University of Southern California Dissertations and Theses

The effect of treadmill running on dendritic spine density in two neurodegenerative disorders: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease and CAG₁₄₀ kn...

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The effect of treadmill running on dendritic spine density in two neurodegenerative disorders: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease and CAG₁₄₀ kn...

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THE EFFECT OF TREADMILL RUNNING ON DENDRITIC SPINE DENSITY IN TWO
NEURODEGENERATIVE DISORDERS: 1-METHYL-4-PHENYL-1,2,3,6-
TETRAHYDROPYRIDINE (MPTP) MOUSE MODEL OF PARKINSON’S DISEASE AND CAG140
KNOCK-IN MOUSE MODEL OF HUNTINGTON’S DISEASE

By

William Anthony Toy

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree

DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)

August 2016

Advisory Committee:
Aaron McGee, Ph.D. (chair)
Giselle Petzinger, M.D.
John Walsh, Ph.D.
Christian Pike, Ph.D.
Michael Jakowec, Ph.D.

Table of Contents
Dedication and Acknowledgments ........................................................................................................... 1
Chapter 1: Introduction ............................................................................................................................. 4
Parkinson’s disease ......................................................................................................... 4
Overview .............................................................................................................................................. 4
Direct/Indirect Motor Pathway Model .............................................................................................. 7
MPTP Mouse Model of Parkinson’s Disease .................................................................................. 10
Huntington’s disease ......................................................................................................14
Overview ............................................................................................................................................ 14
Pathology ........................................................................................................................................... 16
Transgenic Mouse Models ............................................................................................................... 18
Dendritic Spine Density ..................................................................................................21
The Dendritic Spine .......................................................................................................................... 21
Dendritic Spine Density .................................................................................................................... 22
Exercise as an Intervention for Neurodegenerative disorders .............................................22
Exercise is the Best .......................................................................................................................... 22
Exercise and Parkinson’s Disease ................................................................................................... 23
Exercise and Huntington’s Disease ................................................................................................ 24
Chapter 2: Treadmill exercise prevents dendritic spine loss in direct and indirect
striatal medium spiny neurons in the 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) mouse model of Parkinson’s disease ..................................................... 25
Introduction ..................................................................................................................25
Material and Methods .....................................................................................................28
Animals ............................................................................................................................................... 28
MPTP-lesioning .................................................................................................................................. 29
Exercise Regimen.............................................................................................................................. 30
Golgi Staining .................................................................................................................................... 31
Biocytin Labeling ............................................................................................................................... 31
Morphological Analyses .................................................................................................................... 32
Immunohistochemistry .................................................................................................................... 33
Statistical analyses............................................................................................................................ 35
Results .........................................................................................................................35

MPTP administration leads to loss of striatal DA ......................................................................... 35
Intensive treadmill exercise attenuates initial running speed deficit of MPTP-
lesioned mice ..................................................................................................................................... 37
Exercise increases dendritic spine density in DA-D1R- and DA-D2R-containing
MSNs in MPTP Mice. ......................................................................................................................... 39
Exercise increases PSD-95 expression in MPTP-lesioned mice .................................................. 42
Exercise increases synaptophysin expression in MPTP-lesioned mice ...................................... 42
Exercise increases dendritic arborization in MPTP-lesioned mice .............................................. 44
Exercise does not offset distal pruning of DA-D2R MSN dendrites in MPTP-
lesioned mice ..................................................................................................................................... 46
Discussion .....................................................................................................................47
Conclusion ....................................................................................................................52
Chapter 3: The effect of treadmill exercise on dendritic spine density in the
CAG140 knock-in mouse model of Huntington’s disease ...................................................................... 54
Introduction ..................................................................................................................54
Methods and Materials ...................................................................................................55
Animals and Groups ......................................................................................................................... 55
Exercise Regimen.............................................................................................................................. 56
Golgi Staining .................................................................................................................................... 56
Dendritic Spine Density .................................................................................................................... 57
Western Immunoblotting ................................................................................................................. 59
Statistical Analyses ........................................................................................................................... 61
Results .........................................................................................................................62
Dendritic Spine Density – 4 months .............................................................................................. 63
Dendritic Spine Density – 6 months .............................................................................................. 67
Western Immunoblotting ................................................................................................................. 71
Discussion .....................................................................................................................81
Conclusion ....................................................................................................................83
Chapter 4: Conclusions, Perspectives, Limitations, Other .................................................................. 84
References ................................................................................................................................................. 89

Page 1

Dedication and Acknowledgments
1

This dissertation
2
is dedicated to my wife, Jessica
3
. There is absolutely no way
this dissertative
4
work would have ever been completed without you. I cannot thank
you enough for your support, motivation, and love. We have been through some
amazing times together, and some really awful
5
ones as well. With the completion of
this chapter of my/our lives I can’t wait to take the next steps with you. It’s going to
get crazy! at a slow to moderate pace. No worries.
Mike and Giselle, you have been some of the most inspirational and interesting
people I have ever met. Giselle, your enthusiasm for science and life is contagious and
every time I finish having a conversation with you I feel like a fire has been lit inside me
to do something great. Mike, your wit and humor has made for some of the best
laughs these past few years; also, your depth of knowledge concerning science from
the past millennium is utterly impressive. I’m glad that I can call you both my friend
and look forward to future gatherings filled with music, laughter, and, of course,
barbeque.
Mom and Dad, without hesitation you have supported me throughout every
endeavor that I have undertaken. I cannot tell you enough how grateful I am to have
you as parents.

1
I think the all-caps title and other designations are silly. It makes the text difficult to read and the
content is hardly shout worthy.
2
“Dissertation” is used instead of “Thesis” because the USC Graduate School refers to doctoral students
submitting a dissertation and master’s students submitting a thesis.
3
I was expecting you to throw a random footnote in somewhere. :D
4
Finally, a word I made up that’s actually a bonafide, legitimate, in-the-dictionary word!
5
I added another one for good measure, then took it all away
Page 2

I would like to thank my committee members Dr. Aaron McGee (chair), Dr. John
Walsh, and Dr. Christian Pike for their time, effort, and guidance over these last few
years.
I would like to thank Dr. Ruth Wood for the use of her vibratome (a tissue
cutting device) on which I have sectioned a few too many brains.
My fellow lab members have been invaluable accomplices in this mad world of
science. In no particular I would like to thank Dr. Daniel Stefanko
6
, Matthew Halliday,
Dr. Natalie Kintz, Vivek Shah, Damaris Garcia, Brian Leyshon, William (Bill
7
) Toy,
8
and
Marta Vuckovic.
The Keck School of Medicine really cares about their students and I’m thankful
that it has provided break rooms for the students, especially the one with a 1960s pool
table.
I would also like to thank the people that have indirectly helped me these past
few years. The food service professionals in Seaver dining hall have been good to me
and my belly. The custodial staff of McKibben Annex has been on top of their game
and kept our lab a happy place for years.
I would like to thank my dogs Rena and Ghia. They have no idea that I’m
thanking them, but I did give them an extra treat, so that’s pretty good right?

6
“Dr.” prefix is only for his Ph.D. “Dr. Dr.” will be incorporated after completion of his M.D.
7
I’ve always been called Bill. If you read this footnote, please call me Bill.
8
The Oxford or serial, serials, or Harvard comma will be used throughout this work. I feel it helps with
spoken cadence.
Page 3

I am grateful for the monetary contributions from: NINDS RO1 NS44327, U.S.
Army NETRP (Grant #W81XWH-04-1-0444), Zumberge Foundation of USC, and
Southern California Clinical and Translational Science Institute and University of
Southern California, Keck School of Medicine RR0319, Friends of the USC Parkinson’s
Disease Research Group including George and Mary Lou Boone, Walter and Susan
Doniger, Edna and John Ball, Team Parkinson, Team 4BA, the family of Don Gonzalez
Barrera, and the Roberto Gonzales Family Foundation.

Page 4

Chapter 1: Introduction
The work contained in this dissertation was completed in the hopes of furthering
our understanding of the two neurodegenerative disorders described below. I can only
barely grasp how awful it must be to suffer from one of these diseases, especially
Huntington’s.
If you’ve made it this “far” in, enjoy the footnotes thrown in throughout.

Parkinson’s disease
Overview
Parkinson’s disease (PD) is a progressive, neurodegenerative disorder that
affects 1% of the industrialized world’s population above the age of 60. It is the
second most prevalent neurodegenerative disorder after Alzheimer’s disease (de Lau
and Breteler, 2006) with diagnosis occurring at 50 years of age and older. In addition,
recent studies have shown that prevalence and incidence is increasing dramatically in
developing countries (Liu et al.) and as the aged-population continues to grow (Figure
1) . This is a growing problem which will lead to an increased social (familial) and
economic (health care, employment) burden. By 2040 it is estimated that this cost to
the United States will be upwards of $50 billion (Huse et al., 2005).
Page 5

Figure 1. Incidence of Parkinson’s disease by age and
gender, Kaiser Permanente, 1994–1995. (Van Den Eeden et
al., 2003)

The cause of the disease is not known in about 90% of cases with clearly
identifiable genetic cases being rare (5 to 15%) (Lesage and Brice, 2009). It is believed
that genetic susceptibility plays a minor role to environmental factors. Environmental
factors that have been linked to PD include pesticides, fungicides, herbicides, heavy
metals, industrial solvents, and air pollution (Goldman, 2014). Interestingly, cigarette
smoking is a protective factor against PD (de Lau and Breteler, 2006). Not as
interesting
9
, though still mildly interesting, is that caffeine and alcohol are also
protective factors.

9
How caffeine and alcohol contribute to numerous diseases has been studied. At least once a week on
the KTLA morning show (KM > CP) you’ll hear how one of them will either kill you faster or is made
directly from the fountain of youth.
Page 6

Motor movement dysfunction is the most recognizable feature of the disease
with the cardinal symptoms being postural instability, tremor, rigidity, and slow
movements (Jankovic, 2008). In addition to the cardinal symptoms, other symptoms of
the disease may manifest in time. These include
difficulty swallowing, a flexed/bent over posture, a
shuffling/festinating gait, and freezing.

Figure 2. Illustration of the Parkinson disease by Sir
William Richard Gowers from A Manual of Diseases of
the Nervous System in 1886 showing the characteristic
posture of PD patients.

Non-motor symptoms may also manifest. These include sleep abnormalities,
fatigue, autonomic disturbances, mood disorders,
10
and cognitive dysfunction (Zesiewicz
et al., 2006). Depression, anxiety, apathy, and hallucinations have been reported by
PD patients (Aarsland et al., 2007). These non-motor symptoms can be just as
debilitating as the motor symptoms and contribute to a decreased quality of life for PD
patients as well as their families.
The pathology of the disease has been well characterized. The main gross
pathological finding is the loss of dopaminergic cells of the substantia nigra pars
compacta. Up to 70% of these neurons are lost by the time of death. Histopathology
shows abnormal protein aggregates called Lewy bodies, loss of astrocytes, and

10
This statement was directly copied from the cited article. In the original segment there was no Oxford
comma and I have added one here.
Page 7

activated microglia. Pathophysiological findings show that with the loss of
dopaminergic neurons there is a disruption of dopamine modulated circuits of the basal
ganglia (Obeso et al., 2008). These circuits are the motor, oculo-motor, associative,
limbic, and orbitofrontal. In comparison to the other pathways, the motor pathway has
been studied more extensively because of the profound motor deficits observed in the
disease. However, in recent years the other pathways are gaining more attention due
to increased findings of subtle yet detrimental functioning of these pathways.

Figure 3. Unilateral reduction of tyrosine
hydroxylase (TH) staining in 6-OHDA rat
suggesting a reduction of dopamine in the
striatum (ST) and loss of dopaminergic cells of
the substantia nigra (SN) pars compacta.
(Ciesielska et al., 2011)

Direct/Indirect Motor Pathway Model
Of importance to understand in PD and
for potential treatment is how the brain is
connected and how these circuits attempt to
stay “healthy” in PD, and then their eventual failure. The dopaminergic neurons of the
substantia nigra pars compacta send afferents to the striatum (caudate/putamen) (Albin
Page 8

et al., 1989). These afferents terminate at either medium spiny neurons (MSNs) of the
direct or indirect pathway. The direct pathway MSNs project to the internal globus
pallidus/substantia nigra pars reticulate. The GPi/SNpr then projects to the thalamus,
which projects to the cortex, which projects to the striatum, completing our circuit. The
indirect pathway MSNs project to the external globus pallidus, which projects to the
subthalamic nucleus, which projects to the GPi/SNpr and this circuit follows the same
path back to the striatum as the direct pathway. The direct and indirect pathways play
opposing roles and have been labeled “go” and “no-go,” respectively (Bahuguna et al.,
2015). When direct pathway MSNs are activated there is an increased potential for
movement to occur. When indirect pathway MSNs are activated there is a decreased
potential for movement to occur. In PD, there is an asymmetric activation in the
striatum when DA input is lost. The indirect pathway becomes overactive and
contributes to the motor symptoms that are observed in PD.

Page 9

Figure 4. Schematic representation of the direct/indirect
pathway classical model in the physiological condition and in
Parkinson's disease. (Calabresi et al., 2014)

The classical model described in the previous paragraph has been challenged
recently with new evidence suggesting that a hard divide between the pathways is not
fully correct (Calabresi et al., 2014). Research has shown that direct pathway MSNs
have axon collaterals that not only synapse at the GPi, but also the GPe, suggesting that
the direct pathway may be able to regulate the indirect pathway. It has also been
discovered that heterodimers of D1/D2 receptors exist in MSNs. These DA-receptors
are usually found on direct MSNs (D1 receptor) or indirect MSNs (D2 receptor). With
the existence of these heterodimers, crosstalk between the pathways can occur at the
level of the striatum. Interneurons of the striatum have also been implicated in
allowing for regulation of the opposing pathway through the release of acetylcholine
and nitric oxide.

