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Experience-dependent neuroplasticity of the dorsal striatum and prefrontal cortex in the MPTP-lesioned mouse model of Parkinson’s disease
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Experience-dependent neuroplasticity of the dorsal striatum and prefrontal cortex in the MPTP-lesioned mouse model of Parkinson’s disease
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Content
EXPERIENCE-DEPENDENT NEUROPLASTICITY OF THE DORSAL STRIATUM
AND PREFRONTAL CORTEX IN THE MPTP-LESIONED MOUSE MODEL OF
PARKINSON’S DISEASE
by
Natalie Margaret Kintz
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 2015
Advisory Committee:
Michael Jakowec, PhD
Giselle Petzinger, MD
John Walsh, PhD
Daryl Davies, PhD
Daniel Holshneider, MD
Copyright 2015 Natalie M Kintz
ii
ACKNOWELDGEMENTS
I would like to thank my doctoral adviser Dr. Michael Jakowec and my co-mentor Dr.
Giselle Petzinger for inviting me to join their team in May 2011, and for providing me with the
resources and support to complete my graduate studies. Both Dr. Jakowec and Dr. Petzinger
were incredible mentors, and their dedication and commitment to their work continues to inspire
me. I truly appreciate the time they spent helping me address technical issues and for the
professional guidance they provided along the way. They are both brilliant scientists and I am
honored that I was able to work closely with them over the last few years.
I would also like to thank the other members of my dissertation committee. Dr. John
Walsh, Dr. Daryl Davies, and Dr. Daniel Holschneider always treated me with respect and
showed great enthusiasm for my work. I greatly appreciate the time and energy they dedicated to
serving on my dissertation committee. Their support encouraged me to stay focused and they
helped me progress forward in my scientific career.
In addition, I would like to acknowledge and thank the following for their contributions
to the work presented here: Dr. John Walsh and Garnik Akopian for providing critical guidance
into developing the electrophysiological experiments presented in Chapter 2. Dr. Garnik
Akopian, may he rest in peace, performed the electrophysiological experiments presented in
Chapter 2. Garnik was a very experienced and talented electrophysiologist. It was a great
opportunity to work with him and to learn more about the theoretical and experimental principles
of neurophysiology. Celia Williams for preparing the synaptoneurosomal preparations presented
in Chapter 2. Dr. Ruth Wood for providing invaluable insight in designing mouse behavioral
studies. Dr. Wood also served as a strong female mentor, and I greatly appreciate the time and
energy she devoted into developing a positive and supportive “women in science” environment
iii
at USC. Daniel Stefanko set up the reversal-learning T-maze task discussed in Chapter 3, and
trained James Tavornwattana, my undergraduate mentee, on how to conduct the behavioral task.
James also helped with a variety of molecular experiments over the last four years, and I am very
thankful for all of his support. Dr. Jakowec processed the samples for HPLC and trained me to
conduct the HPLC analysis discussed in both Chapter 2 and Chapter 3. William Toy, Vivek
Shah, Damaris Garcia, and Brian Leyshon provided general laboratory support, technical
guidance, and encouragement.
I was fortunate to work with a few very talented scientists and graduate students. Among
them, Daniel Stefanko and William Toy were fellow graduate students in the lab and they
supported me as a colleague and a friend throughout my entire graduate career. I am thankful
that I was able to work alongside such wonderfully talented, bright, and motivated scientists. In
addition, Dr. Raina Pang was extremely supportive and was my graduate student mentor during
the first few years of my graduate career. Dr. Pang helped me work through technical issues and
she was always there to help me through challenging situations. I would also like to thank Dr.
Jillian Shaw, Panthea Heydari, Madeline Andrews, and Marshall Howland for their friendly
support throughout my graduate career. I am forever grateful for their friendship.
Finally, I would like to thank my parents, David and Susanna, my brothers, Sammy,
Matthew, and Corydon, and my partner, Robin Sargent, for always supporting and believing in
me. I could not have made it through this long and difficult journey without the love and support
of my family, friends and mentors.
Funding for the studies presented in this dissertation came from: (1) the Allemann Merit
Fellowship, Neuroscience Graduate Program (2010-2011 and 2014-2015); (2) the TL1 (Pre-
doctoral) training Award though Southern California Clinical and Translational Science Institute
iv
and University of Southern California, Keck School of Medicine (2012-2013); and (3) a
Graduate Student Research Assistantship/USC Parkinson’s Graduate Research Fellowship
funded by the USC Parkinson’s Disease Research Group including George and Mary Lou
Boone, Walter and Susan Doniger, and the family of Don Gonzalez Barrera (2011-2012 and
2015). These studies would not be possible without the generous support of the Roberto
Gonzales family Foundation and their interest in PD research and the importance of exercise /
healthy lifestyle for patients and families.
v
TABLE OF CONTENTS
LIST OF FIGURES vi
LIST OF TABLES viii
ABSTRACT ix
CHAPTER 1: INTRODUCTION 1
1.1 Parkinson’s Disease 2
1.2 Experience-dependent neuroplasticity: Exercise in PD 9
1.3 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Model of DA-depletion 11
1.4 Dopamine (DA) 12
1.5 Glutamate Neurotransmitter System 16
1.6 Experimental models of learning and memory 19
1.7 Basal Ganglia (BG) Circuit 23
1.8 Prefrontal Cortex 30
1.9 Brief Summary 34
CHAPTER 2: Exercise modifies α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid
receptor (AMPAR) expression in striatopallidal neurons in the 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine-(MPTP)-Lesioned Mouse 36
CHAPTER 3: Exercise Modifies Dopamine Neurotransmission within the Prefrontal
Cortex and Reduces Perseverative Behavior in the MPTP-lesioned Mouse 71
CHAPTER 4: CONCLUSIONS 101
BIBLIOGRAPHY 111
vi
LIST OF FIGURES
Figure 1: Neuropathology in Parkinson’s Disease 4
Figure 2: Exercise and Neuroplasticity in Parkinson’s Disease 10
Figure 3: Four Main Dopamine Pathways in the Brain 13
Figure 4: Distribution of Dopamine Receptors in the Brain 15
Figure 5: Dopamine Signaling Pathways in the Brain 16
Figure 6: Basic Structure of AMPARs 18
Figure 7: AMPAR Dynamics During LTP Induction and Maintenance 20
Figure 8: Diagram of Select Basal Ganglia Circuits 23
Figure 9: Medium Spiny Neuron Cell Types in the Striatum 26
Figure 10: Alterations in the Basal Ganglia Circuit in Parkinson’s Disease 29
Figure 11: Corticostriatal Pathways Involving the PFC and Striatum 31
Figure 12: Homology Between Human and Rodent PFC 32
Figure 13: Study Timeline (Chapter 2) 43
Figure 14: Exercise Normalizes GluA2 and Decreases GluA1 Cell Surface Expression in
D
2
R-MSNs in MPTP Mice
54
Figure 15: Exercise Decreased GluA1 Cell Surface Expression in D
2
R-MSNs 55
Figure 16: Western Immunoblot Analysis of GluA1 and GluA2 Protein Preparations
Enriched for PSD Fractions from Dorsolateral Striatal Tissue
57
Figure 17: Analysis of the Rectification Index of Dorsolateral Striatal D
2
R-MSNs and
D
1
R-MSNs
59
Figure 18: AMPAR Expression 7 Days After MPTP or Saline Administration 61
Figure 19: Analysis of the Rectification Index in D
2
R-MSNs and D
1
R-MSNs Using the
D
2
R-antagonist l-Sulpiride
63
Figure 20: Corticostriatal Expression of Long-Term Depression after 28 days of Exercise 64
vii
Figure 21: HPLC Analysis of Dopamine Levels within the Prefrontal Cortex 82
Figure 22: HPLC Analysis of DA Levels within the Dorsal Striatum 83
Figure 23: Western Immunoblot Analysis of Tyrosine Hydroxylase Protein Preparations
from the PFC
84
Figure 24: Western Immunoblot Analysis of Tyrosine Hydroxylase Protein Preparations
from the Dorsal Striatum
85
Figure 25: Western Immunoblot Analysis of Dopamine D
1
Receptor Protein
Preparations from the PFC
87
Figure 26: Western Immunoblot Analysis of Dopamine D
2
Receptor Protein
Preparations from the PFC
89
Figure 27: Western Immunoblot Analysis of Dopamine D
4
Receptor Protein
Preparations from the PFC
91
Figure 28: Learning Performance During the T-Maze Reversal-Learning task 93
Figure 29: Error Analysis During the T-Maze Reversal-Learning Task 95
Figure 30: Graphic Model Summarizing Chapter 2 Study Results 103
viii
LIST OF TABLES
Table 1: Effects of Dopamine Replacement Therapy on Cognitive Function in
Parkinson’s Disease
8
Table 2 HPLC Analysis of Striatal Dopamine (Chapter 2) 42
Table 3: Summary of Chapter 3 Study Late Time Point Results 107
ix
ABSTRACT
Parkinson’s disease (PD) is the second most common neurodegenerative disorder, for
which there is no disease modifying treatment or cure. PD is characterized as a dopamine (DA)-
deficit disorder, marked by progressive movement-related disability and other non-motor
symptoms. The clinical diagnosis of PD relies largely on the presence of the characteristic motor
symptoms, including resting tremor, bradykinesia, rigidity, and postural instability (Olanow et
al., 2009). In addition to the motor features, cognitive impairment is pervasive in PD and often
involves deficits in executive function, including working memory and behavioral flexibility
(Svenningsson et al., 2012, Martínez-Horta and Kulisevsky, 2015). Most PD treatments aim to
reduce the severity of motor symptoms by restoring DA, however these therapies do not stop,
slow or reverse disease progression, and long-term use is often associated with severe medical
complications (Ahlskog and Muenter, 2001). Furthermore, none of the current treatment
strategies effectively target cognitive disturbances observed in PD (Martínez-Horta and
Kulisevsky, 2015). Since the current treatment strategies are highly unsatisfactory, there is an
urgent need to develop new therapies that drive neuroplasticity and repair in the DA-depleted
brain for the improved treatment of PD.
