474-220). The
stereotaxic needle was lowered into the bore hole and 6-OHDA was injected at a rate of 0.5
µL/min. After full injection of the 6-OHDA, the needles remained in the MFB for 5 minutes.
The needle was then raised at a rate of 0.1 mm per 15 seconds out of the target location. Mice
are then single caged and were given gel carprofen medigel (non-steroidal anti-inflammatory
drug , C15H12ClNO2, Clear H2O). After surgery, mice were allowed to recover for four weeks.
2.3 Rotation Behavior:

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Before testing mice were put into one of two treatment groups: saline (n=15) or DHM (n=16).
Mice were treated 5 days a week for 4 weeks with 5mg/kg I.P. injections of either saline or DHM.
L-DOPA (5.0 mg/kg with 1.25 mg/kg benserazide in 0.9% saline, S.C.; cat. no. PHR1271 L-DOPA
B7283 benserazide, Sigma-Aldrich) and d-Amphetamine (AMPH) (5.0 mg/kg in 0.9%saline, I.P;
cat. no. A-5880, Sigma-Aldrich) induced rotations were tested on 6-OHDA and lesioned mice with
experimentation following in the beginning of the dark cycle before the mice were exercised for 4
weeks. After exercise L-DOPA+DHM (30mg/kg with 0.9% saline I.P. Master Herbs Inc.,
Pomona, CA), L-DOPA+DHM, DHM (30mg/kg), L-DOPA (5mg/kg), and AMPH (5mg/kg)
induced rotations were tested on the same 6-OHDA lesioned mice with experimentation following
in the beginning of the dark cycle. 30 Minutes before testing began all 31 mice were moved into
the behavior room to allow for acclimation. During this period mice were placed in a room with
only red light to prevent disruption of the animals’ natural light/dark cycle. Mice were given 0.2
ml of 0.9% sterile saline subcutaneously and placed in a 5000 mL beaker for baseline rotational
testing. For 10 minutes, ipsi-lateral and contra-lateral rotations were counted for the saline
injections. Then mice were injected with desired compound(s) and ipsi-lateral and contra-lateral
rotations were counted. L-DOPA, L-DOPA+DHM rotations were counted for 90 minutes, whereas
AMPH, AMPH+DHM, and DHM induced rotations were counted for 120 minutes.

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Figure 3: Mice in beakers for L-DOPA+DHM induced rotational behavior
2.4 Scoring of Rotations:
All rotation tests were recorded and scored by researchers who were blinded to the treatment
groups. Rotations were counted only if the mouse performed a tight rotation, appearing as if it was
chasing after its own tail. Both contralateral and ipsilateral rotations were recorded.
2.5 Behavioral Testing:
Mice were given L-DOPA 5mg/kg (S.C) 1.5 hours for behavioral testing
2.51 Exercise:
Mice were exercised on a treadmill for 1 hour 5 days a week for 4 weeks using the following
paradigm:
Treadmill Exercise (Modified from Mark Colt 11/13/19; adapted from Fisher et. al, 2004)
1) Ensure that mice can maintain a speed of 5.0 m/min for five minutes one week before the
exercise paradigm.

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2) Give mice 15 minutes to warm up and then exercise them for 30 minutes, modifying speed
every 5 minutes. Cool down period should also be 15 minutes.

Sample running speeds (during most intense exercise):
0-5 minutes: 5 m/min
5-10 minutes: 8 m/min
10-15 minutes: 13 m/min
15-20 minutes: 15 m/min
20-25 minutes: 18 m/min
25-30 minutes: 20 m/min
Break (5 mins) 5 m/min
30-35 minutes: 20 m/min
35-40 minutes: 20 m/min
40-45 minutes: 18 m/min
45-50 minutes: 13 m/min
50-55 minutes: 8 m/min
55-60 minutes: 5 m/min

3) During the first few days of training, 20 m/min is not attainable. The goal is to push the mice
to what they can feasibly do, increasing max speed every day, until hopefully by the end of 2
weeks, they can maintain 20 m/min. Lesioned animals will determine the maximum pace.
7) Increase treadmill speed and duration when all mice within each group maintain a forward
position on the treadmill for 75% of the running period.
8) Exercise sessions are 60 minutes total, 5 days/week, for 4 weeks, for a total of 30 days of
exercise.