Page 10

Figure 5. Extremely detailed schematic of new basal ganglia
motor circuit pathway hypothesis. (a) Glutamatergic inputs
originating from both the cortex and the thalamus release
glutamate onto striatal neurons. Dopaminergic terminals,
originating from the substantia nigra pars compacta, release
dopamine onto MSNs and different subtypes of striatal
interneurons. In particular, three main subtypes of striatal
interneurons are implicated in the feed forward and parallel
control of striatal circuits. Cholinergic interneurons release
ACh acting on both presynaptic glutamatergic terminals and
postsynaptic MSNs; these interneurons also respond to
dopamine via D1/D5 and D2 receptors. NOS-positive
interneurons produce NO, acting as a retrograde messenger
as well as on MSNs facilitating LTD at the postsynaptic level.
Fast-spiking interneurons release GABA on MSNs, providing
a parallel inhibitory system that controls both direct and
indirect pathway MSNs. MSNs can express either D1-like or
D2-like receptors, as well as D1-D2 heteromeric receptors.
eCBs released from MSNs can act as retrograde messengers
on CB1 cannabinoid receptors located on glutamatergic
terminals. The induction of either LTP or LTD in MSNs
regulates the striatal control on output structures and motor
activation/inhibition. (b) The advanced phase of Parkinson's
disease is caused by a severe dopamine denervation that
leads to the complete loss of striatal synaptic plasticity.
Under this condition, both LTD and LTP of MSNs are lost. As
a consequence of the loss of these forms of plasticity,
variations of output signals from the striatum are absent and
no change in the motor state can be induced. Dopamine
denervation alters the physiological activity of striatal
interneurons as well as the neurochemical signals that
originate from these cells and influence the activity of MSNs.
A2AR, adenosine 2A receptor; CB1R, endocannabinoid
receptor; DA, dopamine; D1R and D2R, dopaminergic
receptors; M1R and M2/4R, muscarinic receptors. (Calabresi
et al., 2014)

MPTP Mouse Model of Parkinson’s Disease
MPTP was first linked to PD by Langston et al. (1983) after four heroin users
were admitted to local hospitals presenting with Parkinsonism. The young patients with
Page 11

Parkinsonism had used a bad batch of synthetic heroin which contained a byproduct
which was identified by Langston et al., as MPTP.
With the discovery of a peripherally injectable chemical that induces
Parkinsonism, scientific research began understanding the cellular and molecular
changes that MPTP elicited. Monkeys were the first animals to be used for this line of
research, but in 1984 (Heikkila et al.) discovered that intraperitoneal injections of MPTP
into a mouse produced similar histological changes seen in monkeys. This discovery
helped increase the gathering pace of knowledge in the model because it removed
constraints that are present when working with monkeys, e.g. cost and ethical issues.
Until recently rats have not been used because they have a great tolerance for the drug
and lesioning is hard to induce, but intranasal administration of MPTP has shown
promise (Prediger et al., 2006).
MPTP is lipophilic allowing for ease in crossing the blood-brain barrier. Once in
the brain MPTP is converted into MPP+ by monoamine oxidase-B in astrocytes. MPP+
then leaves astrocytes and is taken up by dopaminergic cells through the dopamine
transporter. Once inside a cell, MPP+ makes its way into mitochondria were it disrupts
the electron transport chain by disabling complex I. Shutting down this enzyme leads
to a loss of ATP production, increases in intracellular Ca
2+
, free radicals, and eventually
causes cell death.
MPTP can be administered in a chronic or acute schedule to bring about different
stages of PD. An acute schedule produces the greatest loss of DAergic neurons and is
used as a model for the later stages of PD. A lower dose, in a chronic administration
Page 12

schedule, kills off SNpc neurons at a slower rate and is used to model the progression
of the disease (Meredith and Rademacher, 2011).
Our lab utilizes an acute administration schedule (4 injections at 20 mg/kg, 80
mg/kg total) with the C57BL/6J and we (including Dr. Jakowec’s previous work) have
made the following discoveries using this model. MPTP administration leads to a 70%
loss of substantia nigra pars compacta neurons and 90% depletion of striatal dopamine,
similar to what is observed in humans (Jackson-Lewis et al., 1995). Dopaminergic
neurons of the ventral tegmental area are also lost, but not to the same degree of the
SNpc. Silver staining shows that dopaminergic neurons are in the terminal phase of
degeneration by post-injection day 4 and most likely die by necrosis instead of
apoptosis. α-synuclein protein concentrations are unchanged in this acute MPTP model
(Vila et al., 2000). Injection of human neural stem cells shows that these cells can
survive to at least 90 days post-injection (Liker et al., 2003). The transplanted cells can
migrate away from the injection site to areas such as the corpus callosum,
hippocampus, and other regions of the striatum. These cells may also become tyrosine
hydroxylase producing cells. The following paper (Jakowec et al., 2004b) looked at
multiple neurochemical markers and found some surprising results. Three to 4 months
after MPTP-induced striatal dopamine depletion, striatal dopamine levels return to pre-
lesion levels. This increase of striatal dopamine correlates with an increase in striatal
tyrosine hydroxylase protein which is post-translationally modified as evidence by a shift
in its isoelectric point. At 7 days post-lesioning, there is a decrease in the dopamine
transporter, however, by day 90 post-lesioning levels have returned to about 80%.
Page 13

Fisher et al. (2004) was a pivotal paper for the lab. This paper showed that 30 days of
treadmill exercise is able restore lost motor performance in achieved velocity and
endurance. In addition, it was found that exercise reduces dopamine transporter
expression, increases dopamine D2-receptor transcription, but does not alter tyrosine
hydroxylase protein levels. These finds suggest that MPTP-lesioned mice are better
able to utilize what little dopamine they have left by allowing dopamine a longer acting
duration at the synapse. The next paper detailed assessments of other neurochemicals
and motor task performance. It was found that striatal dopamine levels are not altered
by exercise, but that exercise was able to increase performance on the accelerating
rotarod (Petzinger et al., 2007). Further, fast-scan cyclic voltammetry showed a
stimulus-evoked increase in dopamine release. A battery of cognitive and psychiatric
testing revealed no changes in anxiety or depression, impairments in associative
memory and fear conditioning, and high-performance liquid chromatography showed
reduced serotonin levels (Vuckovic et al., 2008). VanLeeuwen et al. (2010a) looked at
how exercise could affect the AMPA receptor. Exercise increased GluA2 subunit
expression and no change in GluA1 subunit expression was observed. Further, there
were electrophysiological changes that occurred in medium spiny neurons. There was a
decrease in the size and amplitude of excitatory postsynaptic currents, as well as a loss
of polyamine sensitive inward rectification. Together these results suggest an increase
in utilization of the GluA2 AMPAR subunit. In another study, MPTP mice ran the same
amount on a voluntary running wheel, and had increased anxiety-like behavior (marble
burying) (Gorton et al., 2010). Exercise was able to reduce anxiety in a different
Page 14

assessment (open arm). In another paper by Dr. Marta, dopamine D2 receptor is
increased by exercise (Vuckovic et al., 2010). Decreased testosterone does not affect
tyrosine hydroxylase staining in the striatum. Working to further our understanding of
AMPAR subunit modulation, Dr. Natalie found that dopamine depletion leads to an
increase in GluA2-lacking AMPARs in indirect pathway medium spiny neurons and that
exercise reverses this effect (Kintz et al., 2013a). And that’s the last paper before my
published article which is reproduced for your viewing pleasure in Chapter 2.

Huntington’s disease
Overview
Huntington’s disease (HD) is a neurodegenerative disorder that presents with
psychological, psychiatric, and motor impairments. The motor symptoms are usually
the first impairments to be noticed. Motor symptoms include chorea
11
(involuntary,
random, jerky movements), rigidity, dystonia, impaired voluntary motor control, and
difficulty with swallowing, chewing, and speaking (Walker, 2007). The cognitive
symptoms are subtler, however research in the past decade has shown that cognitive
impairments occur well before the appearance of motor symptoms (van Duijn et al.,
2007). Further, the level of cognitive dysfunction may help predict the onset of motor
symptoms. Cognitive symptoms include depression (Folstein et al., 1983a), anxiety,
irritability, apathy, and obsessive-compulsive disorders (van Duijn et al., 2007). Other

11
derived from the Greek word χορεία, meaning dance
Page 15

psychological problems include being impulsive, erratic, lack of motivation, loss of
concentration, and difficulty communicating (Folstein et al., 1983b).

Table 1. Symptoms of Huntington’s disease. A better way
to communicate the symptoms of HD than writing a long
winded paragraph. (Pandey, 2013)

HD is caused by a CAG trinucleotide expansion in exon 1 of the huntingtin (HTT)
gene on chromosome 4 (MacDonald et al., 1993). This expansion codes for an
elongated poly-glutamine segment in the huntingtin protein that disrupts normal
huntingtin protein functioning. Full penetrance
12
occurs when the number of CAG
repeats is 40 or greater, 36 to 39 repeats has a reduced penetrance, 27-35 is classified
as intermediate, and 26 or less is classified as normal and will not get the disease
(Walker, 2007). Age of onset is inversely correlated to the number of CAG repeats
(Rosenblatt et al., 2006). Prevalence is estimated between 0.4 to 5.7 per 100,000
(Pringsheim et al., 2012). The autosomal dominant disorder is usually diagnosed in

12
Penetrance is the likelihood that the disease will manifest
Page 16

middle age from involuntary motor movements and cognitive dysfunction. (Martin and
Gusella 1986). Age of onset usually occurs between the ages of 35 and 42 (Brothers,
1964), with 3% of cases occurring before the age of 15 (Bittenbender and Quadfasel,
1962). The prodromal or premanifest period occurs 10-15 years prior to diagnosis and
onset of motor symptoms. During this period, individuals are mainly affected by
cognitive and behavioral problems.

Pathology
13

Gross pathology is characterized by a loss of neurons, initially of the striatum,
followed by the cerebral cortex, hippocampus, substantia nigra, and cerebellum
(Walker, 2007). This loss of neurons can lead to a whole brain weight decrease from
20 to 30% near the end of the disease. Staining of HD affected tissue reveals
additional pathology. There is an increase in astrocytes and activation of microglia
suggesting brain inflammation. There is also the presentation of nuclear inclusion
bodies. It is debated as to what role these nuclear aggregates play in the disease.
Some studies suggest that nuclear inclusions are detrimental to presenting cells, while
others argue that they may be a byproduct of a cell’s defense mechanism against the
disease (Rubinsztein and Carmichael, 2003). Another interesting finding about the
nuclear aggregates is that their presence does not correlate with disease staging
(Gutekunst et al., 1999). Neuronal loss and atrophy lag behind the observed
manifestations of the disease suggesting cellular and molecular dysfunction occurs first

13
Due to my poor writing style, this section continues on and on and on. If it doesn’t and seems well
organized, then my wife did an awesome job of editing it.
Page 17

(Vonsattel et al., 1985), however, striatal cell death may underlie motor deficits
(Montoya et al., 2006). One hypothesis is that mutant huntingtin is post-translationally
modified, resulting in poly-glutamine fragments being cleaved from the protein
(Rubinsztein and Carmichael, 2003). These fragments are then able to interfere with
other proteins due to hydrogen bonding
14
and begin to form protein aggregates
throughout the cell body becoming mechanical obstructions to cellular processes.
Mutated huntingtin also interferes with the normal functioning of the protein which is
not well understood and appears to be involved with many cellular functions.
Huntingtin is required for embryonic development as evidenced by homozygous mHTT
expression being embryonic lethal in mice (Nasir et al., 1995). Huntingtin is able to
modify transcription of other genes (BDNF) (Zuccato et al., 2001). It is found in close
proximity with vesicles and microtubules, and interacts with HIP1, a protein involved
with endocytosis suggesting a role in cellular transport (Velier et al., 1998; Hoffner et
al., 2002). Positron emission tomography has shown that brain metabolism is also
affected in the disease. There is reduced activation of the striatum and anterior motor
projections during motor tasks (Weeks et al., 1997). This decreased
15
metabolism is
correlated with cognitive deficits (Kuwert et al., 1990; Ciarmiello et al., 2006; Ciarmiello
et al., 2012). PET results have also shown decreases of up to 75% of striatal dopamine
receptors 1 and 2 (Sedvall et al., 1994; Turjanski et al., 1995; Ginovart et al., 1997).

14
Glutamine is a polar amino acid
15
When this word was written Noah Syndergaard just threw a 100 mph fastball to Chase Utley. That is
unreal velocity. And upon finishing that statement “Thor” fired a 100 mph strike 3. Crazy! This was the
game where Thor threw behind Utley and was immediately ejected from the game. Utley then went on
to hit to HRs and have 5 RBIs on the day.
Page 18

Transgenic Mouse Models
N-Terminal Transgenic Models:
The N-terminal transgenic mouse lines (e.g. R6/1, R6/2) were the first HD mouse
models to be created. In these lines, the 5’ end of the human HTT gene, which
contains the CAG expansion, is inserted into the mouse genome. From these lines it
was learned that mutant HTT was sufficient to elicit HD-like symptoms and deficits in
mice.
The R6/2 is first rodent model of HD that produces motor deficits by 6 weeks
and death occurring at 13 weeks (Mangiarini et al., 1996). These transgenic mice
express exon 1 of human HTT containing 115 to 150 CAG repeats. The HD features of
this mouse model include chorea-like motor disturbance, involuntary stereotypic
movements, seizures, and tremors. In this mouse line mutant Htt nuclear inclusions
were first observed, which were subsequently discovered in human patients (Landles et
al., 2010). The short-comings of this line include a very short window for intervention
and that neuronal cell loss is not observed (Turmaine et al., 2000). Because the
intervention period is so short, this mouse line may better represent early-onset HD
than the more common adult-onset.

Full-length Transgenic Models:
In these models, bacteria or yeast artificial chromosome of the full-length HTT
gene are transduced into a mouse line. These mouse lines exhibit a slower progression
of symptoms and time-to-death. Because these mice have full-length protein
Page 19

production, more targeted approaches for interventions can be used that have a greater
applicability to humans.
The bacterial artificial chromosome (BAC)-mediated transgenic mouse model
expresses full-length human huntingtin with 97 repeats of a mixed CAG/CAA (Gray et
al., 2008). This line is much slower progressing than the R6/2 model with mice living
until 18 months of age. During this time there are symptoms of motor deficits, synapse
dysfunction, with neuronal cell loss and atrophy. YAC128 lines express 100 to 126
glutamines.

Knock-in Models:
Homologous recombination has been utilized to create knock-in mouse models.
This allowed for precise addition of a specified number of CAG repeats into the mouse
HTT gene or replacement of the wild-type mouse allele with a mutant human allele.
Further, this technique allows for greater control over the number of copies of
huntingtin that end up inserted into the mouse genome. In the above models the
genome insertion number is not always one. These models also better mimic the allele
representation in humans, namely one wild-type and one mutant allele.
The CAG140 mouse model was created in the hopes of developing a model that
better represents the human condition and disease progression. Homologous
recombination was used to knock-in 140 CAG repeats (CAG)138CAACAG) into the mouse
huntingtin gene (Menalled et al., 2003). In this mouse model, there are no observed
motor deficits until 1 year of age (tremor, clasping), though, as early as 1 month of
Page 20

age, HD mice showed a decrease in motor activity in the open field and amount of
rearing. Also, voluntary wheel running is marginally decreased at 4 months and at 6
months greatly decreased (Hickey et al., 2008). We have also found that motor deficits
start to occur around the 12-month mark as evidenced in an analysis of gait that shows
a decrease in stride length (Stefanko et al., 2016). Further, we have also shown
cognitive and psychiatric problems begin as early as 4 months of age (Stefanko et al.,
2016). Others have shown anxiety-like behaviors to develop around 1.5 months in the
light-dark box assessment (Hickey et al., 2008). Taken together, this time course of
disease progression represents well the disease course in humans.
Antibody labeling in the CAG140 mouse against mutant huntingtin of various
brain regions show a progressive increase in the amount of aggregated htt in nuclei and
the neuropil. The striatum and olfactory tubercle show the earliest evidence of
aggregates at 1 month of age and staining intensity increasing to 6 months of age.
Sensory motor cortex, hippocampus, and olfactory bulb staining is observed at 2
months with a small increase in staining by 6 months. Nevertheless, it is still unclear
how the increasing concentrations of mutant huntingtin aggregates affects these mice.
The CAG140 mouse line isn’t perfect as the previous couple of paragraphs may
have conveyed. This mouse line is subject to germline and somatic CAG repeat
instability, requiring occasional genotyping of these mice. Also, the time-course for
tissue loss is not well known, except that it has been reported that 40% of striatal
neuron loss occurs by late-age (20-26 months old) along with tremor and gait
impairment.
Page 21

Dendritic Spine Density
The Dendritic Spine
16

Before we kick off talking about the density of dendritic spines, I should probably
talk just a little bit about what a dendritic spine is
17
. Oh, really quick, a dendrite is a
branch (think tree branch) that extends outward from the cell body of a neuron
18
.
Dendritic spines are small protrusions (less than 1 cubic micrometer) that extend from
the surface of a dendrite. There is a continuum of shapes they come in, but for
labeling purposes thin, mushroom, and stubby are used. These differing shapes
correlate to different ages in the growth and maturation of a dendritic spine.
Dendritic spines act
19
as a post-synaptic point of communication, receiving input
from an upstream neuron’s axon bouton. These mainly receive excitatory input from
other neurons, transducing that signal into excitatory postsynaptic potentials. This
transduction occurs through glutamate receptors (AMPAR or NMDAR) that allow the
cations sodium, potassium, and calcium to move through the pore formed between
their subunits. A dendritic spine may also be modulated by inhibitory input as well.