Importantly, exercise is a promising therapeutic strategy that has been shown to exert
beneficial effects on both motor and cognitive features of PD, but the mechanisms underlying
these benefits are not fully understood (Petzinger et al., 2013). The studies discussed in this
dissertation were designed to help address this gap in knowledge by further investigating the
mechanisms driving experience-dependent neuroplasticity and repair in the DA-depleted brain.
Studies in this dissertation examined experience-dependent neuroplasticity using the 1-methyl-4-
x
phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of DA-depletion, a commonly used
experimental model of PD, and physical exercise in the form of high intensity treadmill running.
Chapter 2 of this dissertation focuses on the molecular and physiological mechanisms by
which exercise promotes plasticity of glutamate neurotransmission within the DA-depleted
dorsolateral striatum, and how these neuroplastic changes relate to motor function in MPTP
mice. Our work specifically focuses on exercise-dependent changes in the alpha-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic-acid (AMPA) type glutamate receptor (AMPAR)
expression in striatal medium spiny neurons (MSNs). AMPARS are fast acting ionotropic
glutamate receptors that play a critical role in modulating experience-dependent neuroplasticity,
and alterations in AMPAR expression have been proposed to underlie synaptic dysfunction and
disease pathophysiology in neurological disorders such as Alzheimer’s disease and drug
addiction (Alt et al., 2006, Conrad et al., 2008, Kessels and Malinow, 2009, Chang et al., 2012a).
Changes in AMPAR expression have also been observed in the DA-depleted brain, and may
contribute to potentiated glutamatergic signaling observed in the striatopallidal (D
2
R-MSNs)
projection pathway in PD (Wullner et al., 1993, Betarbet et al., 2000, Brown et al., 2005, Picconi
et al., 2005, Ba et al., 2006, Ouattara et al., 2010, VanLeeuwen et al., 2010). Our previous work
demonstrating increased GluA2-lacking AMPAR expression within the dorsolateral striatum
after MPTP is consistent with increased glutamatergic neurotransmission observed in the DA-
depleted brain (VanLeeuwen et al., 2010). Our previous work also demonstrates that exercise
improves motor function, and increases total GluA2 subunit expression and the contribution of
GluA2-containing channels within the dorsolateral striatum of MPTP-lesioned mice. However
the relationship between GluA2-lacking AMPAR expression and striatopallidal D
2
R-MSNs has
not been fully defined.
xi
The study discussed in Chapter 2 builds upon our previous work by evaluating whether
the observed changes in dorsolateral striatal AMPAR expression with MPTP and exercise are
specific to the striatopallidal (D
2
R-MSN) or striatonigral (D
1
R-MSN) projection-pathways.
Furthermore, since AMPARs are known to play a key role in modulating neuroplasticity, and
because MPTP has been shown to reduce corticostriatal expression of long-term depression
(LTD), we also evaluated if exercise restores corticostriatal LTD in MPTP-lesioned mice. For
these studies, Drd
2
-eGFP-BAC transgenic mice expressing enhanced green fluorescence protein
under the control of the DA-D
2
receptor (D
2
R) promoter were used to delineate differences in
AMPAR expression between D
2
R-MSNs and D
1
R-MSNs after MPTP and exercise (Gong et al.,
2002, Chan et al., 2012, Nelson et al., 2012).
We found MPTP increased the contribution of GluA2-lacking AMPARs selectively in
D
2
R-MSNs and exercise reversed this effect in MPTP mice. Furthermore, these exercise-induced
changes in AMPAR channels observed in MPTP mice were associated with alterations in GluA1
and GluA2 subunit expression, the restoration of corticostriatal plasticity, and improved motor
performance. These data suggest that changes in dorsolateral striatal AMPAR expression in D
2
R-
MSNs may represent an important mechanism underlying the motor behavioral improvements
seen with effects of exercise in MPTP mice.
Chapter 3 of this dissertation focuses on the molecular mechanisms by which exercise
promotes plasticity of DA neurotransmission within the prefrontal cortex (PFC), and how these
neuroplastic changes relate to cognitive function in MPTP mice. In addition to motor features,
cognitive disturbances are observed in PD and are associated with alterations in PFC function
and DA neurotransmission (Brozoski et al., 1979, Puig et al., 2014, Martínez-Horta and
Kulisevsky, 2015). Exercise may be a promising therapeutic strategy for cognitive impairment in
xii
PD since several studies in healthy aging individuals have demonstrated a significant beneficial
effect of exercise on cognition (Chang et al., 2012b, Hotting and Roder, 2013), and preliminary
evidence suggests exercise improves executive function, specifically behavioral flexibility, in PD
(Tanaka et al., 2009). This current study investigates exercise restores PFC DA levels, modulates
PFC DA-D
1
receptor (D
1
R), D
2
R, and DA-D
4
receptor (D
4
R) levels, and improves behavioral
flexibility during a reversal-learning T-maze task in MPTP-lesioned mice.
We found in MPTP-lesioned mice exercise restored PFC DA levels, reversed the MPTP-
induced increase in PFC D
1
Rs and decrease in D
4
Rs, and induced differential effects on D
2
R
expression which appeared to be inversely related to exercise effects on PFC DA levels. We also
found in MPTP-lesioned mice exercise reduced perseverative behavior, but did not improve
overall performance on a reversal-learning T-maze task. These data suggest exercise, by
restoring PFC DA levels and by modulating PFC DA receptor expression, may remediate some
aspects of executive function, such as behavioral flexibility, but does not improve overall
learning deficits observed in MPTP mice. Future studies will determine if skill-based exercise
paradigms that require higher levels of cognitive engagement during the exercise intervention,
either alone or in conjunction with aerobic training (i.e. treadmill running), are more effective at
improving deficits in executive function observed in MPTP mice.
Collectively, these results highlight the value of preclinical research in animal models of
DA-depletion in providing insight into experience-dependent mechanisms driving plasticity and
repair in PD. Understanding these mechanisms is of key interest because they may provide
insight into new drug targets and/or new therapeutic strategies for the improved treatment of PD
and other disorders of the BG.
1
CHAPTER 1: INTRODUCTION
The goal of the studies comprising this dissertation was further elucidate the experience-
dependent mechanisms by which exercise promotes plasticity and repair in the dopamine (DA)-
depleted brain using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of
DA-depletion, a commonly used experimental model of Parkinson’s disease. Chapter 1 will
provide an introduction to the important concepts underlying the research discussed in this
dissertation. Chapter 2 will describe the molecular and physiological mechanisms by which
exercise promotes plasticity of glutamate neurotransmission within the dorsolateral striatum, and
how these neuroplastic changes relate to motor function in MPTP mice. Chapter 3 will discuss
the molecular mechanisms by which exercise promotes plasticity of DA neurotransmission
within the prefrontal cortex (PFC), and how these neuroplastic changes relate to cognitive
function in MPTP-lesioned mice. Chapter 4 will describe the significance of this dissertation
research, and future considerations. Completion of the studies outlined in this dissertation
furthers our understanding of the role exercise plays in modulating DA and glutamatergic
neurotransmission, as well as cognitive function in the MPTP-lesioned mouse. Results form
these studies highlight the value of preclinical research in animal models of DA-depletion in
providing insight into experience-dependent mechanisms driving plasticity and repair in PD.
These studies are of key importance because they may provide insight into novel drug targets
and/or therapeutic strategies for the improved treatment of PD.