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2.52 Rotarod:
All 31 C57BL/J6 6-ohda lesioned mice were trained on the rotarod to evaluate motor
coordination. Mice are placed on the rotarod twice a day for 2 days to allow for acclimation.
Mice were evaluated on the rotarod once a week for 4 weeks at the end of each exercise program
for that week. Mice are placed on an accelerating rotarod from 0-40 rpm occurring every 30
seconds. Mice remain on the rotarod for 300 seconds or until they fall. Latency to fall (in
seconds) is recorded for each mouse for each trial.

Experimental Design
Figure 4: Illustrating the behavioral paradigms used in my investigations (Created with Biorender.com)

2.6 Dissections and Tissue Collection:
After behavioral experimentation had concluded mice were sacrificed and tissues were collected
for future immunohistochemistry staining (IHC),western blot, and qrt-PCR.
2.61 Immunohistochemistry Staining for TH:

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Mice were anesthetized with avertin (25.0 mg/kg I.P) and cardiac perfusion was performed by
perfusing from the left ventricle with 4% phosphate buffer saline (PBS) at a rate of 8mL/min for
2 minutes. Then for 5 minutes phosphate buffered saline with paraformaldehyde (PFA) at a rate of
5mL/min was used. The brains were removed and placed into a conical containing 40mL of PFA
solution and stored overnight . The next day, each brain was transferred to a 20% sucrose solution
and stored overnight. 24 hours later, each brain was flash freezed with isopentane (C5H12) on dry
ice. Using a cryostat (LEICA CM1900), striatal slices of 25 µm were cut and immersed into PBS
solution. Cut slices were blocked with 4% normal goat serum with TBS+Triton X-100 (0.2%) for
one hour (room temperature) on a nutator. Sections were then stained with tyrosine hydroxylase
primary antibody (1:5000 dilution, cat. no. MAB318, Millipore) with 2% normal goat serum and
TBS+Triton X-100 (0.2%) and then incubated (4°C) for 48 hours. After 48 hours, slices were
washed 3 times for 5 minutes with TBS. Slices were then incubated (room temperature) in a
solution containing IRDye® 800CW secondary antibody (1:2000 dilution, Licor), 2% normal goat
serum, and TBS+Triton X-100 (0.05%). The slices were incubated in the solution for one hour
(room temperature) in a foil and then striatal sections were washed with TBS-Triton X-100 (0.5%)
for 5 minutes followed by TBS for 5 minutes. Slices were analyzed using the Licor Odyssey
Program.
Slides of the brain slices were scanned by the Odyssey Imaging System and the highest resolution
JPEG images were selected. ImageJ was used for fluorescence analysis and the difference in
fluorescence of the left striatum (lesioned side) from the right striatum (intact side) was divided
by the fluorescence of the right striatum to generate the percent difference in fluorescence between
the two sides
Evaluation of lesion percentage analyzed on ImageJ

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(Difference of Non-Lesion and Lesion) ➗(Difference of Non-Lesion and Lesion+Non-Lesion ) ✖ 100

Figure 5: IHC TH stain Lesion Percentage= 98.96%

Figure 6: IHC TH stain Lesion Percentage= 67.9%

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Figure 7: IHC TH stain Lesion Percentage= 11.85%

2.62 Micro dissected Tissue:
Fresh tissue is micro dissected from brain into the following sections (left and right) the dorsal
striatum (from bregma ±2.47,-0.07,2.08) ventral striatum (from bregma ±2.36,-.07,-.077)
prefrontal cortex (from bregma ±1.29,2.33,2.2),hippocampus (from bregma ±0.88, -0.97,2.78)
cortex (from bregma ±3.85,-0.97,2.8) ,and cerebellum (from bregma ±2.4,-5.47,3.37).
Western Blot:
Micro dissected tissue samples are first put in 200uL lysis buffer (50 mM Tris pH 7.41mM EDTA
and100 uM PMSF (phenylmethylsulfonyl fluoride, stock is made in 100% EtOH). Tissue was then
homogenized in sonicator, sonocating for 10s at a time. Tissue was spun down at 14 kxg for 2min
to remove tissue debris. Then the 200uL of mix was removed and the tissue debris was discarded.
Then Bicinchoninic Acid Assay (BCA) analysis was completed to quickly determine total protein
concentration by measuring absorbance at 562 nm and comparing it to a protein standard