16
Beware that this section just rambles on and on and on
17
which may turn out to be quite difficult as Clayton Kershaw and the Dodgers try to take the series from
the Mets. Kershaw is on a Cy Young worthy run in this month of May.
18
I’m not quite sure why I added this sentence. I think it was to help describe these neuronal structures
for my mother, but then I start talking about other structures that I don’t elaborate on…
19
This is the 20,000 word added to this document.
Page 22

Dendritic Spine Density
Dendritic spine density is the quantity of dendritic spines per unit length of
dendrite
20
. This quantitative measure can be used to assess how well integrated a
given neuron is in a circuit and as a gauge of neuronal health. In a healthy brain, an
increase in dendritic spines is observed after training the brain, either with cognitive or
physical tasks (Comery et al., 1995; Spires et al., 2004). As such, an increase in
density is correlated with positive benefits learned in behavioral tasks. On the flip
side
21
, a decrease in dendritic spine density correlates with poor performance on
behavior tasks and in diseases and aging (Levine et al., 1986; Stephens et al., 2005).
In disease states, dendritic spine density can increase. In a mouse model of fragile-X
syndrome there is an increase in thin dendritic spines that never mature into permanent
spines indicating a disruption of synaptic development and elimination. Further
information on dendritic spine density, how it changes in disease states, and can be
modified with exercise is covered in the introductions of Chapters 2 and 3.

Exercise as an Intervention for Neurodegenerative disorders
Exercise is the Best
Exercise is one of the most effective and wide-reaching treatments for
many ailments. Exercise and regular physical activity is one of the most important
things people can do to improve or maintain their health, according to the Centers for

20
Oh no! Game of Thrones is coming on soon and I still have a bunch to write!
21
Ads for the new Ninja Turtles movie are flooding YouTube and this made me think of it.
Page 23

Disease Control and Prevention. Moderate-intensity physical activity
22
, such as brisk
walking (15-20 min/mile), water aerobics, or playing frisbee, for 150 min/week (30
min/business day) is the recommended minimum dosage. Undertaking a physical
activity regimen can positively affect multiple facets of one’s body including: reduction
and control of weight, reduced risk of heart and vascular diseases, reduced risk of type
2 diabetus
23
, improved bone and muscle health, improved mental functioning, and you
might even live longer.
Exercise and Parkinson’s Disease
The effects of various exercise regimens have been evaluated in the context of
Parkinson’s disease. An excellent review on the topic was co-authored by one of my
PIs, Dr. Giselle Petzinger (Speelman et al., 2011)
24
. Goodwin et al., (2008) performed a
meta-analysis on the effects of exercise on PD and found that exercise is able to
improve physical functioning, health-related quality of life, leg strength, balance and
walking, but did not see evidence for reducing fall risk or depression. Other reviews
have noted the following findings: improvement in physical performance and activities
of daily living (Crizzle and Newhouse, 2006), task specific tasks can improve balance,
gait, physical condition (Kwakkel et al., 2007), and reduction in gait hypokenesia
(Mehrholz et al., 2016).

22
At first glance, “moderate-intensity” seems like an ambiguous term, but actually has been defined by
public health experts as burning 3.5 to 7 calories per minute.
23
Isn’t it just a little funny when diabetes is pronounced the way I spelled it?
24
I can’t believe this review came out in 2011. That seems so far away.
Page 24

Exercise and Huntington’s Disease
The effects of exercise in Huntington’s disease have not been as well described
as in PD. However, some studies have been conducted that show exercise’s promise in
helping people with HD. Proprioceptive neuromuscular facilitation stretching can bring
about improvements in balance and gait (Mirek et al., 2015). A multidisciplinary study
utilizing exercise and occupational therapy reported that volumetric increases in gray
matter correlated with improved verbal learning and memory (Cruickshank et al., 2015).
Executive functioning can be improved with rhythmic exercises and drumming (Metzler-
Baddeley et al., 2014). Physical activity in the form of playing the video game Dance
Dance Revolution can lead to improvements of gait metrics (Kloos et al., 2013). The
studies briefly mentioned have all been smaller, pilot studies that have showed promise
in understanding the role exercise may play in HD.

Page 25

Chapter 2: Treadmill exercise prevents dendritic spine loss in
direct and indirect striatal medium spiny neurons in the 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of
Parkinson’s disease

This chapter was produced in collaboration with the following individuals:
Dr. Giselle M. Petzinger, Brian J. Leyshon, Dr. Garnik K. Akopian, Dr. John P. Walsh,
Matilde V. Hoffman, Dr. Marta G. Vučković, and Dr. Michael W. Jakowec.

Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative disorder
characterized by the loss of midbrain dopaminergic neurons leading to the depletion of
striatal dopamine (DA). Individuals with PD manifest both cognitive and motor deficits
including balance instability, gait dysfunction, slowness, tremor, and rigidity. While DA
replacement therapy remains a cornerstone of treatment, its efficacy diminishes over
time. Currently, there is no known cure for PD. A major interest of research in PD is to
find treatments that have the capacity to modify disease progression or better prevent
disease onset. For example, epidemiological studies have suggested that intensive
exercise, especially in males, over a lifetime can influence the occurrence of PD (Chen
et al., 2005). Over the last decade a number of investigators have demonstrated
exercise to the beneficial for the treatment of PD especially for the treatment of gait
Page 26

and balance (Petzinger et al., 2010; Speelman et al., 2011; Petzinger et al., 2013). In
animal models of PD, exercise has been shown to have the capacity to be both
neuroprotective, by preventing the loss of DA in lesion models (Gerecke et al., 2010),
and neurorestorative, providing enhancement in DA neurotransmission leading to
reversal of motor deficits (Fisher et al., 2004; Petzinger et al., 2007). These
observations, along with ongoing basic and clinical research, are beginning to
investigate the potential role of exercise in modifying disease progression in individuals
with PD as well as in animal models of DA-depletion. Towards this goal, a major focus
of our lab has been to elucidate the underlying molecular mechanisms by which
exercise affects neuroplasticity including synaptic structure and function within the
injured basal ganglia in neurotoxin animal models of PD.
In PD and in neurotoxin animal models, the depletion of striatal DA leads to
alterations in basal ganglia neurotransmission that manifest in a number of different
ways. For example, the striatopallidal (indirect) projection medium spiny neurons
(MSNs) become hyperactive and are thought to underlie the onset of akinesia (slowness
of movement) (Calabresi et al., 1997). In addition, corticostriatal projections targeting
striatal MSNs also display aberrant glutamatergic neurotransmission as demonstrated
through electrophysiological techniques (Cepeda et al., 1989; Pisani et al., 2005).
Therefore, it is not surprising that striatal MSNs manifest these changes in
neurotransmission by alterations in the dendritic spine number and dendritic
arborization, the primary morphological correlates of synaptic neurotransmission. For
example, studies using Golgi staining have shown a reduction in dendritic arborization
Page 27

and spine density in the caudate nucleus and putamen in tissues from patients with PD
(McNeill et al., 1988; Stephens et al., 2005). Similar findings have been made in both
the 6-hydroxydopamine (6-OHDA) rat and the 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) nonhuman primate models of DA-depletion where spine
density changes have been reported (Ingham et al., 1989; Ingham et al., 1993; Villalba
and Smith, 2010).
The purpose of this study was to determine if intensive exercise in the form of
treadmill running leads to a reversal of both dendritic spine loss and the reduction in
dendritic arborization in a mouse model of DA-depletion. This speculation is largely
based on the fact that we observe exercise enhanced alterations in both glutamate and
DA neurotransmission in the MPTP-lesioned mouse model (Kintz et al., 2013a). Since
dendritic spine density and branching are influenced by experience including
environmental enrichment and exercise in several regions of the mammalian brain we
were interested in knowing if exercise leads to a similar outcome (Comery et al., 1995;
Eadie et al., 2005; Leggio et al., 2005; Stranahan et al., 2007). We also took advantage
of the Drd2-eGFP-BAC transgenic mouse strain (Chan et al., 2012; Nelson et al., 2012)
in conjunction with biocytin injection methods that allowed us to delineate dopamine D1
receptor (DA-D1R)-containing MSNs and dopamine D2 receptor (DA-D2R)-containing
MSNs since some models of DA-depletion show preferential loss of dendritic spine
density selectively on DA-D2R-containing MSNs (Day et al., 2006). Utilizing the MPTP
mouse model of DA-depletion with either Golgi staining or biocytin labeling we
examined the effects of 6 weeks of intensive treadmill running on dendritic spine
Page 28

density and arborization of MSNs within the dorsolateral striatum, a region responsible
for motor control and a site where we have documented neuroplastic changes in
response to exercise (Petzinger et al., 2007).

Material and Methods
Animals
Twelve C57BL/6J young adult (8 to 10 weeks old) male mice from Jackson Labs
(Bar Harbor, Maine) and 52 young adult (8 to 10 weeks old) male Drd2-eGFP-BAC mice
(Tg(Drd2-EGFP)118Gsat/Mmnc) supplied from the Mutant Mouse Regional Resource
Center of NIH (MMRRC) program at the University of California, Davis (Gong et al.,
2003) were used for this study. We have established a colony of Drd2-eGFP-BAC mice
that have been backcrossed into C57BL/6J mice in our lab at least 10 times to enhance
genomic stability and validate comparison of outcomes with those derived from
C57BL/6J mice. Male hemizygous mice Drd2-eGFP-BAC mice continually backcrossed
onto C57BL/6J mice were used for this study. Both groups of C57BL/6J and Drd2-eGFP-
BAC mice were randomly assigned to one of four treatment groups including: (i) saline,
(ii) saline+exercise, (iii) MPTP, and (iv) MPTP+exercise. Mice were group housed with a
reverse light cycle (lights off 7 a.m. to 7 p.m.) with ad libitum access to food and water.
Experimental procedures were approved by the University of Southern California’s
Institutional Animal Care and Use Committee and conducted in accordance with the
National Research Council Guide for the Care and Use of Laboratory Animals (DHEW
Publication 80-23, 2011, Office of Laboratory Animal Welfare, DRR/NIH, Bethesda, MD).
Page 29

All efforts were made to minimize animal suffering and the number of animals used to
achieve statistical significance.
Some concern has been raised about the Drd2-eGFP-BAC mouse line and the
possibility that this BAC line does not express the physiology and associated behavior
seen in C57BL/6J mice (Kramer et al., 2011). However, several reports indicate that
Drd2-eGFP-BAC mice backcrossed to C57BL/6J mice display both normal behavior and
DA neurotransmission (Taverna et al., 2008; Chan et al., 2012; Nelson et al., 2012). In
our hands no differences were detectable between Drd2-eGFP-BAC mice and C57BL/6J
mice in maximum running velocity on the treadmill, normal striatal DA-levels, amount of
DA-depletion, or degree of substantia nigra pars compacta dopaminergic cell death
resulting from systemic injections of MPTP in our striatal lesioning protocol (Jackson-
Lewis et al., 1995; Kintz et al., 2013a).

MPTP-lesioning
Half of the C57BL/6J and half of the Drd2-eGFP-BAC mice were administered 4
intraperitoneal injections of MPTP at 20 mg/kg (free-base, Sigma, St. Louis, MO) at 2-
hour intervals. Remaining mice received intraperitoneal injections of saline. This
lesioning regimen results in 90-95% loss of striatal dopamine and 65 to 70% loss of
nigrostriatal dopaminergic neurons (Jackson-Lewis et al., 1995; Jakowec et al., 2004a).
Drd2-eGFP-BAC mice on the C57BL/6J background had DA-depletion and nigrostriatal
cell death indistinguishable from wild-type C57BL/6J mice (Kintz et al., 2013a). In this
study, striatal DA levels of Drd2-eGFP-BAC mice were assessed by high-performance
liquid chromatography (HPLC) analysis. HPLC analysis was conducted 5 days after
Page 30

lesioning, corresponding to the start of the treadmill exercise regimen, and at 42 days
after lesioning, corresponding to the completion of the treadmill exercise regimen.

Exercise Regimen
1 week before the start of the treadmill exercise regimen (2 days before MPTP
lesioning), 8 to 10 week old C57BL/6J and Drd2-eGFP-BAC mice that could maintain a
forward position on the 45 cm treadmill belt for 5 minutes at 5.0 m/min were randomly
assigned to the 4 groups to ensure that all animals performed similarly on the treadmill
task prior to MPTP-lesioning. The treadmill used in these studies was a Model EXER-6M
Treadmill manufactured by Columbus Instruments (Columbus, Ohio). A non-noxious
stimulus (metal beaded curtain) was used as a tactile incentive to prevent animals from
drifting back on the treadmill. The treadmill exercise regimen was conducted as
previously described (Fisher et al., 2004). Briefly, exercise was initiated 5 days following
MPTP or saline administration, a time point after cell death is complete, and continued 5
days/week for a total of 6 weeks of exercise (corresponding to a final of 42 days after
MPTP-lesioning). Exercised mice started at a velocity of 10.0 m/min, which increased to
24.0 m/min by the final week. These velocities were adjusted (either increased or
decreased) such that all mice within a group could maintain a forward position on the
treadmill for 75% of the running period. At the end of each week, the average
achieved velocities were calculated to produce a time course of improvement in running
velocity. All mice not exercised were given access to an immobile treadmill for an
equivalent amount of time as the running mice.

Page 31

Golgi Staining
A total of 12 C57BL/6J mouse brains were processed using the Golgi stain. After
the final session of running, the C57BL/6 mice were euthanized by cervical dislocation
and decapitated to allow extraction of the brain. Golgi-Cox staining (PK401, FD
NeuroTechnologies, Ellicott City, MD) was used to visualize neurons. Tissue was
impregnated for 2 weeks, sectioned in the coronal orientation in 120 μm thick sections,
and processed according to the manufacturer’s specifications. A representative Golgi
stained MSN and high magnification images of dendrites from our groups can be found
in Figure 3b and 3c.

Biocytin Labeling
Drd2-eGFP-BAC mice allowed for the differentiation of the two types of MSNs
located in the dorsolateral striatum; DA-D2R-containing MSNs express eGFP and
fluoresce green while DA-D1R-containing MSNs do not. 400 μm thick (spanning 0.20
mm to 0.60 mm anterior to bregma), coronal sections were obtained from a recently
sacrificed mouse. MSNs were filled with a solution containing 0.5% biocytin (B4261,
Sigma-Aldrich, St. Louis, Missouri) using a sharp micropipette. Diffusion of biocytin was
assisted with hyperpolarizing pulses for 20 minutes. Tissue sections were fixed in Tris-
buffered saline (TBS) containing 4% paraformaldehyde, pH 7.2, cryoprotected in TBS
with 30% sucrose, and then sectioned in a cryostat at 60 μm thickness. MSNs were
visualized using a protocol based on Wilson and Sachdev (2004) which utilizes the
DAB/HRP reaction (PK-4000, Vector Laboratories, Burlingame, CA). A representative
Page 32

DA-D1R MSN and high magnification images of dendrites from our groups can be found
in Figure 4a and 4b. A representative DA-D2R MSN and high magnification images of
dendrites from our groups can be found in Figure 4c and 4d.