2
1.1 Parkinson’s Disease
Parkinson’s disease (PD) is the second most common neurodegenerative disorder, for
which there is no disease modifying treatment or cure. PD, or Paralysis Agitans, was first
formerly described in 1817 by James Parkinson’s, a British physician, in his publication titled
“An essay on the shaking palsy” (García-Montes, 2012). Today, PD affects approximately 1-3%
of the general population at age 65 (Kowal et al., 2013). Age is the greatest risk factor for PD,
and the incidence of PD is expected to double by 2030 due to the increasing age of the general
population (Kowal et al., 2013). Although limited data, the incidence of PD appears to be higher
in men than women, and highest among Hispanics, followed by non-Hispanic Whites, Asians
and Blacks (Wirdefeldt et al., 2011, Goldman, 2014).
The etiology of PD is not well understood, but likely involves both genetic and
environmental factors. Approximately 15 genes and genetic loci have been associated with PD,
including mutations in the alpha synuclein gene (SNCA, PARK1), parkin gene (PARK2), DJ-1
gene (PARK7), PINK1 gene (PARK6), and LRKK2 gene (PARK8), (Singleton et al., 2013).
However, only a small portion of clinical cases can be linked to genetic mutations (Goldman,
2014).
A number of environmental factors have also been associated with PD. For example,
epidemiological studies suggest exposure to environmental toxicants such as pesticides, solvents,
metals, and other pollutants is associated with an increased risk of PD. Interestingly, cigarette-
smoking and caffeine intake are associated with reduced risk of PD. In addition, exercise, the use
of non-steroidal anti-inflammatory medications, and the use of dihydropyridine calcium (Ca
2+
)
channel-blocking drugs have been associated with reduced risk of PD (Goldman, 2014). In
general, it is believed that genetic and environmental factors negatively influence a number of
3
disease related processes, including, mitochondrial dysfunction, oxidative stress, microglial
activation and inflammation, proteasomal impairment, aggregation of alpha-synuclein protein
and impaired autophagy, that result in similar neuropathological and clinical consequences
(Goldman, 2014).
1.1.1 Neuropathology:
The key pathological features of PD include (1) the progressive loss of dopaminergic
neurons in the substania nigra pars compacta (SNpc) and the subsequent loss of dopaminergic
innervations to the striatum, a region in brain that regulates motor control, and (2) the presence
of ubiquitinated alpha-synuclein inclusions termed Lewy bodies and Lewy neurites. Dopamine
(DA) released in the striatum targets D
1
receptor (D
1
R) medium spiny neurons (MSNs) of the
direct pathway (D
1
R-MSNs, also called striatonigral pathway) and D
2
receptor (D
2
R) MSNs of
the indirect pathway (D
2
R-MSNs, also called the striatopallidal pathway) to regulate striatal
signaling and motor control (discussed in more detail below). Consequently, loss of DA alters
striatal plasticity and basal ganglia (BG) circuitry, and ultimately is believed to give rise to the
typical motor symptoms of PD (Olanow et al., 2009) (Figure 1).
In addition to deficits in DA neurotransmission, aberrant glutamatergic neurotransmission
is observed in PD. Glutamate neurotransmitter systems are also intimately involved in the brain
circuitry responsible for regulating motor control, and the related motivational and cognitive
processes. Consequently, pathological changes in glutamate neurotransmission may also
contribute to the behavioral disturbances observed in PD.
4
Figure 1: Neuropathology in Parkinson’s disease. (A) Schematic representation of the
nigrostriatal pathway (in red) in a healthy brain. It is composed of melanin-containing
dopaminergic neurons within the SNpc (see arrows), which send dopaminergic input (thick solid
red lines) to the BG and synapse in the striatum (in rodents, the dorsolateral striatum is
comparable to the posterior putamen and the dorsomedial striatum is comparable to the anterior
caudate in humans). The photograph depicts normal melanin pigmentation in SNpc neurons (see
arrows). (B) Schematic representation of the diseased nigrostriatal pathway in PD (in red). In
PD, there is marked degeneration of DA neurons within the SNpc (the photograph demonstrates
depigmentation of melanin, see arrows), and the corresponding loss of DA within the striatum,
particularly the putamen (dorsolateral striatum in rodents, dashed red line). (C)
Immunohistochemical labeling of Lewy bodies, in a dopaminergic SNpc neuron.
Immunostaining with an antibody against alpha-synuclein reveals a Lewy body (black arrow)
with an intensely immunoreactive central zone surrounded by a faintly immunoreactive
peripheral zone (left photograph). Conversely, immunostaining with an antibody against
ubiquitin yields more diffuse immunoreactivity within the Lewy body (right photograph). Figure
and legend from: (Dauer and Przedborski, 2003)
5
1.1.2 Clinical Characteristics:
The clinical diagnosis of PD relies largely on the presence of the characteristic motor
symptoms including resting tremor or shaking, bradykinesia (slowed movement), akinesia (loss
of voluntary movement) rigidity or stiffness of the limbs due to resistance of movement, and
postural instability (Olanow et al., 2009). Motor symptoms develop gradually and the rate of
decline varies widely across patients (Poewe, 2006).
Habit-based (stimulus response, S-R) motor learning is also impaired in PD. Motor
learning is defined as a practice-related change or improvement in motor performance. The
initial phase of motor skill learning involves the activation of circuits involved in reward-based
and goal-directed learning (Redgrave et al., 2010), including the ventral striatum (vStr) and
frontal cortex. Extended training of a motor skill involves a shift from goal-directed to habit-
based (S-R) learning, which is critically dependent on the dorsal striatum (dStr). In the context of
PD, DA depletion preferentially impacts the dStr, and consequently, impairs habit-based
learning. Individuals with PD sustain increased cognitive effort during motor learning tasks
because the ability to shift from goal-directed to habit-based learning is impaired (Redgrave et
al., 2010, Petzinger et al., 2013).
In addition to motor dysfunction, a broad spectrum of non-motor features, including
autonomic, sensory, sleep, cognitive and affective disturbances are increasingly recognized as
common manifestations of PD. Non-motor features of PD often appear before motor deficits.
There is also emerging evidence that these non-motor features often overshadow motor
impairments in later stages of the disease and may become a primary source of disability,
reduced quality of life, and economic burden (García-Montes, 2012, Vlagsma et al., 2015). The
6
work discussed in Chapter 3 of this dissertation will focus on cognitive impairments of the
executive function subtype in PD.
Cognitive impairment is pervasive in PD, and ranges from mild deficits in specific
domains of cognitive functioning to dementia (Svenningsson et al., 2012). Cognitive deficits,
much like motor deficits, typically worsen with increasing disease duration. The frequency of
cognitive dysfunction in patients with PD without clinical evidence of dementia is estimated to
be ~30% at the time of diagnosis. Moreover, the frequency of dementia in PD is estimated to be
~ 20% at the time of diagnosis, with a cumulative prevalence of up to 80% after 20 years of
follow-up (Martínez-Horta and Kulisevsky, 2015). The mechanisms underlying cognitive
disturbances in PD have not been fully elucidated, but likely involve complex interactions
between a number of brain regions and neurotransmitter systems.
Mild cognitive impairment (MCI), particularly of the executive function subtype, is the
earliest, and most prominent cognitive feature in PD, which transitions to dementia, increased
fall risk, and poor quality of life (Martinez-Horta et al., 2013, Pagonabarraga et al., 2013).
Executive function is a set of processes that include working memory, visuospatial skills,
attention, and behavioral flexibility, and is critical for the initiation, planning, regulation and
monitoring of goal-directed, adaptive behavior (Martínez-Horta and Kulisevsky, 2015).
Behavioral flexibility is particularly impaired in PD, and it manifests as diminished ability to
generalize a learned motor task and transfer it into a different contextual setting (Sawamoto et al.,
2008, Darvas et al., 2014). Deficits in executive function lead to problems in daily functioning,
and result in loss of independence and increased psychosocial and economic burden in patients
with PD, caregivers, and health providers. The prefrontal cortex (PFC) along with its connections
to the striatum is an important brain region sub-serving executive function. Furthermore, DA
7
plays a key neuromodulatory role in the PFC, and aberrant DA neurotransmission is believed to
contribute to executive function deficits observed in PD (Brozoski et al., 1979, Puig et al., 2014,
Martínez-Horta and Kulisevsky, 2015). There remains, however, an important need to better
understand the mechanisms underlying PFC dysfunction in the DA-depleted brain.
1.1.3 Limitations to current treatment strategies
PD symptom severity increases as the disease progresses, which translates into significant
adverse effects on health-related quality of life, and enhanced burden on healthcare systems and
caregivers (Kowal et al., 2013). Current clinical treatment strategies aim to reduce the severity of
motor symptoms, however these treatments do not stop, slow or reverse disease progression, and
long-term use is often associated with severe medical complications (Shook and Jackson, 2011).