18
absorption vs. concentration curve. BCA analysis was completed in triplicates for each sample
with 50-part A to 1-part B for 200uL working solution per sample plus 10uL of sample per well.
The standard BCA curve consisted of 125,250,500,750,1000,1500,1750,and 2000 ug/mL as
standards. The samples diluted 1:1 with sample loading buffer (4x Laemmli Sample Buffer Cat.
#1610747) with a minimum volume of 12uL and 20ug of protein per sample. Then samples were
boiled (80°C) ,cooled on ice, and then loaded into wells(well were rinsed with a running buffer
(1X Tris/Glycine) before loaded ). Gel ran at 200V for 1hr and was checked every 15 minutes and
stopped when the bands reached the bottom of the gel. Gel was then transferred to a
Polyvinylidene fluoride (PVDF) membrane using a Bio-Rad Trans-Blot Turbo. Gel was then
blocked for 1hr with Bio-Rad EveryBlot Blocking Buffer (Cat #12010020). Then gel was
incubated (4°C) on rotisserie overnight with desired primary (9.550mL Wester Blocking Buffer
+0.05mL 20% Tween-20 to get final volume .1% Tween- 20+ specified uL of each primary
antibody). The next day the gel was washed 4 times for 5 min in 10mL TBS + .1% Tween 20.
Then it was incubated (room temperature) on the rotisserie for 30min with secondary (9.95mL
Western Blocking Buffer +0.05mL 20% Tween-20+ specified uL of each 2ndary antibody).
Lastly, the gel was washed 4 times for 5 min with 10mL TBS + .1% Tween 20. Western blot was
analyzed using the Licor Odyssey Program and fluorescence of the protein band was analyzed with
ImageJ.
qRT-PCR:

mRNA from each sample was extracted and isolated using the RNeasy kit (Qiagen) and Zymo
Direct-zol RNA MiniPrep (Cat. #R2051). Then the purity of the isolated RNA was assessed using
a 1:10 dilution on the spectrophotometer and A230, A260, and A280 (protein absorbance at values
230nm, 260nm, and 280nm) values as well as the calculated concentration was recorded. Relative

19
purity of RNA can be assessed by the ratio of A260/A280; a ratio of 1.8-2 is generally considered
acceptably pure and should have no issue. The reverse transcription of RNA to complementary
DNA (cDNA) was completed using the PCRBIO cDNA Synthesis Kit from Genesee (Cat #: 17-
700). Then in RNAse/DNAse free PCR tubes (we use 8-strip tubes from Olympus), 1μl of 20x
RTase, 4μl of 5x cDNA Synthesis Mix, 200ng of of RNA, and RNAse-free water up to 20μl was
prepared and put into the thermal cycler (Perkin Elmer GeneAmp PCR System 2400) using the
following settings: 30 min at 42°C, 15 min at 85°C, then slowly cool to 4°C. Then cDNA, gene(s)
of interest, and qPCRBIO SyGreen Mix with low ROX is used to make a master mix with the
following 10μl SyGreen, 7.5μl nuclease-free H2O, 0.25μl forward primer, 0.25μl reverse primer.
The master mix with gene specific primers are quantitatively amplified using a thermal cycler
(eppendorf realplex Mastercycler). The comparative Ct method was used to determine difference
in gene expression and values were normalized to expression levels of B-Actin.
Study #2
2.7 Animals

49 eight-week-old male C57 mice were purchased from Jackson Laboratories (Bar Harbor, Me).
Mice were kept on a 12h light/dark cycle food and water available ad libitum.
The mice were separated into treatment groups (saline or MPTP). The MPTP mice received four
i.p. injections (20mg/kg) of 5 mg/mL MPTP (MPTP-HCl, Sigma) over the course of two days.
Five days after the last injection (the time it takes for MPTP attenuated dopaminergic toxicity)
behavior experiments were started. Experiments were carried out under the accordance with the
Institutional Animal Care and Use Committee of the University of Southern California.