Morphological Analyses
For Golgi stained tissue, MSNs located in the dorsolateral striatum between 0.14
mm to 0.62 mm anterior to bregma (Figure 3a), with the following properties were
analyzed based on (i) soma that was round or ovoid with a diameter of 11 to 20 μm, (ii)
three to eight visible primary dendrites, (iii) and sparse dendritic spines on primary
branches with an increase in dendritic spines from second or third order branches
onward (Rafols et al., 1989). High magnification image of representative dendrites is in
Figure 1c. Dendritic spine density was calculated by dividing a MSN’s traced dendritic
length by its dendritic spine count total along those traced dendrites. Dendritic length
was obtained by tracing dendrites of a MSN starting about 30 μm away from the soma,
a distance where spines are abundant, until the dendrite was cut off or at 15 μm from
the terminus, an area were spine density tapers off. Spine counts were obtained by
manually counting dendritic spines along the traced dendrites. This analysis was
performed on Golgi stained and biocytin filled MSNs. Complete tracings of biocytin filled
MSNs allowed for utilization of the Sholl analysis to determine branching characteristics
of a neuron at distances away from the soma (Sholl, 1953). Sholl analysis was only
performed on biocytin filled cells. All morphological data was collected at 80x or 120x
magnification with an Olympus Cooperation BX50 microscope (Shinjuku, Tokyo, Japan)
Page 33

using a QImaging QIClick camera (Surrey, British Columbia, Canada) to acquire images
that were analyzed using BIOQUANT Life Science 2011 V 11.2.60 (BIOQUANT Image
Analysis Corporation, Nashville, TN) or Fiji (Schindelin et al., 2012). Data were
gathered by an individual blinded to the experimental conditions.

Immunohistochemistry
12 Drd2-eGFP-BAC mice (3 per experimental group) were used for
immunohistochemical analysis. Immunohistochemistry was performed on 20 μm-thick
slices containing the striatum between 0.14 mm to 0.62 mm anterior to bregma. 8
tissue sections per animal were washed 3 times for 15 minutes in TBS pH 7.2 and
blocked for 1 hour at room temperature in 6% normal goat serum (NGS) and in TBS
with 0.05% Triton X-100 (TX). The following antibodies were used to detect PSD-95
and synaptophysin: mouse anti-PSD-95 IgG (1:2500, EMD Millipore, Billerica, MA),
rabbit anti-synaptophysin IgG (1:10000, Epitomics, Burlingame, CA), and biotinylated
goat anti-mouse IgG (1:5000, Vector Laboratories, Inc., Burlingame, CA). Antibody
specificity was validated by subjecting sections to the same IHC protocol but without
the addition of primary and/or secondary antibody. Primary antibodies were diluted in
TBS with 2% NGS and 0.05% TX. Following incubation in the primary antibody solution
overnight at 4°C, tissue was washed three times in TBS. The tissue was then incubated
in biotinylated antibody solution consisting of TBS with 2% NGS and 0.05% TX
overnight at 4°C and subsequently rinsed 3 times in TBS. From this point on the tissue
was protected from light. Sections were then incubated with Cy5-conjugated
Page 34

streptavidin (1:5000, Rockland Immunochemicals, Inc., Gilbertsville, PA) and Alexa
Fluor 594-conjugated goat anti-rabbit IgG (1:10000, Invitrogen, Grand Island, NY)
diluted in TBS with 3% NGS and 0.05% TX for 1 hour at room temperature. Sections
were mounted on gelatin-subbed slides, dried, and then coverslipped using Vectashield
Mounting Medium with DAPI (Vector Laboratories, Inc., Burlingame, CA).
Representative high magnification images of PSD-95 and synaptophysin staining can be
found in Figure 5a and 5b.
Immunofluorescence intensity was captured at 100x magnification with an
Olympus BX61 microscope (Shinjuku, Tokyo, Japan) equipped with a Disk Scanning Unit
(spinning disk confocal) and 100 W mercury light source (U-LH100HG) using a
Hamamatsu Photonics ORCA-R2 camera (Hamamatsu, Japan) and analyzed using
Metamorph Advanced 7.7.2.0 (Molecular Devices, LLC, Sunnyvale, CA). One z-stack was
taken from the dorsolateral striatum of each brain slice and deconvolved using
Metamorph Autoquant Deconvolution software to remove background noise. The frame
of best focus, defined as the frame in which the largest portion of the fluorescing areas
of the image is in focus, was then selected from the stack. In order to account for
striatal bundles, cell bodies, or unfocused areas, data was collected from three regions
of interest placed randomly on areas of fluorescing tissue. A threshold was
automatically performed using Metamorph’s internal algorithm, which thresholds for the
brightest pixels in the right tail of the pixel brightness histogram, leaving out the
majority of pixels in the image. Regional statistics were then collected via the
"Integrated Morphometry" function of Metamorph.
Page 35

Statistical analyses
SPSS Statistics 21 (IBM, Armonk, NY) was used to compare means of data
acquired. For HPLC analysis, a Mann-Whitney t-test was performed for comparisons
between MPTP versus saline treated mice: (1) day 5 post-lesion MPTP non-exercised,
(2) day 42 post-lesion MPTP non-exercised, (3) day 42 post-lesion MPTP exercise. A
one-way ANOVA was performed to compare differences in striatal DA across the MPTP
groups: (1) day 5 post-lesion MPTP, (2) day 42 post-lesion MPTP non-exercised, (3) day
42 post-lesion MPTP+exercise. Tukey’s honest significance test was used to perform
post hoc analyses of the dendritic spine density data. Fisher’s least significant
difference was used to perform post hoc analyses for data from the Sholl Analysis and
immunohistochemistry. Figures were made with GraphPad Prism 5 (GraphPad
Software, Inc., San Diego, CA) and GIMP (GNU Image Manipulation Program,
www.gimp.org)

Results
MPTP administration leads to loss of striatal DA
In the non-exercised Drd2-eGFP-BAC mice, striatal DA was significantly reduced
(p = 0.002) at 5 days post-lesioning by 88.2% in MPTP (43.8 ± 8.5 ng/mg protein ±
SEM) compared to saline mice (372 ± 38) and still significantly reduced (p = 0.001) at
42 days post-lesioning by 86.3% in MPTP (57.4 ± 5.7) compared to saline mice (419 ±
27). In the exercised Drd2-eGFP-BAC mice, striatal DA was significantly reduced at 42
Page 36

days post-lesioning by 86.0% in MPTP+exercise (50.7 ± 10.2) compared to
saline+exercise mice (361 ± 20). No significant difference in DA levels was observed
between day 5 post-lesion MPTP non-exercised, day 42 post-lesion MPTP non-
exercised, and day 42 post-lesion MPTP+exercise mice (F(2,19) = 0.594, p = 0.562).
No significant difference in DA levels was observed between day 5 post-lesion saline
non-exercised, day 42 post-lesion saline non-exercised, and Day 42 post-lesion
saline+exercise mice (F(2,20) = 0.846, p = 0.444). Importantly, Drd2-eGFP-BAC mice
and C57BL/6 mice were similarly sensitive to MPTP. HPLC analysis of C57BL/6 mice
lesioned with MPTP showed a decline in striatal DA compared to saline mice at similar
levels (VanLeeuwen et al., 2010a; Kintz et al., 2013a). Striatal dopamine levels
remained unchanged in both the saline and MPTP-lesioned groups during the 6-week
period of these studies.

Figure 1: MPTP-lesioning leads to early DA depletion which is
sustained out to 42 days post-lesion. Striatal dopamine levels of
Drd2-eGFP-BAC mice are reduced at 5 days after MPTP-lesioning.
This loss is sustained in these mice even after 30 days of treadmill
exercise (42 days post-lesion). DA levels of control mice were
unchanged after 30 days of treadmill exercise.
Page 37

Intensive treadmill exercise attenuates initial running speed deficit of MPTP-lesioned
mice
Similar to our previous findings (Fisher et al., 2004; Petzinger et al., 2007), our
recent cohort of MPTP+exercise mice needed 5 weeks of intensive treadmill running to
no longer be significantly different than saline+exercise mice in their achievable
treadmill velocity. The time course of improvement in running velocity is shown in
Figure 2. The week 1 assessment showed that MPTP+exercise mice (9.5 ± 1.0, meters
/ minute ± SEM) were significantly slower (p < 0.05) than saline+exercise mice (14 ±
1.4). However, the week 5 assessment showed that MPTP+exercise mice (21 ± 1.2)
were no longer significantly different (p > 0.05) than saline+exercise mice (23 ± 0.6)
and this restoration of motor ability persisted through week 6.

Figure 2: MPTP mice are able to achieve running velocities no
different than control mice after 5 weeks of treadmill exercise.
MPTP-lesioning initially leads to the failure of mice to achieve
running velocities similar to control mice. After 5 weeks of
Page 38

treadmill running there was no longer a significant difference
between the groups showing that MPTP mice are no longer
impaired. The asterisks represent statistical significance at p <
0.05 “*”.

Exercise increases MSN spine densities in MPTP-lesioned mice
A two-way ANOVA of Golgi stained MSNs within the dorsolateral striatum
revealed a statistically significant difference between the groups (F(3,49) = 23.3, p <
0.001) as well as significant effects of exercise (F(3,49) = 45.6, p < 0.001) and lesion
(F(3,49) = 15.78, p < 0.001), with an interaction between the variables (F(1,49) =
4.56, p = 0.038).
Post hoc analyses (Figure 3d) revealed the following differences between specific
treatment groups. MPTP-lesioning led to a significant decrease (p = 0.001) of total
dendritic spine densities of MSNs compared to saline controls (MPTP 5.47 ± 0.30, n =
9; Saline 7.45 ± 0.21, n = 10; mean spines / 10 μm ± SEM). Exercise led to a
significant increase (p < 0.001) in spine densities observed between the MPTP groups
(MPTP+Ex 8.36 ± 0.46, n = 10). Exercise also led to a significant increase (p = 0.004)
in spine densities between the saline groups (Saline+Ex 8.96 ± 0.23, n = 24). There
were no significant differences in total spine density between MPTP+exercise and either
of the saline groups (Saline p = 0.264; Saline+Ex p = 0.475).

Page 39

Figure 3: Analysis of Dendritic Spine Density with Golgi Staining.
Panel a shows a representative coronal section of the mid-
striatum processed using the Golgi stain. The outline (white)
delineates the dorsolateral striatum where MSN dendrites were
sampled. Panel b shows a representative high magnification
image of a single Golgi stained MSN indicating dendritic arbor and
dendritic spine density. The scale bar represents 25 microns. “cc”
– corpus callosum. Panel c shows representative dendrites from
MSNs of all four groups. Note the reduction in dendritic spine
density with MPTP and an increase in spine density with exercise.
The scale bar represents 10 microns. Panel d graphically depicts
the quantitative data from all four groups. Note the significant
increase in dendritic spine density with exercise in both the saline
and MPTP mice. The asterisks represent statistical significance at
either p < 0.05 “*” or p < 0.01 “**”.

Exercise increases dendritic spine density in DA-D1R- and DA-D2R-containing MSNs in
MPTP Mice.
Analysis of DA-D1R-containing MSNs
In order to further delineate exercise effects on dendritic spine densities in MSNs
of both the DA-D1R- direct and the DA-D2R-indirect pathways, individual MSNs were
electrophoretically filled with biocytin. A two-way ANOVA of DA-D1R-containing MSNs
revealed a statistically significant difference between the groups (F(3,10) = 8.41, p =
0.004) with an exercise effect (F(3,10) = 5.65, p = 0.039), a trend towards a lesion
Page 40

effect (F(3,10) = 3.93, p = 0.076), and an interaction between the variables (F(3,10) =
14.8, p = 0.003).
Post hoc analyses revealed (Figure 4e) the following differences between specific
treatment groups. MPTP mice (5.08 ± 0.61, n = 3; mean spines / 10 μm ± SEM) had
significant decreases (p = 0.021) in dendritic spine densities compared to saline-
injected controls (8.39 ± 0.35, n = 2). MPTP+exercise mice (8.62 ± 0.58; n = 5) had
significantly greater (p = 0.003) spine densities than MPTP mice. There was not a
statistically significant difference in spine densities between MPTP+exercise mice and
either of the saline groups (Saline p = 0.993; Saline+Ex 7.56 ± 0.29, n = 4, p = 0.434).
Also, there was no significant difference (p = 0.774) between the saline groups.

Analysis of DA-D2R-containing MSNs
A two-way ANOVA of DA-D2R-containing MSNs revealed a statistically significant
difference between the groups (F(3,16) = 4.49, p = 0.018) with a lesion effect (F(3,16)
= 4.81, p = 0.043), and an interaction between the variables (F(3,16) = 5.78, p =
0.029), but not an exercise effect (F(3,16) = 2.85, p = 0.111).
Post hoc analyses (Figure 4f) revealed the following differences between specific
treatment groups. MPTP mice (6.42 ± 0.86, n = 5; mean spines / 10 μm ± SEM) had
significant decreases (p = 0.032) in dendritic spine densities compared to saline mice
(9.25 ± 0.72, n = 4). MPTP+exercise mice (8.94 ± 0.35, n = 6) had significantly more
spines than MPTP mice (p = 0.034). There was no significant difference in spine
densities between MPTP+exercise mice and either of the saline groups (Saline p =
Page 41

0.984; Saline+Ex 8.81 ± 0.50, n = 6, p = 0.999). There was no significant difference
(p = 0.962) between saline mice groups.

Figure 4: Analysis of Dendritic Spine Density with Biocytin filled
DA-D1R or DA-D2R MSNs. Panel a shows a representative image
of a DA-D1R-containing MSN filled with biocytin and processed for
visualization. The scale bar represents 25 microns. Panel b shows
representative images of dendrites from biocytin-filled DA-D1R-
containing MSNs in each of the four groups. Note the reduction of
dendritic spines with MPTP and an increase in dendritic spine
density with exercise. The scale bar represents 10 microns. Panel
e graphically depicts the quantitation of these data showing a
significant reduction in dendritic spine density with MPTP and its
increase with exercise in DA-D1R-containing MSNs. Panel c shows
a representative image of a DA-D2R-containing MSN filled with
biocytin and processed for visualization. The scale bar represents
25 microns. Panel d shows representative images of dendrites
from biocytin-filled DA-D2R-containing MSNs in each of the four
groups. Note the reduction of dendritic spines with MPTP and an
increase in dendritic spine density with exercise. The scale bar
represents 10 microns. Panel f graphically depicts the
quantitation of these data showing a significant reduction in
dendritic spine density with MPTP and its increase with exercise in
DA-D2R-containing MSNs. The asterisks represent statistical
significance at either p < 0.05 “*” or p < 0.01 “**”.

Page 42

Exercise increases PSD-95 expression in MPTP-lesioned mice
A two-way ANOVA of PSD-95 expression in the dorsolateral striatum revealed a
statistically significant difference between the groups (F(3,8) = 14.2, p = 0.001) with
an exercise effect (F(3,8) = 24.0, p = 0.001), a lesion effect (F(3,8) = 18.2, p = 0.003),
but no interaction between the variables (F(3,8) = 0.246, p = 0.633).
Post hoc analyses (Figure 5c) revealed the following differences between specific
treatment groups. PSD-95 expression in MPTP mice (75.9 ± 1.63, n = 3; relative % of
saline ± SEM) was significantly lower (p = 0.010) than expression in saline controls
(100 ± 6.53, n = 3). MPTP+exercise mice (103 ± 5.42, n = 3) had significantly
greater (p = 0.005) expression than MPTP mice. Saline+exercise mice (122 ± 5.26, n
= 3) had significantly greater expression than all other groups (Saline p = 0.014; MPTP
p < 0.001; MPTP+Ex p = 0.028).