The most common clinical treatment is DA replacement therapy using the precursor for DA, L-
3,4-dihydroxyphenlalanine (levodopa, or L-DOPA). L-DOPA administration increases DA
concentrations in the brain, and improves motor symptoms in PD patients during the early stages
of the disease. However, chronic L-DOPA treatment is associated with severe medical
complications, such as dyskinesia (involuntary motor fluctuations) (Schrag and Quinn, 2000). In
fact, approximately 30% of PD patients experience dyskinesia’s after 4-6 years of L-DOPA
treatment, and this risk approaches 90% after 9 years of L-DOPA treatment (Ahlskog and
Muenter, 2001). Furthermore, the effectiveness of L-DOPA subsides after prolonged treatment,
leading to periods of “wearing-off” in which the therapeutic benefit of L-DOPA diminishes
during or toward the end of a dose (Obeso et al., 2000).
In addition to these motor-related complications, DA-replacement therapy has not been
shown to consistently improve cognitive disturbances in PD, particularly early in disease
progression. As mentioned above, there is preferential loss of DA in the dStr, and its related
8
projections such as the dorsal lateral PFC (which plays a role in executive function). However,
the vStr and its related projections such as the medial PFC (which plays a role in decision
making) are relatively spared during the early and middle stages of PD. The dose of DA
replacement drugs used to mitigate the motor disturbances observed in PD can lead to excessive
stimulation of the brain regions, like the vStr that are relatively spared (i.e. maintain relatively
normal levels of DA) in early PD. Thus, while DA replacement therapy may exert beneficial
effects on some cognitive functions like set-shifting, it has deleterious effects on other cognitive
functions like decision-
making (Table 1).
Since the current
treatment options are highly
unsatisfactory, there is an
urgent need to develop new
therapeutic strategies that
modify disease progression
and/or improve symptomatic
treatment of PD without
inducing adverse effects.
Importantly, exercise is a promising therapeutic strategy that has been shown to exert beneficial
effects on both motor and cognitive features of PD, but the mechanisms underlying these
benefits are not fully understood.
Table 1: Effects of DA-replacement therapy on cognitive
function in PD
Cognitive Benefit
Set-shifting
Working memory
Spatial working memory
Cognitive
Deterioration
Concurrent learning
Probabilistic reversal learning
Decision making
Visual hallucinations
No effect
Attentional set shifting
Task switching-abstract rules
Pattern/spatial recognition memory
Associative learning
Verbal Memory
(Kehagia et al., 2010, Martínez-Horta and Kulisevsky, 2015)
9
1.2 Experience-dependent neuroplasticity: Exercise in PD
As mentioned briefly above, experience-dependent neuroplasticity refers to the process
by which the brain encodes new experiences and learns new behaviors. Neuroplasticity
encompasses a wide range molecular (i.e. receptor density, neurotransmitter release), structural
(i.e. dendritic spine formation), physiological (i.e. cell signaling), circuit (i.e. functional
connectivity, blood flow), and behavioral (i.e. motor performance, cognitive function) processes.
These neuroplastic processes are modified in response to a number of different experiences,
including: development, learning, exercise/physical activity, emotional experience, stress, injury
and/or trauma. Studies in this dissertation will examine experience-dependent neuroplasticity in
response to two aspects: brain injury (DA-depletion using the MPTP-lesioned mouse model of
PD), and physical exercise in the form of high intensity treadmill exercise.
Exercise is a general term used to define a “type of physical activity involving planned,
structured and repetitive bodily movement conducted for the purpose of improving or
maintaining one or more aspects of physical fitness,” (American College of Sports Medicine,
2013). Exercise includes a wide variety of physical activities that target different health-related
(cardiorespiratory endurance, body composition, muscular strength, muscular endurance, and
flexibility) and skill-related (agility, coordination, balance, power, reaction time and speed)
components of physical fitness (American College of Sports Medicine, 2013).
Substantial research suggests that exercise exerts beneficial effects in both individuals
with PD and in rodent models of DA-depletion. It is important to note that the type, duration, and
intensity of exercise paradigms vary dramatically across different research studies. In general,
both health-related exercise training protocols (i.e. aerobic training), and skill-related exercise
paradigms have been shown to exert beneficial effects on motor behavioral outcomes (i.e. gait
10
performance, including velocity, stride length, cadence, postural stability, gait rhythmicity, and
joint excursion), brain health (i.e. blood flow, trophic factors, and immune system), and
cognitive function (Petzinger et al., 2013). These beneficial effects of exercise have been
discussed in detail by others and are summarized in Figure 2 (Petzinger et al., 2013). However,
the mechanisms underlying the beneficial exercise effects in PD are not fully elucidated. The
studies discussed in this dissertation were designed to help address this gap in knowledge by
investigating exercise-dependent changes in DA and glutamate neurotransmission (receptor
levels, DA levels), corticostriatal plasticity (dStr LTD), and cognitive function (behavioral
flexibility using a reversal-learning T-maze task) in MPTP-lesioned mice.
Figure 2: Exercise and neuroplasticity in Parkinson’s disease. Research suggests that both goal-
based (skill-related) exercise and aerobic exercise exert beneficial neuroplastic effects in PD.
These neuroplastic effects include changes in synaptic strength and general brain health, which
may alter neural circuitry and ultimately improve motor and non-motor features of PD. Figure
from: Petzinger et al., 2013
11
1.3 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Model of DA-depletion
For these studies, we used the MPTP-lesioned mouse model of DA depletion, a
commonly used experimental model of PD. MPTP neurotoxicity, and it’s relation to PD, was
first realized in 1982, when several young drug users developed a rapidly progressive
parkinsonian syndrome caused by intravenous use of an analog of the narcotic merperidine
(Demerol) that was contaminated by MPTP. Further research isolated the MPTP compound and
determined that it induces similar biochemical, histological and pathological states as idiopathic
PD. For example, MPTP administration produces similar motor disturbances to those observed in
PD, such as tremor, rigidity, bradykinesia, and postural instability. Furthermore, MPTP
administration selectively degenerates dopaminergic neurons in the SNpc, thereby depleting
striatal DA.
The neurotoxic effects of MPTP in the brain are initiated by the 1-methyl-4-
phenylpyridinum ion (MPP+). After crossing the blood brain barrier, MPTP is metabolized by
monoamine oxidase B (MAO-B) in non-dopaminergic cells (i.e. glial cells) into its active
metabolite MPP+. MPP+ is released into the extracellular space, where it actively accumulates in
dopaminergic neurons in the SNpc via the DA transporter (DAT). MPP+ is further concentrated
in the mitochondria of these DA neurons, where it inhibits mitochondrial complex I activity,
which impairs mitochondrial function, decreases oxygen consumption and ATP production,
disrupts ion homeostasis, and increases free radical production and oxidative stress (Blandini and
Armentero, 2012). Furthermore, research suggests the selective vulnerability of the
dopaminergic neurons in the SNpc in PD and MPTP-models of DA-depletion is linked to the
exclusively high expression of DAT mRNA relative to other midbrain dopamine cell
populations, such as the ventral tegmentum area (VTA). Taken together, the MPTP mouse
12
serves as a good model to investigate plastic changes involving DA neurotransmission in the
DA-depleted striatum, and its related cortical connections at the molecular, cellular and
physiological levels.
1.4 Dopamine (DA)
3-hydroxytyramine (dopamine, DA) is a catecholaminergic neurotransmitter that
generally exerts actions on neuronal circuitry by modulating fast-acting neurotransmitter systems
including glutamate and γ- aminobutyric acid (GABA). Catecholamine’s are derived from the
amino acid tyrosine, and include epinephrine (adrenaline), norepinephrine (noradrenaline), and
DA. L-Phenylalanine is converted into L-tyrosine by the enzyme phenylalanine hydroxylase
(PAH), which in turn is converted to L-DOPA by the enzyme tyrosine hydroxylase (TH). L-
DOPA is subsequently converted into DA by the enzyme aromatic L-amine acid decarboxylase.
DA itself is used as the precursor for norepinephrine and epinephrine. TH is the rate-limiting
enzyme in DA synthesis, and is often used as a molecular marker for DA levels in brain samples
(Daubner et al., 2011).
DA is stored in vesicles via vesicular monoamine transporter (VMAT-2). Upon
stimulation, vesicles within the nerve terminals fuse with the presynaptic terminal and release
DA into the synaptic cleft, where it either activates DA receptors, or is removed from the
synaptic cleft by DAT. DA is metabolized by monoamine oxidase (MAO), aldehyde
dehydrogenase (ALDH) and catechol-O-methyl transferase (COMT) into 2 main metabolites:
DOPAC (3,4-Dihydroxyphenyl-acetic acid) and HVA (homovanillic acid). DA levels, and DA
metabolites can be measured via High-performance liquid chromatography (HPLC).
13
1.4.1 Dopamine Pathways in Brain
There four major DA pathways in the brain including (1) the nigrostriatal pathway, (2)
mesocortical pathway, (3) mesolimbic pathway, (4) tuberoinfundibular pathway (Figure 3). The
nigrostriatal pathway is involved in the coordination of motor movement and is of key interest in
PD. It originates in DA neurons in the SNpc, and projects to the striatum (Smith and Villalba,
2008). The mesolimbic pathway is associated with pleasure, reward and goal directed behavior.