20
Experimental Design:

Figure 8: Illustrating behavioral paradigms used in this experiment (Created with Biorender.com)

2.8 Behavioral Testing :
Before testing mice were put into five treatment groups MPTP treated saline (n=11), saline treated
saline (n=16), MPTP treated DHM (n=9), MPTP treated L-DOPA (n=9), and MPTP treated L-
DOPA+DHM (n=9)
Mice were treated 5 days a week for 2 weeks with 5mg/kg I.P. injections of either saline or DHM

2.81 Rotarod:
all 31 C57BL/J6 MPTP lesioned mice were trained on the rotarod to evaluate motor coordination
and motor learning. Mice are placed on the rotarod twice a day for 2 days to allow for
acclimation. Mice were evaluated on the rotarod for 5 trials with a 90 second break in between

21
each trial . Mice are then placed on an accelerating and reversal rotarod from 0-40 rpm occurring
every 30 seconds. Mice remain on the rotarod for 200 seconds or until they fall. Latency to fall
(in seconds) is recorded for each mouse for each trial.
2.82 Novel Object(NOR):
(NOR) test was used to evaluate alterations in their learning and memory. On Day 1 mice are
placed into a white box with bedding for 5 min. On Day 2 mice are placed back into the box with
two copies of the same object, either glass beaker or large blue Lego, and allowed to explore the
box for 10 minutes while being recorded on a video camera. If a mouse does not explore the
object for 20 seconds they are removed from the analysis. 24hr later the mice are placed into the
box with one Lego and one beaker. The sides of the familiar versus novel object are alternated
and randomized, mice are allowed to explore the box for 10 min. while being recorded via video
camera .
The scorer of the videos is blind to the mice being tested. Using a stopwatch, the video was
watched and the mouse’s behavior in relation to the two objects was assessed. Each time a
mouse interacts with an object the time is recorded. An interaction is counted if the animal’s
nose is within 1cm of the object and/or directed at the object. It does not count if the animal is
climbing on top of the object. Using the time recorded for interaction with novel and familiar
objects, each mouse is rated based on their discrimination index, which is calculated as follows:
𝐷𝐼 =
𝑇 𝑁
−𝑇 𝐹 𝑇 𝑁 + 𝑇 𝐹 (where Tn = time with novel object; Tf = time with familiar)

2.9 Dissections and Tissue Collection:
Fresh tissue is micro dissected from brain for qRT-PCR and western blot into the following
sections (left and right) the dorsal striatum (from bregma ±2.47,-0.07,2.08) ventral striatum (from

22
bregma ±2.36,-.07,-.077) prefrontal cortex (from bregma ±1.29,2.33,2.2) , hippocampus (from
bregma ±0.88, -0.97,2.78) cortex (from bregma ±3.85,-0.97,2.8) ,and cerebellum (from bregma
±2.4,-5.47,3.37).
Study # 3
2.10 Animals:
The 36 mice utilized in the experiments were eight-week-old male C67BL/6J mice purchased
from Jackson Laboratories (Bar Harbor, Me) ). Mice were kept on a 12h light/dark cycle food
and water available ad libitum. Experiments were carried out under the accordance with the
Institutional Animal Care and Use Committee of the University of Southern California.
2.11 Behavioral Testing: Sucrose preference
All 36 male C67BL/6J mice were evaluated for anhedonia via the sucrose preference test. The
mice were separated into four treatment groups: L-dopa (n=9), Saline (n=9), L-DOPA+DHM
(n=9), and DHM (n=9). Mice were given two water bottles in their group cage on day 0. On day
1 refill and record the amount of water consumed from each bottle. On day 2 repeat this action
and then at 7pm remove all fluids from the cage. On day 3 move mice into single housed cages at
7am and allow mice to drink for 4 hours with two bottles in their cage, one bottle with water and
the other with 2% sucrose. The sides of the bottles were randomized, and consumption recorded.

2.12 Statistical Analysis
Statistical analysis was performed using Prism-GraphPad 8 to see if there were any significant
effects between the treated 6-OHDA animals versus untreated 6-OHDA animals, as well as MPTP
injected versus saline injected animals. Significance was set at p < 0.05. Two-way ANOVA and

23
was used to assess the effects of DHM on lesioned animals. ANOVA significant main effects and
interactions were further analyzed using a post hoc Tukey’s test.
3. Results