Exercise increases synaptophysin expression in MPTP-lesioned mice
A two-way ANOVA of synaptophysin expression in the dorsolateral striatum
revealed a statistically significant difference between the groups (F(3,8) = 5.97, p =
0.019) with an exercise effect (F(3,8) = 16.7, p = 0.004), but no lesion effect (F(3,8) =
1.00, p = 0.346), nor interaction of the variables (F(3,8) = 0.235, p = 0.641).
Post hoc analyses (Figure 5d) of synaptophysin expression in MPTP mice (89.8 ±
8.88, n = 3; relative % of saline ± SEM) was not significantly different (p = 0.324) than
saline-injected controls (100 ± 7.05, n = 3). However, exercise led to a significantly
greater (p = 0.012) expression of synaptophysin in MPTP+exercise (121 ± 2.47, n = 3)
Page 43

versus MPTP mice. MPTP+exercise mice had greater expression than saline-injected
controls that was trending towards significance (p = 0.061). Exercise also led to a
significantly greater expression (p = 0.035) in the saline-injected controls (Saline+Ex
125 ± 7.14, n = 3).

Figure 5: Immunohistochemical staining of synaptic
proteins PSD-95 and synaptophysin. Panel a shows
representative immunohistochemical staining of
dorsolateral striatal tissues from all four groups with an
antibody against PSD-95 protein (red) and the nuclear
stain DAPI (blue). The insert boxes in each panel are
images at a higher magnification showing distinct
puncta (synaptic staining). The scales bars in each set
of panels represent 10 microns. Quantitation of
immunofluorescence for PSD-95 is shown in Panel c
indicating a decrease in the number of synapses with
MPTP. With exercise there is a significant increase in
the number of synapses in both saline and MPTP mice.
Panel b shows representative immunohistochemical
staining of dorsolateral striatal tissues from all four
groups with an antibody against synaptophysin protein
(red) and the nuclear stain DAPI (blue). Quantitation of
immunofluorescence for synaptophysin is shown in
Panel d indicating a significant increase in the number
of synapses in exercised mice compared to their non-
exercised counterparts. The asterisks represent
statistical significance at either p < 0.05 “*” or p <
0.01“**”.

Page 44

Exercise increases dendritic arborization in MPTP-lesioned mice
Analysis of DA-D1R-containing MSNs
A two-way ANOVA of the distal sections (defined as 80 to 140 μm from the
soma) of the areas under the Sholl curves yielded a statistically significant difference
among the DA-D1R-containing MSN groups (F(3,19) = 4.68, p = 0.013). A statistically
significant effect of exercise was observed (F(3,19) = 8.67, p = 0.008). The effect of
lesion was trending towards significance (F(3,19), p = 0.093) and an interaction
between the variables was trending towards significance as well (F(3,19), p = 0.087).
The Sholl curve can be found in Figure 6a.
Post hoc analyses (Figure 6b) revealed that distal sections that MPTP+exercise
mice (151 ± 11.8, n = 5; arbitrary units ± SEM) had significantly greater arborization
than all other groups (Saline 100 ± 6.90, n = 4, p = 0.007; Saline+Ex 112 ± 6.46, n =
8, p = 0.014; MPTP 99.6 ± 8.82, n = 6, p = 0.003).
A two-way ANOVA of the proximal sections (defined as 10 to 70 μm from the
soma) did not reveal a significant difference among the groups (F(3,19) = 0.162, p =
0.921).

Analysis of DA-D2R-containing MSNs
A two-way ANOVA of proximal sections (defined as 10 to 70 μm from the soma)
of the area under the Sholl curves of DA-D2R-containing MSNs revealed statistically
significant differences between the groups (F(3,17) = 3.10, p = 0.05). There was a
statistically significant interaction between the variables (F(3,17) = 9.14, p = 0.008).
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We observed no significant effects of exercise and MPTP alone (Exercise F(3,17) =
0.21, p = 0.652; Lesion F(3,17) = 0.001, p = 0.979). The Sholl curve can be found in
Figure 6d.
Post hoc analyses (Figure 6e) of proximal sections revealed that saline mice (100
± 9.91, n = 5; arbitrary units ± SEM) had greater arborization than MPTP mice (75.0 ±
13.3, n = 4) that was trending towards significance (p = 0.059). MPTP+exercise mice
(104 ± 8.09, n = 5) had significantly greater (p = 0.034) arborization that MPTP mice.
Saline mice also had greater arborization than exercised controls (Saline+Ex 73.7 ±
4.94, n = 7) that was trending towards significance (p = 0.068).
A two-way ANOVA of the distal sections (defined as 80 to 140 μm from the soma) did
not yield a significant result (F(3,17) = 0.702, p = 0.564).

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Figure 6: Sholl analysis of Dendritic Arborization. Panel a shows
quantitation of the mean number of intersections from the soma
in biocytin filled DA-D1R-containing MSNs from all four groups.
Panel b shows area under the curve quantitation of DA-D1R-
containing MSNs for proximal (10 to 70 μm) and distal (80 to 140
μm) sections of the Sholl analysis. Note significant differences of
the distal section between MPTP+exercise mice and all other
groups. Panel d shows quantitation of the mean number of
intersections from the soma in biocytin filled DA-D2R-containing
MSNs from all four groups. Panel e shows area under the curve
quantitation of DA-D2R-containing MSNs for proximal (10 to 70
μm) and distal (80 to 140 μm) sections of the Sholl analysis. Note
a significant increase in the proximal section of MPTP+exercise
mice compared to MPTP mice. Panel c shows the quantitative
data for the longest dendrite of DA-D1R-containing MSNs as an
indicator of dendritic pruning. There is no difference in longest
dendrite length in DA-D1R-containing MSNs. Panel f shows the
quantitative data for the longest dendrite in DA-D2R-containing
MSNs. Note MPTP leads to pruning of the dendritic arbor in DA-
D2R-containing MSNs. The asterisks represent statistical
significance at either p < 0.05 “*” or p < 0.01“**”.

Exercise does not offset distal pruning of DA-D2R MSN dendrites in MPTP-lesioned mice
A two-way ANOVA of DA-D2R-containing MSNs of MPTP mice did not show a
significant difference between the groups (F(3,18) = 2.06, p = 0.141). However, the
ANOVA revealed a statistically significant effect of lesion (F(3,18) = 5.97, p = 0.025).
Post hoc analyses (Figure 6f) showed that MPTP mice (136 ± 6.0, n = 7; μm ± SEM)
had significantly shorter (p = 0.044) dendrites than saline-injected controls (Saline 157
± 5.4, n = 4). There were no other significant differences between the groups. Also,
there were no significant differences in longest dendrite lengths among the DA-D1R-
containing MSN groups (Figure 6c).

Page 47

Discussion
Intensive treadmill exercise results in an increase in overall dendritic spine
density within the striatum of the MPTP mouse model. DA-depletion led to a decrease
in spine density of both DA-D1R- and DA-D2R-containing MSNs of the direct and indirect
pathways, respectively, while exercised mice did not show this loss in either pathway.
Treadmill running has been shown to increase dendritic spine density within the
striatum in a hemorrhagic stroke model in the rodent (Takamatsu et al., 2010);
however, to the best of our knowledge this is the first report of an exercise induced
increase in spine density in an animal model of PD. Increased striatal dendritic spine
density has also been reported in the context of environmental enrichment in non-
injured, intact animals. Specifically, Comery et al. (1995) reported that rodents
exposed to 30 days of a complex environment demonstrated a 30% increase in spine
density of MSNs. In this report we also observed an increase in dendritic spine density
in striatal MSNs in saline+exercise mice supporting the fact that exercise has a positive
effect on basal ganglia connectivity in the healthy and intact brain. In addition, exercise
and/or environmental enrichment have been shown in healthy animals to increase spine
density in extra-striatal regions including the CA3 hippocampal neurons, layer III
pyramidal of the cortex, and cerebellar Purkinje cells (Comery et al., 1995; Eadie et al.,
2005; Leggio et al., 2005; Stranahan et al., 2007).
We found that the effects of exercise on spine density were associated with
increased expression of two synaptic proteins, PSD-95 and synaptophysin, in MPTP
mice. PSD-95, a structural anchoring protein, is localized to the post-synaptic terminal,
Page 48

while synaptophysin, a vesicle associated protein, is localized to the pre-synaptic
terminal. Several studies have reported that synaptogenesis occurs in the context of
exercise and motor skill learning (Black et al., 1990; Takamatsu et al., 2010; Pagnussat
et al., 2012). In our study the exercise induced increase in PSD-95 and synaptophysin
expression along with the increase in dendritic spine density is consistent with increased
synaptogenesis in the dorsolateral striatum. This may then underlie improved motor
performance with exercise that we have previously reported in the MPTP-lesioned
mouse model (Fisher et al., 2004; Petzinger et al., 2007; VanLeeuwen et al., 2010a;
Kintz et al., 2013a).
Sholl analysis, a measure of neuronal dendritic arborization, revealed that
exercise led to an increase in arborization of both pathways in MPTP-lesioned mice.
These effects were specific to either proximal (DA-D2R) or distal (DA-D1R) segments of
the dendritic field. In addition, there was no effect of exercise on arborization in saline
animals. No effect of exercise was observed on the longest dendritic length in any
group. Consistent with our findings, exercise increased arborization of striatal neurons
in a model of intracerebral hemorrhagic stroke with no effects on arborization in sham
animals (Takamatsu et al., 2010). Interestingly, exercise effects on spine density and
arborization within the hippocampus are similar to those observed within the striatum.
Specifically, while exercise led to a general and widespread increase in spine density
within many areas of the hippocampus, the exercise effects on gross morphological
changes to dendritic arborization were limited to a small number of regions (Stranahan
et al., 2007).
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Dendritic spine loss after DA-depletion has been reported to occur either
exclusively in DA-D2R-containing MSNs in rodents or in both DA-D1R- and DA-D2R-
containing MSNs in monkeys. For example, Day et al. (2006) reported a selective loss
of spine density in DA-D2R-containing MSNs of the indirect pathway in both reserpine
and 6-hydroxydopamine (6-OHDA) rodent models of DA-depletion. Our findings differ
from Day et al. (2006) in that we observed spine density changes in both DA-D1R and
DA-D2R-containing MSNs utilizing the MPTP-lesioned mouse. However, our findings are
in agreement with Villalba et al. (2010) where loss of spine density in both DA-D1R- and
DA-D2R-containing MSNs were observed several months after MPTP-lesioning in non-
human primates. Differences that may account for variability between studies could be
due to a number of parameters including the neurotoxin lesioning paradigm (type of
toxin, dosage, and route of administration), the time between lesion and analysis of
tissue, and the animal model with respect to age, sex, or species.
In this study, we observed that DA-depletion led to a decrease in dendritic spine
density 42 days after MPTP administration. Published studies using a number of DA-
depleting agents suggest that spine loss may take several days to weeks to occur after
lesioning: 6-OHDA (12 days) (Ingham et al., 1989), reserpine (5 days) (Day et al.,
2006), MPP
+
(2 weeks) (Neely et al., 2007). In light of these reports and our current
findings we hypothesize that the most parsimonious mechanism by which exercise may
alter dendritic spine density is by reversing spine loss. Since DA-depletion leads to the
loss of dendritic spines and our exercise protocol was started after MPTP-induced cell
death and spine loss had occurred, exercise may then reverse this effect by promoting
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new spine formation. This interpretation is based on our observation that exercise
promotes dendritic spine formation in both saline+exercise and MPTP+exercise mice. In
future studies we propose to investigate mechanisms involved in dendritic spine
formation including a precise analysis of spine density in a time course fashion as well
as investigate underlying dynamic changes (nascent versus mature) that may occur in
models of DA-depletion and exercise.
There are a number of potential mechanisms by which exercise may modulate an
increase in spine density within the striatum. One mechanism could be through
neurotrophic factors (Cotman and Berchtold, 2002). Exercise has been shown to
elevate the expression of a number of neurotrophic factors, including brain-derived
neurotrophic factor, and these factors are known to regulate spine formation.
Alternatively, exercise effects on spine density may be through glutamate modulation
(Calabresi et al., 1997). Exercise has been reported to alter glutamate
neurotransmission, including AMPA (alpha-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid) receptor subtypes in DA-depleted rodent models. Exercise
induced alterations in glutamatergic neurotransmission may serve to mitigate
excitotoxicity due to DA-depletion. Other receptors that are known to be modulate
spine loss in the context of DA-depletion, such as the L-type calcium channel may play
a role in exercise mediated effects on spine density (Day et al., 2006). Finally, exercise
may lead to changes in a number of factors affecting general brain health including the
activation of astrocytes or microglia, alterations in the permeability of the blood brain
barrier, increased vasculature (angiogenesis), and blood flow, all of which can influence
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neurotrophic factor expression or delivery and provide an environment promoting spine
growth and spine maintenance (Li et al., 2005; Yang et al., 2007; Al-Jarrah et al., 2010;
Kawanishi et al., 2010; Bernardi et al., 2013).

A question not examined in this study, though highly relevant, is how does exercise
modulate MSN dendritic spine morphology with dopamine-depletion? To our
knowledge, no studies have investigated the dynamics of spine morphology on striatal
MSNs in the context of exercise. However, other groups have reported the effects of
exercise on spine morphology in other regions of the brain. Gonzalez-Burgos et al.
(2011) reported that in non-injured adult rat cerebellar Purkinje cells, 4 weeks of
treadmill running led to increased quantities of stubby, mushroom, and wide dendritic
spine subtypes compared to non-exercised controls. In the hippocampus, Stranahan et
al. (2007) reported that average dendritic spine surface area of CA1 pyramidal neurons
decreased in rats that had access to a running wheel compared to sedentary controls,
suggesting that exercise led to an increase in filopodia-like spines. As a future study we
plan to determine more precisely potential morphological changes that may take place
on dendritic MSNs and to determine if any changes underlie the behavioral benefits
seen with intensive exercise in the dopamine-depleted striatum.
A limitation of our study was that we did not observe an increase in the dendritic
spine density in saline+exercise compared to saline sedentary groups using biocytin
labeling. This is in contrast to analysis of Golgi stained MSNs where we observed an
exercise induced increase in spine density in intact saline animals. While the
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discrepancy between these two labeling methods is not clear one potential reason may
be due to differences in the mechanisms by which Golgi impregnation and biocytin
labeling differ. For example, Golgi impregnation requires tissues to be prefixed in
formaldehyde (or a related fixative) prior to deposition of the silver chromate
precipitate. This is in contrast to biocytin filling of striatal MSNs after
electrophysiological recording or patch clamping where biocytin (a 372 molecular weight
amide of biotin conjugated to L-lysine) diffuses into a live cell from the recording
pipette. A number of studies have reported differences in neuron morphology when
comparing biocytin labeling and Golgi staining. For example, Coleman et al. (1992)
using biocytin infusion showed extensive dendritic morphology of the immature cat
lateral geniculate nucleus which was in contrast to reports by Mason (1983) who
indicated a lack of dendrites using Golgi impregnation in this same region. Similar
discrepancies have been reported in a number of brain regions using these two stains
to reveal dendritic morphology (Conley and Wilson, 1992; Bannister and Larkman,
1995; Li et al., 2012).