It originates from DA neurons in the VTA, and projects to the limbic area via the vStr (Nucleus
accumbens, NAcc). The mesocortical pathway is associated with motivational and emotional
responses. It also originates from DA cells in the VTA, and projects to the frontal cortex
(anterior cingulate cortex, entorhinal cortex, PFC, olfactory bulb). The tuberoinfundibular
pathway regulates the secretion of prolactin by the pituitary gland and is involved in maternal
behavior. It originates from DA cells in the hypothalamus (arcuate nucleus) and projects to the
pituitary gland (Beaulieu and Gainetdinov, 2011).
Figure 3: Four Main Dopamine Pathways in the Brain: (1) nigrostriatal pathway (in blue, SNpc
compacta to the dorsal striatum (2) mesolimbic (in red, VTA to limbic area via the vStr/NAcc,
(3) mesocortical (in grey, ventral tegmentum area to frontal cortex), and (4) tuberoinfundibular
(in green, hypothalamus to pituitary). Figure from: (Leucht et al., 2011)
14
These DA cells, and their respective pathways, play a key role in a number of important
central nervous system functions including motor control, arousal, cognition, reward, sleep,
attention, and learning (Beaulieu and Gainetdinov, 2011). Perturbations to the DA-system are
linked to a number of disorders including, PD, Huntington’s disease, schizophrenia, ADHD, and
Tourette’s syndrome. Important to the work discussed herein, loss of DA neurons in the SNpc,
and the corresponding depletion of nigrostriatal DA, is believed to contribute to the motor and
cognitive features of PD.
1.4.2 Dopamine Receptors
DA exerts physiological affects by activating G-protein coupled DA receptors (D
1
R,
D
2
R, D
3
R, D
4
R, D
5
R) (Beaulieu and Gainetdinov, 2011). DA receptors are divided into two
major classes based on structural, pharmacological, and signaling properties: D
1
-like receptors
(D
1
R and D
5
R subtypes), and D
2
-like receptors (D
2
R, D
3
R and D
4
R subtypes) (Thompson et al.,
2010) (Figure 4). The work discussed in this dissertation will focus on the D
1
R and the D
2
R
because they are the most abundantly expressed DA receptors in brain (Jaber et al., 1996, Lidow
et al., 2003, Araki et al., 2007), and on the D
4
R because it is the dominant D
2
-like receptor in the
PFC (Jaber et al., 1996, Falzone et al., 2002, Puig et al., 2014).
D
1
Rs and D
2
Rs are expressed at highest levels in the nigrostriatal, mesolimbic, and
mesocortical areas such as the dStr, vStr/NAcc, olfactory bulb and frontal cortex, and at lower
levels in the hippocampus, hypothalamus, SN and VTA. The D
4
R is expressed at lowest levels in
the brain, however it is highly expressed in the PFC. It is also expressed at lower levels in the
amygdala, GP, hippocampus, and hypothalamus (Jaber et al., 1996, Beaulieu and Gainetdinov,
2011, Brichta et al., 2013).
15
Figure 4: Distribution of Dopamine Receptors in the Brain. The work in this dissertation focuses
on the D
1
R, D
2
R and D
4
R. D
1
R and D
2
R expression levels are highest in the dStr and the
vStr/NAcc, and at lower levels in the hippocampus, hypothalamus, frontal cortex, SNpc and the
VTA. The D
4
R is expressed at lowest levels in the brain. However, D
4
R expression levels are
highest in the PFC, and at lower levels in the hippocampus and hypothalamus. Image From:
(Brichta et al., 2013)
1.4.3 Dopamine Signaling Pathways
The D
1
- and D
2
-like receptors differ functionally in the intracellular signaling pathways
they modulate. DA signaling exerts many downstream effects, however DA receptor functions
are often associated with the regulation of cyclic AMP (cAMP) and protein kinase A (PKA)
signaling. D
1
-like receptors couple to Gα
s/olf
proteins to activate adenylate cyclase (AC), and
subsequently stimulate cAMP production, and enhance PKA activation. On the other hand D
2
-
like receptors couple to Gα
i/o
proteins that inhibit AC, and thus decrease cAMP and PKA activity
(Beaulieu and Gainetdinov, 2011). PKA exerts actions on several substrates including cAMP
response element-binding protein (CREB), ionotropic glutamate receptors (i.e. AMPA
receptors), GABA receptors, and certain ion channels (voltage-gated potassium, K
+
, sodium, Na
+
and calcium, Ca
2+
channels) (Gerfen et al., 1990; Kebabian & Calne, 1979). Of these substrates,
16
the 32-kDa dopamine and cAMP-regulated phosphoprotein (DARPP-32) is perhaps the most
extensively studied molecules involved in DA receptor signaling. DARPP-32 is a multifunctional
phosphoprotein that modulates cell signaling in response to several different neurotransmitters,
including DA. This pathway is of particular interest because it has been shown to modulate
glutamate receptors (Figure 5). Briefly. D
1
R stimulation drives synaptic GluA1 insertion, and
enhances Glu2-lacking AMPAR expression. Conversely, D
2
R stimulation reduces GluA1
synaptic insertion, and restores GluA2-containing AMPAR expression. AMPARs will be
discussed in more detail below.
Figure 5: Dopamine Signaling Pathways in the Brain. Dopamine D
1
- and D
2
-like signaling exerts
opposing effects on intracellular signaling pathways that regulate a number of downstream
targets including GluA1 surface expression. Green lines=stimulatory signaling, red
lines=inhibitory signaling, Green circles=PKA-dependent phosphorylation sites
(DARPP32thr34; GluA1ser845). AC= adenylate cyclase; PKA= protein kinase A, DARRP32=
32-kDa dopamine and cAMP-regulated phosphoprotein
1.5 Glutamate Neurotransmitter System
Glutamate is a key excitatory neurotransmitter in the central nervous system, and aberrant
glutamatergic neurotransmission has been described in PD. Glutamate receptors are classified
into two main types: ionotropic (AMPA, NMDA and Kainite) and metabotropic receptors
D
1
-like
(D
1
, D
5
)
D
2
-like
(D
2
, D
3,
D
4
)
AC
PKA
DARPP32
Thr
34
P
PP-1
GluA1
Ser
845
P
GluA1
GluA1
GluA2-lacking
AMPARs
= stimulatory signaling
= inhibitory signaling
P
= PKA-dependent
phosphorylation site
17
(mGluRs). The work herein will focus on the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid receptor (AMPAR) type glutamate receptor because it plays a key role in modulating
experience-dependent neuroplasticity, and alterations in AMPAR expression have been linked to
other neurological disorders (Alt et al., 2006, Conrad et al., 2008, Kessels and Malinow, 2009,
Chang et al., 2012). Changes in AMPAR expression have also been observed in the DA-depleted
brain, and may contribute to potentiated glutamatergic signaling observed in PD (Wullner et al.,
1993, Betarbet et al., 2000, Brown et al., 2005, Picconi et al., 2005, Ba et al., 2006, Ouattara et
al., 2010, VanLeeuwen et al., 2010).
1.5.1 AMPA-type Glutamate Receptors (AMPARs)
AMPARs mediate most fast, excitatory neurotransmission in the central nervous system
and are believed to underlie many complex behaviors such as learning and memory. AMPARs
are tetramers comprised of various combinations of four different subunit proteins: GluA1,
GluA2, GluA3 and GluA4. AMPARs primarily exist as heteromers, however AMPARs can form
homomers especially if one subunit is over expressed (Nakagawa, 2010). All subunits share
similar structural organization: extracellular N terminus (N terminus domain, NTD and ligand
binding domain, LBD), intracellular C terminus (CTD) and an ion channel domain consisting of
four hydrophobic membrane-spanning domains (M1-M4). Three of these membrane domains
actually span the membrane (M1, M3 and M4), while M2 is a re-entrant membrane loop that
faces the cytoplasm (Figure 6) (Dingledine et al., 1999, Nakagawa, 2010).
18
Subunit composition, particularly the
presence or absence of GluA2, dictates
important electrophysiological channel
properties including calcium permeability
(Ca
2+
) and rectification (Rossmann et al.,
2011). GluA2 subunits are post-
transcriptionally edited to contain an
arginine (R) residue instead of the
genomically encoded glutamine residue (Q)
at position 607 of the M2 membrane loop region (Q/R site). Q/R editing is unique to the GluA2
subunit, and it occurs in 95% of GluA2 mRNA transcripts in the brain (Isaac et al., 2007). Q/R
editing of GluA2 changes the biophysical properties of the subunit because arginine introduces a
positive charge to the pore-lining M2 region. The positive charge in the pore renders the channel
impermeable to divalent cations like Ca
2+
and prevents channel block by endogenous
intracellular polyamines (Cull-Candy et al., 2006, Isaac et al., 2007). GluA2 subunits serve as the
dominant phenotype in AMPARs, thus GluA2-containing AMPARs (i.e. GluA2-GluA1
heteromers) are impermeable to Ca
2+
, exhibit reduced single-channel conductance, and display a
linear current-voltage (I-V) relationship (Pellegrini-Giampietro et al., 1997). Conversely, GluA2-
lacking channels (i.e. GluA1 homomers or GluA1-GluA3 heteromers) are permeable to Ca
2+
,
exhibit high single channel conductance, and display inwardly rectifying I/V relationships
(rectification) at depolarized membrane potentials due to a voltage-dependent block by
endogenous intracellular polyamines (Rossmann et al., 2011). Under normal conditions, GluA2-
lacking receptors represent a very small percentage of neuronal AMPARs (Mansour et al., 2001).