3.1 DHM in a 6-OHDA Model:

I began my testing using a 6-OHDA unilateral lesion model where I tested 31 mice with
exercise and compared the effect of DHM (n=16) versus saline controls (n=15). As illustrated in
Figure 10, both DHM as well as DHM + exercise significantly decreased L-DOPA and AMPH
induced rotations. It is hypothesized that DHM, a positive allosteric modulator of GABAAR, may
be changing GABAergic tone or may be potentially working to help functionality of intact
synapses leading to a decrease in rotational behavior. Rotational behavior relies on changes in
homeostatic plasticity on the lesioned side of the striatum or an influx of dopamine on the intact
side (Konieczny et al. 2017). It is possible that DHM lessens neuronal injury leading to a
reduction of the expression of postsynaptic DA receptors, a compensatory response seen in
lesioned models of PD, causing a reduction in rotations (Navntoft et al. 2016). In the lesioned
system, the severity of functional deficits is generally linked to the degree of striatal denervation
(Dreyer et al. 2014). DHM may lessen DA denervation changing the functional deficits seen in
the PD model (Dreyer et al. 2014). In addition, improvement has been shown using positive
modulators of GABAAR signaling in mouse models of neurological disorders that present
insufficient inhibitory tone (Jacob et al. 2019).
Exercise was shown to enhance both L-DOPA and AMPH induced rotations in a 6-
OHDA unilateral model. Exercise increases striatal D2 and D3 receptor availability and enables
a downregulation of DAT causing a change in DA signaling and in this study resulted in an
increase in DA mediated rotations (Fisher et al. 2013, Petzinger et al. 2007) .

24

Figure 9: DHM reduced L-DOPA induced rotations L-DOPA (5 mg/kg)-induced rotational behavior is significantly
attenuated in DHM and DHM+ exercise treated mice. DHM (5 mg/kg) significantly decreases L-DOPA’s effect on the number of
contralateral turns in wildtype (WT) mice. Values on the y-axis represent the mean number of contralateral turns per 10-minute
interval Æ SEM from 31 DHM treated WT. **p < 0.05, versus L-DOPA+ saline -treated WT mice, before exercise ***p< 0.001
versus after exercise Bonferroni post hoc test. n=31 male C57/B6 6-OHDA lesioned mice (before and after exercise).

As illustrated in Figure 10, rotations were decreased due to both the chronic administration of
DHM versus saline mice and acute administration of DHM versus saline seen in drug-induced
rotational behavior.

25

Figure 10: DHM reduced L-DOPA induced rotations when administered chronically and acutely 31 Male C57/B6 6-
OHDA lesioned mice L-DOPA induced rotational behavior (before and after exercise) lesioned mice separated into treatment
groups n=16 DHM treated mice n=15 Saline treated mice (DHM 5mg/kg daily or saline 5mg/kg daily)

26

Figure 11: DHM reduced AMPH induced rotations AMPH (5 mg/kg)-induced rotational behavior is significantly attenuated
in DHM and DHM+ exercise treated mice. DHM (5 mg/kg) significantly decreases AMPH’s effect on the number of ipsilateral
turns in wildtype (WT) mice. Values on the y-axis represent the mean number of ipsilateral turns per 10-minute interval Æ SEM
from 31 DHM treated WT. ***p < 0.001, versus L-DOPA+ saline -treated WT mice, before exercise ***p< 0.001 versus after
exercise Bonferroni post hoc test. n=31 male C57/B6 6-OHDA lesioned mice (before and after exercise).
In the 6-OHDA model a significant change was not seen in rotarod between treatment groups
(saline versus DHM) as illustrated in Figure 12 . A change may have not been captured in the
particular paradigm used or there was a ceiling effect from the 4 weeks of exercise all the mice
received. Ceiling effect as a result of 4 weeks of exercise has been shown in other behavior
paradigms such as water maze protocols and tone conditioning stimulus tests (Robison et al.
2018, Baruch, Swain and Helmstetter 2004).

Figure 12: DHM versus saline treated mice did not show a significant change in rotarod in a 6-OHDA model with exercise
31 male C57/B6 6-OHDA lesioned mice n=16 DHM treated mice n=15 Saline treated mice. Accelerating rotarod and reversal
with max speed 40 RPM. Y axis indicating latency to fall in seconds x axis indicating trial # over 4 weeks with 3 trials once a
week.