Conclusion
In conclusion, this study demonstrates an exercise induced increase on dendritic spine
density and arborization in an animal model of PD. Increased expression of proteins
involved in synaptic connectivity observed in the MPTP mouse model may contribute to
exercise related benefits in motor skill learning and performance in the DA-depleted
state. Future studies will examine if changes in spine morphology are associated with
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alterations in the electrophysiological properties of striatal MSNs and the exercise-
dependent mechanisms involved in spine maintenance and/or formation. In addition,
findings from these studies may be translated to clinical exercise studies in individuals
with PD that investigate exercise related alterations in motor circuit connectivity within
the dorsal basal ganglia using fMRI. Finally, exercise induced alterations in spine
density help support the potential role of exercise in modifying disease progression in
individuals with PD through neuroplastic changes including synaptogenesis and motor
circuitry.

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Chapter 3: The effect of treadmill exercise on dendritic spine
density in the CAG140 knock-in mouse model of Huntington’s
disease

This chapter was produced in collaboration with the following individuals:
Dr. Daniel P. Stefanko, Damaris N. Garcia, Dr. Giselle M. Petzinger, and Dr. Michael W.
Jakowec.

Introduction
Huntington’s disease (HD) is an autosomal dominant, neurodegenerative disease
that presents with psychological, psychiatric, and motor dysfunction and impairments.
A CAG (cytosine-adenine-guanine) expansion in the huntingtin gene is the cause of the
disease and full penetrance occurs when the number of repeats is 40 or greater. Life
expectancy is approximately 20 years from the time of diagnosis (Folstein, 1989) which
typically occurs in middle age, with onset being inversely related to increased numbers
of glutamine residues.
Currently, there is no cure for the disease with treatment consisting of managing
symptoms. Motor behavior difficulties can be attenuated with intervention from
physical, occupational, and speech therapists. Some psychiatric symptoms can be
treated with medications, however other cognitive issues (executive function,
processing speed, attention) are unaffected by current therapies and none are able to
slow or stop the progression of the disease.
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Exercise has been shown to be beneficial in other neurodegenerative diseases
(Alzheimer’s, Parkinson’s) and cases of brain insult (stroke, traumatic brain injury) in
both humans and experimental animal models. We have recently shown that exercise
is able to delay cognitive and mood disturbances in the CAG140 mouse model of HD
during the disease’s prodromal phase (Stefanko et al., 2016). The underlying cellular
and molecular changes producing behavioral improvements in CAG140 mice remains
unknown. In this study, we assess how exercise effects dendritic spine density in
neocortical and neostriatal regions in the CAG140 mouse model of HD.

Methods and Materials
Animals and Groups
The CAG140 knock-in mouse model of Huntington’s disease was used. This KI
mouse contains a chimeric mouse/human exon 1 with 140 CAG repeats inserted into
the murine huntingtin gene (Menalled et al., 2003). Mice were used from our in-house
colony that is maintained through heterozygous pairings and annual backcrossing with
C57BL/6J mice.
Male, homozygous CAG KI and wildtype littermates were randomly assigned to
one of four groups: wildtype with no exercise (WT-NoEx); wildtype with exercise (WT-
Ex); CAG140 KI with no exercise (HD-NoEx); or CAG140 KI with exercise (HD-Ex). These
mice were then exercised for either 4 or 6 months. 4-month animal numbers were WT-
NoEx n = 9, WT-EX n = 10, HD-NoEx n = 10, HD-Ex n = 7. 6-month animal numbers
were WT-NoEx n = 5, WT-Ex n = 4, HD-NoEx n = 5, HD-Ex n = 3.
Page 56

Mice were group housed with a reverse light cycle (lights off from 7 a.m. to 7
p.m.) and had ad libitum access to food and water. Experimental procedures were
approved by the University of Southern California’s Institutional Animal Care and Use
Committee (IACUC) and were conducted in accordance with the National Research
Council Guide for the Care and Use of Laboratory Animals (DHEW Publication 80-23,
2011, Office of Laboratory Animal Welfare, DRR/NIH, Bethesda, MD). All efforts were
made to minimize animal suffering and to reduce the number of animals used to
achieve statistical significance.

Exercise Regimen
28 day old mice were exercised on a motorized treadmill (EXER-6M, Columbus
Instruments, Columbus, Ohio) 40 minutes/day, three days/week, following our
previously reported regimen (Stefanko et al., 2016). During the first week, mice were
introduced to the treadmill at a gentle pace of 8 meters/minute and carefully observed
to ensure they could tolerate the exercise. The second week velocity was 10 m/min
and over the next 4 months the treadmill speed was gradually increased to a final
velocity of 20 m/min. Mice in the 6-month group were exercised for an additional 2
months at 20 m/min.

Golgi Staining
At the end of the four-month or six-month exercise regimen, mice were
sacrificed and their brains extracted. Neurons were stained using the Golgi-Cox method
Page 57

(PK401, FD NeuroTechnologies, Ellicott City, MD) following the manufacturer’s
instructions. Brains were impregnated for one week and coronally sectioned (100 um
thick) using a Vibratome 1000 Plus Sectioning System.

Dendritic Spine Density
Dendritic spine density was quantified in the following locations and types of
neurons: prelimbic cortex (+2.0 bregma) layers 2/3 and 5, apical dendrites of pyramidal
neurons; anterior striatum (+1.1 bregma), dorsolateral, dorsomedial, and ventral,
medium spiny neurons; middle striatum (-0.1 bregma), dorsolateral, dorsomedial, and
ventral, medium spiny neurons; hippocampus (-2.8 bregma) CA1, apical and basal
dendrites of pyramidal neurons; visual cortex (-2.8 bregma), apical dendrites of
pyramidal neurons. These regions were chosen to coincide with blood flow changes
that were discovered between groups (Wang et al., 2016) with the visual cortex acting
as a control region, as reported findings from other HD papers.
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Figure 6.2. Locations were dendritic spine density was
assessed. Panel A is at +1.98 mm bregma, The grey oval
is the prelimbic cortex. Panel B is the anterior striatum at
+1.10 mm bregma. The red circle is the dorsomedial
region, the green circle is the dorsolateral region, the blue
circle is the ventral region. Panel C is the middle striatum
at -0.1 mm bregma. The circles are the same regions as in
Panel B. Panel D contains the CA1 region of the
Hippocampus, maroon oval and the primary visual cortex,
grey oval. Images are from The Mouse Brain (2001) by
Paxinos and Franklin.

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Figure 6.3. Representative Golgi method stained slices
corresponding to the schematics in Figure 6.3

Western Immunoblotting
Western immunoblotting was used to determine the relative expression of
proteins within the dorsal striatum, ventral striatum, and prefrontal cortex (N = 9
animals per group per time point). Tissue was dissected from the dorsal and ventral
striatum and was immediately frozen and stored at -80
o
C until use. Tissue samples
were disrupted by sonication in 300 μl of homogenization buffer (50mM Tris pH 7.4,
150 mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate, and 1/100 dilution protease
inhibitor cocktail (Cat#: 539134; Calbiochem/EMD Millipore, Billerica, MA)). Protein
Page 60

concentration was determined by the BCA method (Pierce; Rockford, IL). The
immunoblotting technique used was previously described (Jakowec et al., 2004b;
VanLeeuwen et al., 2010b; Vuckovic et al., 2010; Kintz et al., 2013b) with slight
modifications. Briefly, samples were separated on a precast PAGEr Tris-glycine gel
(Lonza Rockland Inc; Rockland, ME) and were transferred to nitrocellulose membranes
in Towbin buffer. Nitrocellulose membranes were washed with PBS, and then blocked in
LI-COR blocking buffer (LI-COR, Inc.; Lincoln, Nebraska) for one hour at room
temperature. Membranes were then exposed to the primary antibody solution overnight
at 4
o
C. Primary antibodies used in this study include: PSD-95 (1:500
25
, Cat#: SC-
56412
26
, Santa Cruz Biotechnology; Dallas, TX), and synaptophysin (1:500
27
, Cat#: SC-
654
28
, Santa Cruz Biotechnology; Dallas, TX). Membranes were washed three times in
PBS+0.05% Tween, and exposed to the secondary antibody solution for two hours at
room temperature. Secondary antibodies used in this study include: Goat anti-Mouse
IgG IRDye 800 CW (1:10,000, Cat#: 926-32210, LI-COR Inc.; Lincoln, Nebraska) and
Goat anti-Rabbit IgG IRDye 680 LT (1:15,000; Cat#: 926-68021, LI-COR Inc.; Lincoln,
Nebraska). Nitrocellulose membranes were scanned with an Odyssey Infrared Imaging
System 3.0 (LI-COR, Inc.; Lincoln, Nebraska). Densiometric quantification of
immunopositive bands was determined with Image Studio Software Version 3.1.4 (LI-
COR, Inc.; Lincoln, Nebraska), and expressed as relative optical density (O.D.). The
O.D. of each band was quantified relative to the O.D. of Beta-actin, serving as the

25
Real concentration is unknown at this time
26
Catalog number is unknown
27
Real concentration is unknown
28
Catalog number is unknown
Page 61

protein loading control. For comparison across groups, the relative O.D. levels for each
sample was compared to the averaged value of WT sedentary mice analyzed on the
same blot and normalized. Data are presented as mean ± SEM.

Statistical Analyses
Two-way ANOVAs followed by post hoc analyses using Tukey’s honest
significance test were used to compare means (SPSS Statistics 21, IBM, Armonk, NY).
Figures were made with GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA)
and GIMP (GNU Image Manipulation Program, www.gimp.org) and Paint (Microsoft
Corporation, Redmond, WA

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Results

Figure 7. Graphical representation of dendritic spine
density. A significant increase in dendritic spine density was
observed on apical dendrites of Layer 2/3 prelimbic cortex
pyramidal neurons. Data is mean spines / 10 um ± SEM. “*
p < 0.05”

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Dendritic Spine Density – 4 months
Prelimbic Cortex – Layers 2/3
A two-way ANOVA of apical dendritic spine density of pyramidal neurons showed
a statistically significant difference between the groups (F(3,32) = 4.45, p = 0.010) and
an exercise effect (F(3,32) = 11.4, p = 0.002). There was no interaction between the
variables (F(3,32) = 1.17, p = 0.287) nor a genotype effect (F(3,32) = 0.394, p =
0.535).
Post hoc analyses showed a statistically significant difference (p = 0.024)
between WT-Ex mice (7.76 ± 0.36, n = 10; spines / 10 um ± SEM) and HD-NoEx mice
(6.19 ± 0.33, n = 10). Exercise led to a statistically significant increase (p = 0.022) in
dendritic spines in HD-Ex mice (7.94 ± 0.49, n = 7) compared to HD-NoEx mice. WT-
NoEx mice (6.86 ± 0.40, n = 9) were not different from the other groups.

Prelimbic Cortex – Layer 5
A two-way ANOVA of apical dendritic spine density of pyramidal neurons did not
show a difference between the groups (F(3,32) = 2.46, p = 0.081). Dendritic spine
densities were as follows: WT-NoEx (6.35 ± 0.40), WT-Ex (7.76 ± 0.36), HD-NoEx
(6.19 ± 0.33), and HD-Ex (7.94 ± 0.49).

Striatum – Anterior, Dorsolateral
A two-way ANOVA of medium spiny neuron dendritic spine density showed a
statistically significant difference between the groups (F(3,32) = 9.69, p < 0.001) with
Page 64

an interaction between the variables (F(3,32) = 18.7, p < 0.001), an exercise effect
(F(3,32) = 7.35, p = 0.011), but not a genotype effect (F(3,32) = 1.318, p = 0.259).
Post hoc analyses showed a statistically significant increase of dendritic spine
density in HD-NoEx mice (8.96 ± 0.20, p = 0.002) and WT-Ex mice (9.27 ± 0.20,
p < 0.001) compared to WT-NoEx mice (7.88 ± 0.14). HD-Ex mice (8.64 ± 0.24) were
not different from the other groups.

Striatum – Anterior, Dorsomedial
A two-way ANOVA of medium spiny neuron dendritic spine density showed a
statistically significant difference between the groups (F(3,32) = 6.15, p = 0.002) with
an interaction between the variables (F(3,32) = 10.1, p = 0.003), an exercise effect
(F(3,32) = 6.99, p = 0.013), but not a genotype effect (F(3,32) = 0.101, p = 0.752).
Post hoc analyses showed that exercise led to a statistically significant increase
(p = 0.001) of dendritic spine density in WT-Ex (8.60 ± 0.21) compared to WT-NoEx
mice (7.36 ± 0.20). HD-NoEx mice (8.1 ± 0.21) and HD-EX mice (7.99 ± 0.22) were
not different from the other groups.

Striatum – Anterior, Ventral
A two-way ANOVA of medium spiny neuron dendritic spine density showed a
statistically significant difference between the groups (F(3,32) = 2.93, p = 0.049) with
an exercise effect (F(3,32) = 6.13, p = 0.019). There was not an interaction of the
Page 65

variables (F(3,32) = 1.82, p = 0.186) nor a genotype effect (F(3,32) = 0.568,
p = 0.457).
Post hoc analyses showed that exercise led to a statistically significant increase
(p = 0.040) of dendritic spine density in WT-Ex mice (9.38 ± 0.22) compared to WT-
NoEx mice (8.29 ± 0.35). HD-NoEx mice (8.89 ± 0.22) and HD-Ex mice (9.21 ± 0.35)
were not different from the other groups.

Striatum – Middle, Dorsolateral
A two-way ANOVA of medium spiny neuron dendritic spine density did not show
a difference between the groups (F(3,32) = 0.471, p = 0.704). Dendritic spine
densities were as follows: WT-NoEx (8.19 ± 0.31), WT-Ex (8.69 ± 0.26), HD-NoEx
(8.46 ± 0.28), and HD-Ex (8.43 ± 0.42).

Striatum – Middle, Dorsomedial
A two-way ANOVA of medium spiny neuron dendritic spine density did not show
a difference between the groups (F(3,32) = 2.33, p = 0.093). Dendritic spine densities
were as follows: WT-NoEx (8.05 ± 0.32), WT-Ex (8.53 ± 0.21), HD-NoEx (7.62 ± 0.25),
and HD-Ex (8.2 ± 0.25).

Striatum – Middle, Ventral
A two-way ANOVA of medium spiny neuron dendritic spine density did not show
a difference between the groups (F(3,32) = 0.204, p = 0.893). Dendritic spine
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densities were as follows: WT-NoEx (8.49 ± 0.31), WT-Ex (8.78 ± 0.32), HD-NoEx
(8.58 ± 0.22), and HD-Ex (8.74 ± 0.37).

Hippocampus – CA1, Apical
A two-way ANOVA of pyramidal neuron apical dendritic spine density showed a
statistically significant difference between the groups (F(3,32) = 4.44, p = 0.010) with
an exercise effect (F(3,32) = 10.1, p = 0.003). There was no interaction of the
variables (F(3,32) = 0.033, p = 0.857) nor a genotype effect (F(3,32) = 1.95,
p = 0.172).
Post hoc analyses showed a statistically significant difference (p = 0.008)
between WT-Ex mice (8.63 ± 0.30) and HD-NoEx mice (7.09 ± 0.19). WT-NoEx mice
(7.62 ± 0.35) and HD-Ex mice (8.22 ± 0.52) were not different from the other groups.