Figure 6. Basic structure of AMPARs
highlighting the Q/R editing site (*= Q/R
site). Figure from (Dingledine et al., 1999)
19
However, synaptic activity modulates AMPAR channel expression, and the dynamic regulation
of these channels is believed to underlie important processes including learning and memory.
1.6 Experimental models of learning and memory
Years ago, Hebb postulated that changes in synaptic potentiation and depression were the
cellular basis of learning (Hebb, 1949). He claimed, "when an axon of cell A is near enough to
excite a cell B and repeatedly or persistently takes part in firing it, some growth process or
metabolic change takes place in one or both cells such that A’s efficiency, as one of the cells
firing B, is increased” (Hebb, 1949). Hebb's postulate serves as the basis for Hebbian plasticity.
Hebbian plasticity can be defined as, "rapid, long-lasting changes in synapse strength, in which
consistently correlated pre- and postsynaptic activity drives synapse strengthening, and weakly
correlated activity drives synapse weakening" (Feldman, 2009). Synaptic changes observed in
Hebbian forms of plasticity are rapid, input specific and associative and therefore reinforce
synaptic connections that are active with a specific set of stimuli and weaken synaptic
connections that are not active during a specific set of stimuli. Hebb's postulate is supported by
experimental evidence of two forms of activity-based learning: long-term-potentiation (LTP) and
long-term-depression (LTD).
1.6.1 Long-term potentiation:
Long-term potentiation, or LTP, is defined as a "rapid, long-lasting increase in synaptic
strength induced by a specific neural activity pattern; usually brief, strongly correlated pre- and
postsynaptic activity" (Feldman, 2009), and represents the prime experimental model of learning
and memory in neuronal circuits. Correlated firing between pre and postsynaptic neurons
increases the synaptic strength between the two neurons and enhances the likelihood that pre-
synaptic firing will cause the post-synaptic cell to fire. In other words, after LTP is induced, the
20
induction threshold for subsequent rounds of potentiation is successively reduced and this
process could theoretically push synaptic strength to the limit or, on the other hand, to zero
(Caporale & Dan, 2008; Turrigiano & Nelson, 2004).
Figure 7: AMPAR Dynamics During LTP Induction and Maintenance. Under basal conditions,
GluA2-lacking receptors represent a very small percentage of neuronal AMPARs. During LTP
induction, GluA2-lacking AMPARs are transiently expressed at synaptic sites. During LTP
expression (also referred to as LTP maintenance), GluA2-lacking receptors are gradually
replaced by GluA2-containing AMPARs. Figure from: (Man, 2011)
Experimental LTP can involve many different mechanisms depending on brain region,
cell type, developmental stage, and induction protocol. Importantly, research suggests LTP
induction and maintenance involve changes in AMPARs (Figure 7). LTP induction involves the
transient expression of GluA2-lacking AMPARs. After LTP induction, GluA2-lacking receptors
are gradually replaced by GluA2-containing AMPARs. It is important to emphasize that while
GluA2-containing receptors have been shown to replace GluA2-lacking receptors, GluA2-
containing receptors are not required for the induction of LTP. In fact, GluA2 mutant mice
display enhanced LTP (Jia et al., 1996). Thus, GluA2 AMPAR insertion may actually represent a
homeostatic mechanism to counteract increased Ca
2+
permeability caused by GluA2-lacking
AMPAR insertion. Presynaptic mechanisms have also been proposed to induce LTP. For
example, nitric oxide (NO) can act as a retrograde signaling molecule to increase presynaptic
21
neurotransmitter release (Malenka and Bear, 2004). However, the work herein will focus on
post-synaptic changes in AMPAR expression.
1.6.2 Long-term depression:
Long-term depression, or LTD, is defined as "rapid, long-lasting decrease in synaptic
strength induced by a specific neural activity pattern; usually sustained, weakly correlated pre-
and postsynaptic activity" (Feldman, 2009). LTD is widely expressed in the CNS, and the
mechanisms underlying LTD may vary dramatically depending on the location and stimulation
protocol used to elicit LTD. Many different forms of LTD have been identified including:
NMDA-dependent LTD, mGluR-dependent LTD, and CB1-dependent LTD. As mentioned
above, the work herein will focus on post-synaptic changes in striatal LTD, and more specifically
how these changes relate to changes in AMPAR expression.
Post-synaptic changes in AMPAR expression are also observed in LTD, and are
associated with intracellular CA
2+
signaling. The mechanism by which intracellular Ca
2+
induces
LTD is not fully elucidated, but likely involves interactions with protein phosphatases, which
enzymatically cleave a phosphate group from a substrate. Ca
2+
can activate protein phosphatases
like calcineurin (CN) and protein phosphatase 1 (PP1). CN regulates internalization of both
AMPA and NMDA receptors. CN also mediates actin depolymerization and thus may contribute
to morphological changes observed in spines after LTD-induction. Furthermore, CN exerts
effects by disinhibiting PP1. PPI regulates gene expression by dephosphorylating and thus
inactivating CREB. PP1 also promotes activity-dependent endocytosis of AMPARs via
dephosphorylaiton of GluA1 at ser-845 (a PKA substrate, see Figure 5) (Pöschel & Stanton,
2007), and interfering with GluA1 dephosphorylation can block LTD (Lee et al., 2003)(Malenka
& Bear, 2004). Overall, LTD involves Ca2+ and the subsequent internalization of AMPARs,
22
which results in decreased neuronal excitability and synaptic strength. Striatal plasticity will be
discussed in more detail below.
1.6.3 Homeostatic Plasticity:
Homeostatic plasticity is a negative feedback mechanism that elicits compensatory
changes in excitatory and inhibitory signaling to regulate synaptic strength and stabilize neuronal
firing (Turrigiano, 2011). Briefly, homeostatic plasticity is a mechanism by which neurons
and/or circuits constrain runaway potentiation or depression induced by LTP or LTD.
Homeostatic plasticity infers that neurons and/or circuits can sense activity levels, compare these
levels with some internal set point and then modify synaptic properties to stabilize firing
(Turrigiano, 2011). Neurons and/or circuits can regulate synaptic activity by: (1) modulating
intrinsic excitability, (2) regulating excitatory/inhibitory synaptic strength or synapse number, or
(3) adjusting the threshold in which other forms of plasticity (i.e. LTP/LTD) can be induced
(Abraham, 2008; Turrigiano, 2008)
In summary, LTP/LTD changes manifest on a short time scale, are highly localized
(primarily at the level of a single synapse) and operate in a positive feedback manner. If left
unchecked, Hebbian plasticity could drive synaptic signals towards their maximum or minimum
values. However, this phenomenon of pushing synaptic strength to the limit or to zero is not
observed. Rather, there appears to be homeostatic mechanisms that mediate Hebbian learning
(Turrigiano, 2011). These experimental forms of learning and memory reflect neuroplastic
changes in the potentiation/depotentiation of synaptic strength, and are associated with changes
in DA and glutamate neurotransmission. The work herein will focus on the potential of exercise
to restore neuroplasticity, namely striatal forms of LTP and LTD in MPTP-mouse model of DA-
depletion.
23
1.7 Basal Ganglia (BG) Circuit
As mentioned above, loss of DA input coupled with increased glutamatergic drive in the
striatum alters basal ganglia (BG) circuitry, which is believed to adversely affect motor and
cognitive functions in PD. The BG is a group of subcortical nuclei involved in coordinating
information from sensorimotor, motivational, and cognitive brain areas to control behaviors such
as movement and motor learning. The BG is comprised of the striatum (in primates this consists
of the caudate nucleus and putamen), the external and internal segments of the Globus Pallidus
(GPe and GPi respectively), the subthalamic nucleus (STN), and the substania nigra pars
reticulata (SNr) (Blandini et al., 2000) (Figure 8). In the classic model of the basal ganglia, the
striatum is the main input nuclei, and the GPi and SNr are the principal output nuclei. Input to
the striatum is processed in two main pathways: (1) a monosynaptic ‘direct’ pathway to the
GPi/SNr that facilitates movement; (2) a polysynaptic indirect pathway to the GPi/SNr via the
GPe and the STN that inhibits motor movement. The GPi/SNr neurons project to several
thalamic nuclei, which in turn innervate prefrontal, premotor, and motor cortical areas (Redgrave
et al., 2010).