27

Figure 13: DHM versus Saline Trial 1 out of 3 weeks 1-4 in a 6-OHDA model Trial 1 of week 2 and week 3 showed DHM
treated mice performed better on the rotarod (latency to fall(s)) versus saline treated mice p=0.089 and p=0.085 n=31 male
C57/B6 6-OHDA lesioned mice (n=16 DHM treated mice and n=15 saline treated mice). Accelerating rotarod and reversal with
max speed 40 RPM . Y axis indicating latency to fall in seconds x axis indicating trial #

28
Figure 14: DHM versus Saline Trial 2 out of 3 weeks 1-4 in 6-OHDA model n=31 male C57/B6 6-OHDA lesioned mice
(n=16 DHM treated mice and n=15saline treated mice). Accelerating rotarod and reversal with max speed 40 RPM. Y axis
indicating latency to fall in seconds x axis indicating trial #

Figure 15: DHM versus Saline Trial 3 out of 3 weeks 1-4 in 6-OHDA model n=31 male C57/B6 6-OHDA lesioned mice
(n=16 DHM treated mice and n=15 saline treated mice). Accelerating rotarod and reversal with max speed 40 RPM. Y axis
indicating latency to fall in seconds x axis indicating trial #

29

Figure 16: Average Latency of all 3 trials ;DHM versus saline treated mice did not show a significant change in rotarod in
a 6-OHDA model with exercise 31 male C57/B6 6-OHDA lesioned mice n=16 DHM treated mice n=15 Saline treated mice.
Accelerating rotarod and reversal with max speed 40 RPM. Y axis indicating latency to fall in seconds x axis indicating trial #
over 4 weeks with 3 trials once a week.

I utilized Western blots to confirm the lesions of 6-OHDA mice staining for TH, the rate
limiting enzyme of catecholamine biosynthesis, on both the dorsal striatum right side and the
dorsal striatum left side resulted in the bands pictured below (Figure 18 and 19). The right side
is the intact or non-lesioned hemisphere and the left side is the lesioned hemisphere. The
western blot membranes representing the lesioned side were then analyzed using ImageJ and the
percent lesioned were recorded. The difference in TH staining between the two sides of the
striatum indicates that the right dorsal striatum (intact side) does not express a decrease in TH

30
protein and the left dorsal striatum (lesioned side) does express a decrease in TH protein
representing a successful lesion of 30%-98% in 11 mice.

Figure 17: TH stains of MFB 6-OHDA lesioned mice non lesioned hemisphere Western blot of TH stained proteins of 11
Male C57/B6 6-OHDA lesioned mice on (PVDF) membrane analyzed using LI-COR

31
Figure 18: TH stains of MFB 6-OHDA lesioned mice lesioned hemisphere Western blot of TH stained proteins of 11 Male
C57/B6 6-OHDA lesioned mice on (PVDF) membrane analyzed using LI-COR optical density of TH normalized to the non-
lesioned hemisphere (percentage of lesion of 11 mice)

3.2 Wild Type Mice Sucrose Preference and DHM:

Using 36 wild type (WT) C57/B6 mice sucrose preference (SFT) was used to test for hedonic
responses to the following treatment: saline (n=12), DHM (n=12), and L-DOPA+DHM (n=12).
Anhedonia is a relative lack of pleasure in response to a formerly rewarding stimuli (such as
sucrose or saccharin) and a cardinal hallmark of several forms of depression (Mateus-Pinheiro et
al. 2014). Sucrose preference can be used to accurately characterize psychiatric disorders or
anhedonia responses to treatment. When mice were treated with DHM or DHM+L-DOPA, the
total amount of sucrose drank 4.8mL and 5.5mL respectively and the total amount of volume
drank 3.4mL and 3.8mL respectively were decreased compared to the saline treated groups.
However, the percent of sucrose drank did not change between the three treatment groups.
These findings suggest that anhedonia behavior in male mice was unaltered, but a treatment
effect trend appeared in the total amount of sucrose and liquid drank.