Hippocampus – CA1, Basal
A two-way ANOVA of pyramidal neuron basal dendritic spine density showed a
statistically significant difference between the groups (F(3,32) = 4.28, p = 0.012) with
an exercise effect (F(3,32) = 6.55, p = 0.015) and a genotype effect (F(3,32) = 4.35,
p = 0.045). There was no interaction of the variables (F(3,32) = 0.450, p = 0.507).
Post hoc analyses showed a statistically significant difference (p = 0.007)
between WT-Ex mice (8.10 ± 0.41) and HD-NoEx mice (6.64 ± 0.23). WT-NoEx mice
(7.09 ± 0.24) and HD-Ex mice (7.24 ± 0.31) were not different from the other groups.

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Visual Cortex – Layer 5
A two-way ANOVA of dendritic spine density on pyramidal neurons showed a
statistically significant difference between the groups (F(3,32) = 4.34, p = 0.011) with
an interaction of the variables (F(3,32) = 5.12, p = 0.031), an exercise effect
(F(3,32) = 6.97, p = 0.013), but not a genotype effect (F(3,32) < 0.001, p = 0.999).
Post hoc analyses showed a statistically significant difference (p = 0.006)
between WT-NoEx (6.61 ± 0.26) and WT-Ex mice (8.00 ± 0.24). HD-NoEx
(7.25 ± 0.30) and HD-Ex (7.36 ± 0.33) were not different from the other groups.

Dendritic Spine Density – 6 months
Prelimbic Cortex – Layers 2/3
A two-way ANOVA of pyramidal neuron apical dendritic spine density did not
show a difference between the groups (F(3,13) = 0.851, p = 0.491). Dendritic spine
densities were as follows: WT-NoEx (7.14 ± 0.46, n = 5), WT-Ex (5.87 ± 0.92, n = 4),
HD-NoEx (6.25 ± 0.39, n = 5), and HD-Ex (6.02 ± 0.91, n = 3).

Prelimbic Cortex – Layer 5
A two-way ANOVA of pyramidal neuron apical dendritic spine density did not
show a difference between the groups (F(3,13) = 0.106, p = 0.955). Dendritic spine
densities were as follows: WT-NoEx (6.72 ± 0.38), WT-Ex (6.63 ± 0.60), HD-NoEx
(6.36 ± 0.46), and HD-Ex (6.44 ± 0.79).

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Striatum – Anterior, Dorsolateral
A two-way ANOVA of medium spiny neuron dendritic spine density did not show
a difference between the groups (F(3,13) = 0.656, p = 0.593). Dendritic spine
densities were as follows: WT-NoEx (8.36 ± 0.29), WT-Ex (8.95 ± 0.18), HD-NoEx
(8.55 ± 0.44), and HD-Ex (7.99 ± 0.93).

Striatum – Anterior, Dorsomedial
A two-way ANOVA of medium spiny neuron dendritic spine density did not show
a difference between the groups (F(3,13) = 0.959, p = 0.441). Dendritic spine
densities were as follows: WT-NoEx (8.43 ± 0.63), WT-Ex (8.43 ± 0.25), HD-NoEx
(7.87 ± 0.57), and HD-Ex (7.08 ± 0.84).

Striatum – Anterior, Ventral
A two-way ANOVA of medium spiny neuron dendritic spine density showed a
statistically significant difference between the groups (F(3,13) = 4.01, p = 0.032) with
a genotype effect (F(3,13) = 11.7, p = 0.005), but not an interaction of the variables
(F(3,13) = 1.36, p = 0.264) or an exercise effect (F(3,13) = 0.346, p = 0.522).
Post hoc analyses showed a statistically significant difference (p = 0.047) between WT-
Ex mice (8.78 ± 0.20) and HD-Ex mice (6.96 ± 0.70). WT-NoEx mice (8.55 ± 0.29)
and HD-NoEx mice (7.66 ± 0.40) were not different from the other groups.

Page 69

Striatum – Middle, Dorsolateral
A two-way ANOVA of medium spiny neuron dendritic spine density did not show
a difference between the groups (F(3,13) = 1.27, p = 0.327). Dendritic spine densities
were as follows: WT-NoEx (8.07 ± 0.28), WT-Ex (8.73 ± 0.36), HD-NoEx (7.99 ± 0.53),
and HD-Ex (7.47 ± 0.40).

Striatum – Middle, Dorsomedial
A two-way ANOVA of medium spiny neuron dendritic spine density did not show
a difference between the groups (F(3,13) = 1.98, p = 0.166). Dendritic spine densities
were as follows: WT-NoEx (7.50 ± 0.21), WT-Ex (8.49 ± 0.31), HD-NoEx (7.48 ± 0.49),
and HD-Ex (7.11 ± 0.55).

Striatum – Middle, Ventral
A two-way ANOVA of medium spiny neuron dendritic spine density did not show
a difference between the groups (F(3,13) = 1.54, p = 0.251). Dendritic spine densities
were as follows: WT-NoEx (8.90 ± 0.26), WT-Ex (8.92 ± 0.49), HD-NoEx (7.96 ± 0.45),
and HD-Ex (8.48 ± 0.07).

Hippocampus – CA1, Apical
A two-way ANOVA of pyramidal neuron apical dendritic spine density showed a
statistically significant difference between the groups (F(3,13) = 3.70, p = 0.040) with
Page 70

an exercise effect (F(3,13) = 6.28, p = 0.026), but not an interaction of the variables
(F(3,13) = 0.677, p = 0.426) or a genotype effect (F(3,13) = 3.04, p = 0.105).
Post hoc analyses showed a statistically significant difference (p = 0.034)
between WT-Ex mice (7.56 ± 0.50) and HD-NoEx mice (5.44 ± 0.41). WT-NoEx mice
(6.72 ± 0.52) and HD-Ex mice (7.10 ± 0.49) were not different from the other groups.

Hippocampus – CA1, Basal
A two-way ANOVA of pyramidal neuron basal dendritic spine density did not
show a difference between the groups (F(3,13) = 2.91, p = 0.074). Dendritic spine
densities were as follows: WT-NoEx (6.88 ± 0.59), WT-Ex (7.56 ± 0.89), HD-NoEx
(4.90 ± 0.83), and HD-Ex (5.28 ± 0.24).

Visual Cortex – Layer 5
A two-way ANOVA of pyramidal neuron apical dendritic spine density showed a
statistically significant difference between the groups (F(3,13) = 4.17, p = 0.028) with
an interaction of the variables (F(3,13) = 7.77, p = 0.015), a genotype effect
(F(3,13) = 5.70, p = 0.033), but not an exercise effect (F(3,13) = 0.474, p = 0.503).
Post hoc analyses showed a statistically significant difference (p = 0.023)
between WT-Ex (8.27 ± 0.71) and HD-Ex mice (5.61 ± 0.39). WT-NoEx (6.48 ± 0.14)
and HD-NoEx (6.69 ± 0.57) were not different from the other groups.

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Western Immunoblotting

Figure 8. Western immunoblotting revealed a reduction in
PSD-95 expression located in the dorsal striatum of HD-NoEx
mice starting a 4 months and continuing out to 6 months.
This reduction in expression is rescued with exercise. No
differences where seen between the groups for
synaptophysin expression.
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Figure 9. Western immunoblotting revealed a reduction in
PSD-95 expression located in the dorsal striatum of HD-NoEx
mice starting a 4 months and continuing out to 6 months.
This reduction in expression is rescued with exercise. No
differences where seen between the groups for
synaptophysin expression.
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Figure 10. Western immunoblotting of prefrontal cortex at
2, 4, and 6 months did not show any differences in protein
expression between the groups for the synaptic markers
PSD-95, and synaptophysin.

Western immunoblotting was conducted to examine the effect of exercise on
PSD-95 and synaptophysin in the prefrontal cortex and the dorsal and ventral striatum
in mice in all 4 groups at 2, 4, and 6 months of age. Measurements were normalized to
sedentary WT controls. While we observed no change in the concentrations of the
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synaptophysin, we observed a significant decrease in the concentration of the PSD-95
in both the dorsal and ventral striatum in sedentary HD KI animals as compared to
sedentary WT controls at 4 and 6 months, which was rescued by long-term treadmill
exercise. There was no significant difference in prefrontal cortex PSD-95 concentration
between sedentary HD KI and sedentary WT animals, and exercise had no significant
effects on PSD-95 levels as well.
Specifically, at 2 months of age, a two-way ANOVA analysis of sedentary and
exercised HD KI and WT mice comparing PSD-95 concentration in the dorsal striatum
showed no statistically significant difference between all 4 groups (F(3,36) = 0.04, p
=0.99). There was no statistically significant effect of genotype (F(1,36) = 0.06, p =
0.81) or exercise (F(1,36) = 0.02, p = 0.88) and no statistically significant interaction
between the variables (F(1,36) = 0.04, p = 0.85). Interestingly, at both 4 and 6
months of age there was a significant decrease in PSD-95 concentration in the dorsal
striatum. At 4 months of age, a two-way ANOVA analysis of sedentary and exercised
HD KI and WT mice comparing PSD-95 concentration in the dorsal striatum showed a
statistically significant difference between all 4 groups (F(3,36) = 14.14, p < 0.001) as
well as significant effects of genotype (F(1,36) = 17.05, p < 0.001) and exercise (F(1, 36) =
8.35, p < 0.01) and a significant interaction between the variables (F(1,36) = 16.27, p <
0.001). Post hoc analysis revealed the following differences between specific treatment
groups. Sedentary HD KI mice had a statistically significant decrease (p < 0.001) in
PSD-95 concentration compared to sedentary WT mice (HD KI, 0.83 ± 0.02; n = 9;
compared to WT, 1.00 ± 0.01; n = 9). Exercise led to a statistically significant increase
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(p < 0.001) in PSD-95 concentration between the HD KI mice (HD KI + Exercise, 0.97
± 0.03; n = 9) compared to HD KI sedentary mice. Exercise did not have a statistically
significant effect (p = 0.99) between WT mice (WT + Exercise, 0.98 ± 0.02; n= 9).
There was no statistically significant change in PSD-95 concentration between KI +
Exercise and either WT sedentary or WT+ Exercise mice (WT p = 0.99; WT + Exercise
p = 0.99).
Similarly, at 6 months of age, a two-way ANOVA analysis of sedentary and
exercised HD KI and WT mice comparing PSD-95 concentration in the dorsal striatum
showed a statistically significant difference between all 4 groups (F(3,36) = 12.56, p <
0.001) as well as significant effects of genotype (F(1,36) = 5.66, p < 0.05) and exercise
(F(1,36) = 20.21, p < 0.001) and a significant interaction between the variables (F(1,36) =
11.80, p < 0.01). Post hoc analysis revealed the following differences between specific
treatment groups. Sedentary HD KI mice had a statistically significant decrease (p <
0.001) in PSD-95 concentration compared to sedentary WT mice (HD KI, 0.82 ± 0.03; n
= 9; compared to WT, 1.00 ± 0.01; n = 9). Exercise led to a statistically significant
increase (p < 0.001) in PSD-95 concentration between the HD KI mice (HD KI +
Exercise, 1.06 ± 0.05; n = 9) compared to HD KI sedentary mice. Exercise did not
have a statistically significant effect (p = 0.99) between WT mice (WT + Exercise, 1.03
± 0.02; n= 9). There was no statistically significant change in PSD-95 concentration
between KI + Exercise and either WT sedentary or WT+ Exercise mice (WT p = 0.86;
WT + Exercise p = 0.99).
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There was no significant difference in synaptophysin levels in the dorsal striatum
between WT and HD KI animals at 2, 4, and 6 months. At 2 months of age, a two-way
ANOVA analysis of sedentary and exercised HD KI and WT mice showed no statistically
significant difference in synaptophysin concentration between all 4 groups (F(3,36) =
0.34, p =0.80). There was no statistically significant effect of genotype (F(1,36) = 0.71, p
= 0.41) or exercise (F(1,36) = 0.34, p = 0.56) and no statistically significant interaction
between the variables (F(1,36) = 0.27, p = 0.61). At 4 months of age, a two-way ANOVA
analysis of sedentary and exercised HD KI and WT mice showed no statistically
significant difference in synaptophysin concentration between all 4 groups (F(3,36) =
2.38, p =0.09). There was no statistically significant effect of genotype (F(1,36) = 1.13, p
= 0.30) and no statistically significant interaction between the variables (F(1,36) = 0.03,
p = 0.87). There was a significant effect of exercise (F(1,36) = 5.67, p < 0.05). Similarly,
at 6 months of age, a two-way ANOVA analysis of sedentary and exercised HD KI and
WT mice showed no statistically significant difference in synaptophysin concentration
between all 4 groups (F(3,36) = 2.23, p =0.10). There was no statistically significant
effect of genotype (F(1,36) = 0.15, p = 0.70) and no statistically significant interaction
between the variables (F(1,36) = 0.20, p = 0.66). There was a significant effect of
exercise (F(1,36) = 6.33, p < 0.05).
As in the dorsal striatum, at 2 months of age, a two-way ANOVA analysis of
sedentary and exercised HD KI and WT mice comparing PSD-95 concentration in the
ventral striatum showed no statistically significant difference between all 4 groups
(F(3,36) = 0.52, p =0.67). There was no statistically significant effect of genotype (F(1,36)
Page 77

= 0.07, p = 0.80) or exercise (F(1,36) = 1.23, p = 0.27) and no statistically significant
interaction between the variables (F(1,36) = 0.27, p = 0.60). Interestingly, at both 4 and
6 months of age there was a significant decrease in PSD-95 concentration in the ventral
striatum. At 4 months of age, a two-way ANOVA analysis of sedentary and exercised
HD KI and WT mice comparing PSD-95 concentration in the ventral striatum showed a
statistically significant difference between all 4 groups (F(3,36) = 7.24, p < 0.001) as well
as significant effects of genotype (F(1,36) = 5.08, p < 0.05) and a significant interaction
between the variables (F(1,36) = 10.71, p < 0.01). There was no significant effect of
exercise (F(1, 36) = 1.77, p = 0.19). Post hoc analysis revealed the following differences
between specific treatment groups. Sedentary HD KI mice had a statistically significant
decrease (p < 0.001) in PSD-95 concentration compared to sedentary WT mice (HD KI,
0.82 ± 0.05; n = 9; compared to WT, 1.00 ± 0.01; n = 9). Exercise led to a
statistically significant increase (p < 0.05) in PSD-95 concentration between the HD KI
mice (HD KI + Exercise, 0.97 ± 0.02; n = 9) compared to HD KI sedentary mice.
Exercise did not have a statistically significant effect (p = 0.97) between WT mice (WT
+ Exercise, 0.94 ± 0.02; n= 9). There was no statistically significant change in PSD-95
concentration between KI + Exercise and either WT sedentary or WT+ Exercise mice
(WT p = 0.99; WT + Exercise p = 0.99).
Similarly, at 6 months of age, a two-way ANOVA analysis of sedentary and
exercised HD KI and WT mice comparing PSD-95 concentration in the ventral striatum
showed a statistically significant difference between all 4 groups (F(3,36) = 9.159, p <
0.001) as well as significant effects of genotype (F(1,36) = 6.72, p < 0.05) and exercise
Page 78