Figure 8: Diagram of Select Basal Ganglia (BG) Circuits. The striatum is the main input nuclei,
and the Globus Pallidus (GPi) and Substantia Nigra pars reticulata (SNr) are the main output
nuclei of the BG. The striatum receives glutamatergic input from the cortex and the thalamus,
24
and dopaminergic input (not shown in diagram) from the SNpc and VTA. Striatal signaling is
divided into the direct and indirect pathways, which exert opposing effects on the BG circuit.
The direct pathway originates from D
1
R-MSNs that project to the GPi and SNr output nuclei.
The indirect pathway originates from D
2
R-MSNs that project to the external segment of the
Globus Pallidus (GPe), which together with the subthalamic nucleus (STN) project to the to the
GPi and SNr output nuclei. The GPi and SNr project to the thalamus, superior colliculus, and
pendunculopontine nucleus (PPN). Figure from: (Gerfen and Surmeier, 2011)
Even though this “classic” model of the direct and indirect pathway remains valid, it is
now recognized that it represents a subset of the connections between BG nuclei. For example,
anatomical data indicates that the direct pathway also sends information to the GPe, the GPe not
only sends information to the STN but also connects directly to the GPi/SNr, and the STN is now
also considered a major input station to the BG (Redgrave et al., 2010). Nevertheless, the basic
structure will be sufficient for the data discussed herein.
1.7.1 Corticostriatal projection pathways
The striatum is the main input nuclei to the BG, and it can be subdivided at different
regions. These regions appear histologically similar, and are not divided by a sharp anatomical
border, but instead by differences in afferent connections. The major division is the dorsal (dStr,
which is further divided into the dorsolateral and dorsomedial regions) and ventral (vStr, which
is further divided into the nucleus accumbens core and shell) striatum. To start, the dStr receives
dense DA input from the SNpc, whereas the vStr is largely innervated by DA cells in the VTA
(Gerfen and Surmeier, 2011).
With regards to glutamate, the motor and premotor cortices project to the dorsolateral
striatum, the dorsolateral PFC and the lateral orbitofrontal cortex project to the dorsomedial
striatum, and the medial orbital frontal and anterior cingulate cortices enervate the vStr.
Ultimately, these cortical-striatal projection pathways form the associative, sensorimotor and
limbic loops, respectively. The associative circuit (dorsomedial striatum) plays a key role in
25
action selection, and is important for the early phases of skill acquisition (goal-directed
behavior). For example, it processes cognitive aspects regarding the selection of conditioned
stimuli that guide action toward the goal (Tremblay et al., 2015). The dorsolateral striatum is part
of the sensorimotor loop, and is involved in the selection/preparation and execution of
movement. Importantly, the sensorimotor loop is more involved once the skill is established and
the action program becomes more automatic (habit learning). On the other hand, the vStr is part
of the limbic circuit, and plays a role in motivation and reward learning. Specifically, this circuit
processes the motivational processes for goal selection including the expected outcome, and
reward (Macdonald and Monchi, 2011, Shiflett and Balleine, 2011, Tremblay et al., 2015).
It is important to note that there is some functional overlap between the striatal sub-
regions even though some striatal-dependent tasks preferentially activate one striatal sub-region
relatively more than other sub-regions (Cerovic et al., 2013). Furthermore, there is evidence for
interaction between these different circuits via cortico-cortical projections, and thalamic relay
nuclei (Tremblay et al., 2015).
1.7.2 Striatum: Organization and Function
At the cellular level, medium spiny neurons (MSNs) are the main neuronal population in
the striatum, representing 90-95% of all striatal cells. MSNs utilize γ-aminobutyric acid (GABA)
as a neurotransmitter and they express high levels of DA and glutamate receptors (Beaulieu and
Gainetdinov, 2011).
There are two different MSN populations in the striatum, characterized by their axonal
projections and by differential expression of neuropeptides and neurotransmitter receptors in
each cell type: (1) direct pathways MSNs (D
1
R-MSNs, also referred to as striatonigral MSNs)
which preferentially express the D
1
R, substance P and dynorphin and (2) indirect pathway MSNs
26
(D
2
R-MSNs, also referred to as striatopallidal MSNs) that preferentially express the D
2
R,
adenosine A
2A
receptor (A
2A
R), and enkephalin (Figure 9) (Gerfen et al., 1990, Gerfen and
Surmeier, 2011, Gittis et al., 2011). Importantly, D
1
R-MSNs and D
2
R-MSNs respond differently
to DA. D
1
R-MSNs are excited by dopamine and thus increase GABAergic signaling, while D
2
R-
MSNs are inhibited by DA and thus suppress GABAergic signaling.
Functionally, activation of direct or indirect pathway MSNS exert opposing effects on
motor behavior. In brief, direct pathway activation facilitates motor behavior, whereas indirect
pathway activation suppresses it (Kreitzer and Malenka, 2008, Cerovic et al., 2013). Alterations
in striatal signaling observed in PD will be discussed in more detail below.
Figure 9: Medium Spiny Neuron (MSN) Cell Types in the Striatum. D
1
R MSNs, also called
direct pathway or striatonigral pathway MSNs, primarily express D
1
Rs and are activated by DA
to facilitate motor behavior. D
2
R-MSNs, also called indirect pathway or striatopallidal MSNs are
de-activated by DA to facilitate motor behavior. Adenosine A
2A
Rs are selectively expressed on
D
2
R-MSNs in the striatum. D
2
R-MSNs are activated by adenosine A
2A
Rs to suppress motor
behavior.
In addition to MSNs, there are four types of aspiny interneurons in the striatum: (1)
tonically active giant aspiny cholinergic neurons (TANS), (2) fast-spiking GABAergic
Receptor Pathway Result of Activation
D
2
R Indirect
Inhibits D
2
R$MSN(firing
Facilitates behavior
A
2A
R Indirect
Facilitates D
2
R-MSN firing
Suppresses behavior
D
1
R Direct
Facilitates D
1
R-MSN firing
Facilitates behavior
D
2
-MSN
Enkephalin
A
2A
R
D
2
R
D
1
-MSN
Substance P
dynorphin
D
1
R
27
interneurons that express parvalbumin, (3) low-threshold spiking interneurons that express
somatostatin-, neuropeptide Y-, nitric oxide synthase, and nicotinamide adenine dinucleotide
phosphate-diaphorase (NADPH-d) and (4) GABAergic interneurons that express calretinin
(Kawaguchi, 1997, Wu and Parent, 2000). Striatal interneurons receive input from cortical and
thalamic projections and can regulate striatal output by modulating MSNs and other interneurons
(Tepper and Bolam, 2004).
1.7.3 Synaptic plasticity in the Striatum
DA plays a key neuromodulatory role in the expression of LTP and LTD in the striatum.
Calabresi and collaborators first described the role of DA in striatal synaptic plasticity (Calabresi
et al., 1992). They found that the application of D
1
R or D
2
R agonist/antagonists, as well as DA
depletion via 6-hydroxy dopamine (6-OHDA), alters the expression of striatal LTP/LTD. Over
the last two decades, a large body of research has documented the different mechanisms
underlying striatal forms of synaptic plasticity. In brief, both LTP and LTD have been observed
in the striatum, but striatal plasticity is expressed differently in indirect and direct pathway
MSNs.
In the healthy brain, LTD is more commonly expressed in indirect pathway/D
2
R-MSNs
than LTP. In D
2
R-MSNs, LTD of excitatory inputs occurs in response to high frequency
stimulation paired with postsynaptic depolarization, or in response to negative spike timing (i.e.
synaptic stimulation delivered after the MSN spikes). Importantly, successful induction of
indirect-pathway LTD involves the activation of post-synaptic D
2
R’s, and on the lack of
activation of post-synaptic A
2A
Rs (Calabresi et al., 1997, Kreitzer and Malenka, 2007, Shen et
al., 2008, Lerner et al., 2010). Furthermore, induction of LTD in D
2
R-MSNs is linked to
activation of mGluR and L-type Ca
2+
channels, which in turn activate endocannabinoids (Lerner
28
and Kreitzer, 2011). Conversely, induction of LTP in D
2
R-MSNs involves the activation of
A
2A
R, as well as the lack of D
2
R activation. The relative activation of D
2
Rs and A
2A
Rs
modulates the expression of plasticity observed in D
2
R-MSNs. Consequently, high levels of DA
and low levels of adenosine will shift plasticity in D
2
R-MSNs towards LTD, whereas low levels
of DA and high levels of adenosine will promote LTP (Lerner and Kreitzer, 2011).
Unlike indirect pathway MSNs, LTP is the predominate form of plasticity expressed in
direct pathway MSNs/D
1
R-MSNs. The expression of LTP in the direct pathways involves D
1
R
activation. LTD in D
1
R-MSNs is not as well studied. Nevertheless, the mechanisms of LTD
induction are similar to that observed in D
2
R-MSNs, including mGluR activation and L-type
Ca
2+
channel signaling (Lerner and Kreitzer, 2011).