32

(a) (b)
Figure 19: DHM and DHM+L-DOPA decrease total sucrose drank and total volume drank compared to controls 36 DHM
(n=12), Saline (n=12), and DHM+L-DOPA (n=12) WT Male C57/B6 Mice SFT total sucrose and volume drank. (a) DHM
treated mice and L-DOPA+DHM drank less sucrose versus saline treated mice p=0.69 and p=0.96 evaluated using Bonferroni
post hoc test (b) DHM treated mice and L-DOPA+DHM drank less volume versus saline treated mice p=0.57 and p=0.91
evaluated using Bonferroni post hoc test

33
Figure 20: DHM did not change percent sucrose drank compared to control testing anhedonia in sucrose
preference model 36 DHM (n=12), Saline (n=12), and DHM+L-DOPA (n=12) WT Male C57/B6 Mice SFT total
percent sucrose drank
3.2 MPTP Model and DHM:
5 days after 4 injections of 20mg/kg of MPTP or saline, 54 male and female mice were separated
into the following groups (saline/saline n=16), (MPTP saline n=11), (MPTP L-DOPA n=9),
(MPTP DHM n=9). And (MPTP L-DOPA+DHM n=9). They were then tested using a motor
learning rotarod paradigm with two days of acclimation. The female mice did not show a
significant improvement in motor learning with DHM or DHM+L-DOPA compared to other
treatment groups. On the other hand, the male MPTP lesioned mice did show a significant
upward trend p=0.0778 of motor learning improvement with L-DOPA+DHM treatment
compared to all other treatments. In addition, this treatment group (L-DOPA+DHM) exceeded
the saline/saline treated group. There was a significant sex difference between motor rotarod
performance possibly indicating a difference in MPTP lesion deficits between male and female.
Previous studies have shown subtle sexually dimorphic impairments in motor performance and
the importance of estradiol protection for females against striatal loss, but its failure to protect
striatal lesion in males (Antzoulatos et al. 2010, Abraham et al. 2019). Together, the present
findings suggest that DHM in adjunct with L-DOPA is able to alter motor learning behavior and
coordination in males and DHM should be further evaluated as an adjunct therapy for
Parkinson’s disease

34

Figure 21: DHM did not change Female MPTP (20mg/kg) lesioned mice performance on rotarod 27 female C57/B6 MPTP
saline/saline n=16), (MPTP saline n=11), (MPTP L-DOPA n=9), (MPTP DHM n=9). And (MPTP L-DOPA+DHM n=9) lesioned
mice. Accelerating rotarod and reversal with max speed 40 RPM. Y axis representing latency to fall in seconds and x axis
representing trial number.

35
Figure 22: DHM+L-Dopa improved Male MPTP (20mg/kg) lesioned mice performance on rotarod 27 female C57/B6
(MPTP saline/saline n=16 MPTP saline n=11 MPTP L-DOPA n=9 MPTP DHM n=9 MPTP L-DOPA+DHM n=9) lesioned mice.
Accelerating rotarod and reversal with max speed 40 RPM. DHM+L-DOPA treated mice improved performance on rotarod
versus saline p=.0898 Y axis representing latency to fall in seconds and x axis representing trial number.

Figure 23: DHM+L-Dopa improved Male MPTP (20mg/kg) lesioned mice performance on rotarod 27 female C57/B6
(MPTP saline/saline n=16 MPTP saline n=11 MPTP L-DOPA n=9 MPTP DHM n=9 MPTP L-DOPA+DHM n=9) lesioned mice.
Accelerating rotarod and reversal with max speed 40 RPM. Y axis representing latency to fall in seconds and x axis representing
trial number.

36
Figure 24: Female MPTP lesioned (20mg/kg) performed significantly better than male MPTP (20mg/kg) lesioned mice 27
females versus 27 male C57/B6 MPTP lesioned mice. Female mice performed significantly better than male mice **p>0.005.
Accelerating rotarod and reversal with max speed 40 RPM. Y axis representing latency to fall in seconds and x axis representing
trial number.