(F(1,36) = 4.42, p < 0.05) and a significant interaction between the variables (F(1,36) =
14.65, p < 0.001). Post hoc analysis revealed the following differences between
specific treatment groups. Sedentary HD KI mice had a statistically significant decrease
(p < 0.001) in PSD-95 concentration compared to sedentary WT mice (HD KI, 0.84 ±
0.03; n = 9; compared to WT, 1.00 ± 0.003; n = 9). Exercise led to a statistically
significant increase (p < 0.001) in PSD-95 concentration between the HD KI mice (HD
KI + Exercise, 0.99 ± 0.04; n = 9) compared to HD KI sedentary mice. Exercise did
not have a statistically significant effect (p = 0.99) between WT mice (WT + Exercise,
0.96 ± 0.02; n= 9). There was no statistically significant change in PSD-95
concentration between KI + Exercise and either WT sedentary or WT+ Exercise mice
(WT p = 0.99; WT + Exercise p = 0.99).
There was no significant difference in ventrostriatal synaptophysin levels
between WT and HD KI animals at 2, 4, and 6 months. At 2 months of age, a two-way
ANOVA analysis of sedentary and exercised HD KI and WT mice showed no statistically
significant difference in synaptophysin concentration between all 4 groups (F(3,36) =
0.04, p =0.99). There was no statistically significant effect of genotype (F(1,36) = 0.06, p
= 0.81) or exercise (F(1,36) = 0.004, p = 0.95) and no statistically significant interaction
between the variables (F(1,36) = 0.06, p = 0.80). Similarly, at 4 months of age, a two-
way ANOVA analysis of sedentary and exercised HD KI and WT mice showed no
statistically significant difference in synaptophysin concentration between all 4 groups
(F(3,36) = 0.75, p =0.53). There was no statistically significant effect of genotype (F(1,36)
= 1.11, p = 0.30) or exercise (F(1,36) = 0.30, p = 0.59) and no statistically significant
Page 79

interaction between the variables (F(1,36) = 0.87, p = 0.36). At 6 months of age, a two-
way ANOVA analysis of sedentary and exercised HD KI and WT mice showed no
statistically significant difference in synaptophysin concentration between all 4 groups
(F(3,36) = 1.01, p =0.40). There was no statistically significant effect of genotype (F(1,36)
= 0.27, p = 0.61) or exercise (F(1,36) = 0.04, p = 0.85) and no statistically significant
interaction between the variables (F(1,36) = 2.79, p = 0.10).
Unlike in the dorsal and ventral striatum, we observed no difference in PSD-95
concentration in the prefrontal cortex between WT and HD KI animals at 2, 4, and 6
months. Specifically, at 2 months of age, a two-way ANOVA analysis of sedentary and
exercised HD KI and WT mice showed no statistically significant difference in PSD-95
concentration between all 4 groups (F(3,36) = 0.28, p =0.84). There was no statistically
significant effect of genotype (F(1,36) = 0.05, p = 0.82) or exercise (F(1,36) = 0.74, p =
0.40) and no statistically significant interaction between the variables (F(1,36) = 0.06, p
= 0.81). Similarly, at 4 months of age, a two-way ANOVA analysis of sedentary and
exercised HD KI and WT mice showed no statistically significant difference in PSD-95
concentration between all 4 groups (F(3,36) = 0.08, p =0.97). There was no statistically
significant effect of genotype (F(1,36) = 0.001, p = 0.97) or exercise (F(1,36) = 0.16, p =
0.69) and no statistically significant interaction between the variables (F(1,36) = 0.07, p
= 0.79). At 6 months of age, a two-way ANOVA analysis of sedentary and exercised
HD KI and WT mice showed no statistically significant difference in PSD-95
concentration between all 4 groups (F(3,36) = 0.90, p =0.45). There was no statistically
significant effect of genotype (F(1,36) = 0.01, p = 0.91) or exercise (F(1,36) = 2.16, p =
Page 80

0.15) and no statistically significant interaction between the variables (F(1,36) = 0.67, p
= 0.42).
Similarly, there was no difference in synaptophysin levels in the prefrontal cortex
between WT and HD KI animals at 2, 4, and 6 months. At 2 months of age, a two-way
ANOVA analysis of sedentary and exercised HD KI and WT mice showed no statistically
significant difference in synaptophysin concentration between all 4 groups (F(3,36) =
0.91, p =0.45). There was no statistically significant effect of genotype (F(1,36) = 2.56, p
= 0.12) or exercise (F(1,36) = 0.14, p = 0.71) and no statistically significant interaction
between the variables (F(1,36) = 0.03, p = 0.88). Similarly, at 4 months of age, a two-
way ANOVA analysis of sedentary and exercised HD KI and WT mice showed no
statistically significant difference in synaptophysin concentration between all 4 groups
(F(3,36) = 0.16, p =0.92). There was no statistically significant effect of genotype (F(1,36)
= 0.36, p = 0.55) or exercise (F(1,36) = 0.001, p = 0.98) and no statistically significant
interaction between the variables (F(1,36) = 0.10, p = 0.76). At 6 months of age, a two-
way ANOVA analysis of sedentary and exercised HD KI and WT mice showed no
statistically significant difference in synaptophysin concentration between all 4 groups
(F(3,36) = 1.10, p =0.36). There was no statistically significant effect of genotype (F(1,36)
= 0.03, p = 0.87) or exercise (F(1,36) = 3.28, p = 0.08) and no statistically significant
interaction between the variables (F(1,36) = 0.001, p = 0.97).
Taken together, this data shows a decrease in concentration of the postsynaptic
protein PSD-95 in the dorsal and ventral striatum of sedentary HD KI animals as
compared to sedentary WT controls at 4 and 6 months of age. This downregulation is
Page 81

rescued by long-term treadmill exercise in HD KI animals. There was no difference in
PSD-95 concentration in the prefrontal cortex and synaptophysin concentration in the
prefrontal cortex and dorsal and ventral striatum in exercised and non exercised HD KI
and WT animals.

Discussion
This is the first study to assess the effect of exercise on dendritic spine density in
the CAG140 knock-in mouse model of Huntington’s disease. In addition, this study took
a comprehensive look into multiple brain regions known to be affected in HD patients
and HD mouse models.
We have shown for the first time that exercise is able to increase dendritic spine
density in the prelimbic cortex on apical dendrites of layer 2/3 pyramidal neurons,
though this effect does not persist out to 6 months. This increase in dendritic spine
density may be a compensatory mechanism that contributes to the improvement in
mood and cognitive tasks we have recently reported (Stefanko et al., 2016). The
involvement of the prelimbic cortex in psychiatric well-being is evidenced in the Wfs1-
mutant mouse model of depression. The authors found that the disruption of prelimbic
cortex, layer 2/3 pyramidal neurons leads to depression-like behavior (Shrestha et al.,
2015). And numerous studies have shown that proper functioning of the prelimbic
cortex is necessary for normal cognitive behavior (reviewed in Granon and Poucet,
2000). Further, this brain region in HD-NoEx mice has been shown in our group’s
previous work (Wang et al., 2016) to have a reduced amount of blood flow compared
Page 82

to HD-Ex mice, suggesting changes in regional metabolism. Our findings support the
work of other groups that have shown dendritic spine density is closely related to
metabolic markers (Segura et al., 2016; Tigaret et al., 2016; Wu et al., 2016).
Another finding from this study was that 4 month old HD-NoEx mice had a
greater number of dendritic spines than WT-NoEx mice in the dorsolateral anterior
striatum. An explanation for this finding could be that in HD-NoEx mice there is an
increase of aberrant, thin dendritic spines. Though unintuitive, this finding is not
outside the realm of the disease progression. Other studies have shown that early in
the disease, in both humans and rodents, there appears to be a proliferative stage
(Graveland et al., 1985; Ferrante et al., 1991). This outgrowth of both dendrites and
dendritic spines may act as a compensatory mechanism of affected neurons in the
striatum.
There was also a lack of significant findings for most other brain regions. An
explanation for this in the 6-month group analysis could be due to our low number of
animals (3-5) per group. We observed greater variance in the data among these
groups, though this variance could also be contributed to time-dependent changes
between proliferative and degenerative processes.
To better understand dendritic spine density changes in this mouse model of HD,
we have several future studies planned. We will explore what types of dendritic spines
(thin, mushroom, stubby) are present on neurons as well as assess dendritic
arborization metrics. We also plan on determining the time course of dendritic spine
Page 83

changes over a longer duration, potentially out to 18 months. We did not look at 4
month to 6 month changes, as this was not originally planned.

Conclusion
Much work still needs to be conducted to understand the time-course changes
and effect treadmill exercise has on dendritic spine density in the CAG140 mouse model
of Huntington’s disease. However, this study is the first to report that changes do occur
in a region and time-dependent manor. From these findings we have gained insight
into a potential mechanism by which exercise may help individuals with HD.

Page 84

Chapter 4: Conclusions, Perspectives, Limitations, Other
Welcome to the Grand Finale! Thanks for reading! This section is the last, so
pat yourself on the back, but not too hard because you haven’t finished this thing yet
29
.
The purpose of my dissertation was to elucidate how exercise can modify brain
circuits, through dendritic spine density, in two neurodegenerative disorders. And
during this endeavor I, with help from many others, have produced two primary
research papers (one paper is in final editing) that have added to our understanding of
exercise’s role in these diseases and will guide future scientific research.
Chapter 2 detailed how exercise affects dendritic spine density in the MPTP
mouse model of Parkinson’s disease. We found that exercise is able to rescue dendritic
spine density on medium spiny neurons. Further, utilizing a transgenic mouse that
allows for the discernment of the D1 and D2 pathway, we found that both pathways are
affected in the MPTP mouse and that exercise rescues dendritic spine density. These
dendritic spine density results were supported by immunohistochemistry analysis of
synaptic proteins. We also investigated morphological properties of medium spiny
neuron dendritic trees. We found that MPTP leads to a reduction of dendritic branch
intersections using the Sholl analysis and that exercise is able to rescue this observed
deficit. Taken together, these findings support the hypothesis that the beneficial effects
of exercise may be due to modulation of the basal ganglia motor circuitry. Specifically,
exercise is able to induce morphological changes in medium spiny neurons in the
dorsolateral striatum that may underlie the benefits of exercise.

29
The usage of “yet” isn’t proper, but it feels appropriate to add the emphasis it conveys in this sentence.
Page 85

Chapter 3 investigated how exercise effects dendritic spine density in the CAG140
mouse model of Huntington’s disease. In this study we looked at two different time
points to better understand if spine density changes correlate with cognitive and motor
behavior metrics from Stefanko et al., (2016). We found that exercise is able to
increase dendritic spine density compared to non-exercised CAG140 mice in the
prefrontal cortex (prelimbic) an area associated with executive function, attention, and
memory at 4 months, but did not see this difference at 6 months. The rest of the data
didn’t show significant differences among the groups. This is itself a pretty interesting
finding. Numerous reports investing dendritic spine density in humans and mouse
models of the disease have reported that there is a period of proliferation entailing
increases in dendritic spine density and outgrowth of dendrites. I hypothesize that
during our window of investigating dendritic spine density, i.e. at 4 and 6 months, we
are observing neurons in either a normal state or one of proliferation. Our evidence
better supports that neurons are possibly still healthy at these time points, at least as
observed through dendritic spine density. I did observe possible states of proliferation,
seen as an abundance of thin spines on some dendrites, though, in which animals this
occurred, I do not know, because I was blind to the groups. Future studies,
undertaken by the next crop of enthusiastic students, should look to classify what types
of dendritic spines are present in these animals.
A question that intrigued me early in my graduate studies was: what is the time-
course for dendritic spine loss to occur in the MPTP mouse? I hypothesized that
dendritic spine loss was not an immediate effect after dopamine depletion. This
Page 86

hypothesis is supported by the work of others that are mentioned in chapter 2’s
discussion as well as a paper from Ruth Wood’s lab, were a student, Eleni, observed an
increase in dendritic spine density on MSNs at 5 days post-lesioning. I tested this
hypothesis with time points of 5 days and 14 days post-lesioning and found that there
were no differences between saline animals and either of the MPTP-lesioned groups.

Figure 11. No difference is observed
between saline and MPTP animals out to 14
days post-lesioning. Don’t trust the error
bars, they’re wrong, I ran the stats wrong
years ago, and I couldn’t find a corrected
figure in time...

This finding and the reports of others
supports my hypothesis. Of course the next
step would be to continue this project out
further, but I’m graduating, hopefully.
During my studies I also looked to
see if dendritic spine density differed
between C57BL/6J mice and our transgenic D2-GFP mice. I wasn’t expecting to find
anything interesting because the D2-GFP mice are on a C57 background, but it turns
out there is a difference in dendritic spine density. Specifically, D2-GFP mice had a
greater dendritic spine density than regular mice. Unfortunately, it appears this data
has been lost. This finding isn’t the first to show that D2-GFP mice aren’t normal. A
Page 87

report from a few years ago, showed that D2-GFP mice over express the D2 receptor,
which is a very large issue.
This concluding chapter probably should discuss more of the limitations of the
work that was conducted, but I’m getting really tired of writing and need to turn this
thing in soon. But here goes anyways. The entire HD project was never planned out
well and really wasn’t supposed to be the second half of my dissertative work. Because
this project wasn’t well planned, the number of 6 month animals in the groups was low
making it more difficult to observe small differences that may exist between the groups
at this time point. I actually never got to plan out any of my research. I had a few
ideas that I wanted to test, but this never happened for a few reasons that I don’t
really care to get into. I was really interested in testing what role context played in
motor and cognitive assessments in the MPTP-mouse. I thought a good experiment
would be to test rotarod performance on these mice. Mice would learn the rotarod task
in one context, e.g. a room with brown walls, and then be tested in in a different
context, e.g. a room with green walls. To see if they were able to transfer what they
learned in the first room to another room. I hypothesized that they would not perform
was well in the second room. Another set of experiments would add an exercise
regimen to the mix between testing rotarod performance in the first room and second
room. My hypothesis for this set of experiments is that these exercised animals would
perform just as well in the second room as they did in the first room. So why is this
interesting and important. Well a large problem in PD patients is that any therapy, be it
physical, occupational, or speech, that they partake in at a clinic to improve a
Page 88

respective behavior does not translate over to other environments, namely one’s home.
So, if a PD patient learns to walk better and perform ADLs with greater efficiency in a
clinical setting, they aren’t able to walk as well at home and ADLs are more difficult to
complete. So, this proposed experiment would provide evidence that exercise is able
help patients overcome the context-barrier. And this experiment should have been the
second half of this dissertation. But I ran into health problems, equipment problems,
funding problems, etc…
So there you have it, my dissertation. I hope it’s enough to graduate. But,
anyways, pass or fail, I’m headed to Costa Rica
30
right afterwards.

30
maybe…
Page 89

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Abstract (if available)

Abstract Exercise has been shown to be beneficial for Parkinson’s disease (PD). A major interest in our lab has been to investigate how exercise modulates basal ganglia function and modifies disease progression. Dopamine (DA) depletion leads to loss of dendritic spines within the caudate nucleus and putamen (striatum) in PD and its animal models and contributes to motor impairments. Striatal medium spiny neurons (MSNs) can be delineated into two populations, the dopamine D1 receptor (DA-D1R)—containing MSNs of the direct pathway, and dopamine D2 receptor (DA-D2R)—containing MSNs of the indirect pathway. There is evidence to suggest that the DA-D2R-indirect pathway MSNs may be preferentially affected after DA-depletion with a predominate loss of dendritic spine density when compared to MSNs of the DA-D1R-direct pathway in rodents

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Creator Toy, William Anthony (author)

Core Title The effect of treadmill running on dendritic spine density in two neurodegenerative disorders: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease and CAG₁₄₀ kn...

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 07/21/2016

Defense Date 06/07/2016

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

Tag dendritic spine density,Exercise,Huntington's,medium spiny neurons,OAI-PMH Harvest,Parkinson's

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Language English

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Advisor McGee, Aaron (committee chair), Jakowec, Michael (committee member), Petzinger, Giselle (committee member), Pike, Christian (committee member), Walsh, John (committee member)

Creator Email williamtoy@gmail.com,wtoy@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-270850

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