4. Discussion
Dopamine depletion models, including the 6-OHDA and MPTP models used in my
study are commonly used in PD investigations to further understand underlying pathology or to
discover novel interventions for PD. When the number of DA neurons is reduced, there are
fundamental physiological changes in the substantia nigra and striatum. In addition, density of
DA terminals, the sites of DA release and uptake, and longevity of striatal extracellular DA are
significantly different.
In this study both the unilateral 6-OHDA and MPTP models were used to examine
DHM’s ability to modulate DA mediated behaviors. In the 6-OHDA model all 31 mice were
exercised. Exercise as an intervention is shown to stimulate dopamine synthesis and have
neuroprotective effects in PD (Goodwin et al. 2008). The rotational behavior paradigm used in
unilateral 6-OHDA lesioned mice is based on either DA stimulation on the lesioned side, the
higher concentration of released dopamine in the intact striatum resulting in rotation in the
direction ipsilateral to the lesion or hyperstimulation of supersensitive post-synaptic DA
receptors in the lesioned or denervated side resulting in contralateral rotations (Bjorklund and
Dunnett 2019). DHM, a bioactive flavonoid extracted from Hovenia dulcis and a positive
allosteric modulator of GABAAR , reduced both L-DOPA and AMPH rotations as seen in Figure
10,11, and 12 suggesting that DHM may be changing GABAergic tone alleviating excitotoxicity
and dopaminergic cell death or may be reducing denervation changing the degree of lesion.
However, DHM or DHM+L-DOPA induced rotations were not examined in this model without

37
exercise. It is important to see if this result is replicated in a 6-OHDA MFB lesioned mouse
model without exercise.
Additionally, in the MPTP model an improvement in motor learning was seen in male
lesioned mice but not female lesioned mice (Figure 23 and 24) establishing DHM’s ability to
alter motor learning behavior and coordination. The excitatory corticocortical pathway plays an
important role in adaptive motor behaviors. Synaptic transmission namely long-term
potentiation and long-term depression plasticity are thought to underlie motor learning and are
dependent on glutamatergic corticostriatal and dopaminergic nigrostriatal pathways (Calabresi et
al. 2007). GABA and glutamate receptors are the main neurotransmitters in the basal ganglia
and are at synaptic, extra synaptic and presynaptic sites. There are many potential locations for
receptor-neurotransmitter interactions and glutamatergic pathways may be altered through DHM,
a GABAergic modulator, resulting in a motor dependent synaptic change (Sakairi et al. 2020).
Although more work is needed before definitive conclusions can be drawn, previous
work by others and current work from my thesis studies suggests that DHM represents a novel
therapeutic for PD that should be further investigated.

38
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Abstract (if available)

Abstract Parkinson’s Disease (PD) is a neurodegenerative disorder distinguished by dopamine (DA) depletion resulting in motor and cognitive deficits. The current treatment options, DA replacement therapy and DA agonists, provide symptomatic relief, but do not alter or improve the underlying pathology. Dihydromyricetin (DHM), a bioactive flavonoid from Hovenia dulcis, is a positive allosteric modulator of Gamma-amino butyric acid (GABA) receptors that has been linked to modulation of dopamine activity in the Central Nervous System (CNS). For example, a downregulation of GABAergic tone is commonly seen in PD patients and is associated with the loss of nigrostriatal dopaminergic neurons (Magrinelli et al. 2016). DHM potentially regulates GABAergic tone alleviating excitotoxicity and dopaminergic cell death suggesting that DHM represents a possible therapy for PD. My thesis tested this hypothesis by investigating the role of DHM on DA dependent behavior using PD models. Rotational behavior in a 6-hydroxydopamine (6-OHDA) model and rotarod behavioral model in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-OHDA models were used to measure the extent of motor impairment in DHM treated groups versus control groups. DHM versus control group decreased rotational behavior in 6-OHDA model and DHM versus control group improved rotarod performance in an MPTP model, suggesting an improvement in motor deficits caused by a dopamine depletion model in DHM treated mice.

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Creator Halbers, Lila Paige (author)

Core Title Evaluating the effects of dihydromyricetin on dopamine mediated behaviors using a Parkinson's disease model

School School of Pharmacy

Degree Master of Science

Degree Program Clinical and Experimental Therapeutics

Publication Date 04/25/2020

Defense Date 04/24/2020

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

Tag 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,6-hydroxydopamine,central nervous system,dihydromyricetin,Dopamine,dopamine D1 receptor,dopamine D2 receptor,dopamine transporter,gamma-aminobutyric acid receptors,internal globus pallidus,medial fiber bundle,monoamine oxidase-B,novel object recognition,OAI-PMH Harvest,Parkinson's disease,positive allosteric modulator,rotarod test,substantia nigra pars compacta,substantia nigra pars reticulata,sucrose preference test,tyrosine hydroxylase

Language English

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Advisor Davies, Daryl L. (committee chair), Jakowec, Michael (committee member), Liang, Jing (committee member)

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