1.7.4 Striatal Signaling Pathways and Alterations in PD
As mentioned above, activation of direct or indirect pathway MSNS exert opposing
effects on motor behavior. In brief, the direct pathway sends inhibitory GABAergic signals to the
GPi/SNr, which in turn disinhibits the thalamus. The thalamus then sends excitatory
glutamatergic input to the cortex to promote behavior. On the other hand, activation of the
indirect pathway sends inhibitory GABAergic signals to the GPe, which in turn disinhibits the
STN. The STN then sends excitatory signals to the GPi/SNr, which inhibits the thalamus, and
suppresses motor movement(Kreitzer and Malenka, 2008, Cerovic et al., 2013) (Figure 10). In
PD, the loss of striatal DA results in reduced inhibitory signaling from the direct pathway, and
increased excitatory signaling from indirect pathway on GPi/SNr, which in turn results in
increased inhibition of the thalamus, and ultimately the suppression of motor behavior.
Furthermore, D
2
R expression is significantly reduced in both humans with PD, and in
animal models of DA-depletion (Vuckovic et al., 2010b, Fisher et al., 2013). Reduced dStr D
2
R
29
expression contributes to the hyperactivation of the indirect pathway. As mentioned above,
GABAergic signaling is suppressed when DA binds to D
2
Rs. In the context of PD, loss of DA
and D
2
Rs results in increased GABAergic inhibition of the GPe, which ultimately leads to
increased inhibition of the thalamus and the suppression of motor behavior.
Figure 10: Alterations in the Basal Ganglia (BG) Circuit in Parkinson’s disease (PD). In PD,
there is a loss of striatal DA which results in reduced inhibitory signaling from D
1
R-MSNs of the
direct pathway, and increased excitatory signaling from the STN of the indirect pathway on the
GPi/SNr (the main output nuclei of the BG), which results in increased inhibition of the thalamus
from the GPi/SNr, and ultimately loss of motor control.
In addition to alterations in DA signaling, PD is associated with aberrant glutamatergic
neurotransmission. Specifically, DA-depletion leads to potentiated glutamatergic
neurotransmission in indirect pathway MSNs, resulting in an imbalance between the direct and
indirect MSN signaling pathways (Picconi et al., 2005). Furthermore, reduced expression of
corticostriatal LTD observed in D
2
R-MSNs DA-depleted striatum, which is consistent with
reduced D
2
R expression and increased glutamatergic drive in PD.
Importantly, exercise has been shown to improve motor behavior and increase the
expression of striatal D
2
Rs without increasing total striatal DA levels in both patients with PD
and in the MPTP-lesioned mouse. Furthermore, data from rodent models suggest exercise
GPe
Cortex
GPi/SNr
Thalamus
STN
SNpc
Normal
Cortex
Striatum
D
2
D
1
GPi/SNr
Thalamus
GPe
STN
SNpc
PD
Striatum
D
2
D
1
Dopamine
Glutamate
GABA
30
restores GluA2 expression in MPTP mice. However the relationship between GluA2-lacking
AMPAR expression and striatopallidal D
2
R-MSNs has not been fully defined. The data
discussed in Chapter 2 of this dissertation builds upon our previous work by evaluating whether
the observed changes in AMPAR expression with MPTP and exercise are selective to the
striatopallidal (D
2
R-MSNs) projection pathway. Furthermore, the studies discussed in Chapter 2
will evaluate if exercise restores corticostriatal LTD in the DA-depleted striatum.
1.8 Prefrontal Cortex
In addition to motor features, cognitive disturbances are observed in PD and are
associated with alterations in PFC function (Brozoski et al., 1979, Puig et al., 2014, Martínez-
Horta and Kulisevsky, 2015). The PFC, much like the striatum, is often referred to as a single
brain region, but can be broken down into different sub-regions based on different
cytoarchitecture, cytochemistry, connectivity and functional properties including the medial,
lateral and ventral parts (Figure 11). Briefly, the medial PFC is composed of the infralimbic,
prelimbic, anterior cingulate cortices. The lateral PFC is composed of the orbitofrontal cortex
and the dorsal and ventral anterior insular cortices. The ventral PFC is composed of the ventral
orbital and ventral lateral orbital cortices (Zingg et al., 2014).
Furthermore, the different regions of the PFC connect to different parts of the striatum,
and are involved in different behavioral processes. For example the dorsolateral regions involve
executive and cognitive-related functions, whereas the ventromedial portions are related to
emotional content and reward associations (Schubert et al., 2014) (see section 1.7.1 above for
more detail).
31
Figure 11: Corticostriatal Pathways Involving the PFC and Striatum. Modulatory dopaminergic
neurons (blue) project to the dorsal striatum via the SNpc (A9 neurons) and the ventral striatum
and prefrontal cortex (PFC) via the VTA (A10 neurons) in the rodent brain. From the striatum,
inhibitory GABA neurons (green) extend to multiple regions including the thalamus, which has
reciprocal excitatory glutamate connections (red) to the striatum, as well as connections to the
PFC. Prefrontal cortical efferent excitatory glutamate neurons extend to the striatum, NAcc,
SNpc, as well as the VTA. Figure from: (Miller et al., 2013)
It is important to note that defining these areas across species is difficult because of large
interspecies differences in the structural organization of the PFC. These differences have raised
some debate about how well the rodent PFC resembles the primate and the human PFC.
However, several studies have since investigated these issues, and it is now generally well
accepted that the rodent PFC is a homologous structure with similar functions to the primate and
human PFC (Schubert et al., 2014) (Figure 12).
32
Figure 12: Homology Between Human and Rodent PFC. (A) Schematic of human brain in which
Brodman’s area 46 is shaded gray. This region is heavily implicated in executive function. (B)
Schematic of the rodent mPFC, which is the functional neuroanatomical homologue of the
primate dorsolateral PFC. (C) a coronal section that better illustrates some subdivisions of the
PFC, including medial regions (prelimbic- PL, infralimbic-IL, and anterior cingulate cortices)
and lateral regions (orbitofrontal cortex-OFC). From (Bizon et al., 2012)
1.8.1 PFC Deficits in PD
DA plays a key neuromodulatory role in the PFC, and the loss of DA is believed to
contribute to PFC dysfunction in PD (Brozoski et al., 1979, Puig et al., 2014, Martínez-Horta and
Kulisevsky, 2015). Brozoski et al (1979) first reported that DA-depletion in the PFC of monkeys
impaired delayed alternation performance in a manner comparable to surgical ablation of the
frontal lobes (Brozoski et al., 1979). These findings that mesocortical DA regulates cognitive
processes have been replicated in a number of different studies over the last few decades. These
studies suggest DA regulates PFC cognitive processes in an “inverted-U” shaped format, where
too little or too much DA exerts detrimental effects on cognitive performance (Floresco, 2013).
In the context of PD, low levels of DA in the PFC may contribute to cognitive dysfunction,
namely deficits in executive function in working memory and behavioral flexibility. For example,
research conducted in DA-depleted animal models suggests that the loss of DA impairs working
memory and behavioral flexibility, but not spatial reference memory (hippocampal dependent
33
memory) (Tanila et al., 1998, Miyoshi et al., 2002, Decamp and Schneider, 2004, Braga et al.,
2005, Pothakos et al., 2009, Moriguchi et al., 2012, Darvas et al., 2014).
In addition to DA levels, DA signaling is influenced by the type and density of DA
receptors expressed in brain. Importantly, D
1
- and D
2
-like receptors (namely D
2
R and D
4
R) exert
opposing effects on PFC function. D
1
-like receptors are preferentially activated under lower
concentrations of extracellular DA, they increase pyramidal cell excitability, and D
1
Rs are
believed to favor stable working memory performance. Conversely, D
2
-like receptors are
preferentially activated under higher concentrations of extracellular DA, they decrease pyramidal
cell excitability, and D
2
Rs favor higher response flexibility (Trantham-Davidson et al., 2004,
Durstewitz and Seamans, 2008, Floresco, 2013). There remains, however, a significant gap in
knowledge regarding how DA receptor expression changes in the DA-depleted brain.
Exercise has been shown to improve motor function in individuals with PD and may also
be a promising therapeutic strategy for cognitive impairment. For example, several studies in
healthy aging individuals have demonstrated a significant beneficial effect of exercise on
cognition in physically fit individuals (Chang et al., 2012b, Hotting and Roder, 2013).
Preliminary evidence also suggests exercise improves cognitive function in PD, as demonstrated
by improved behavioral flexibility using the Wisconsin Card Sorting Task (WCST) (Tanaka et
al., 2009). However, the mechanisms underlying the beneficial effects of exercise are not fully
understood. The studies discussed in Chapter 3 of this dissertation will investigate if exercise
modulates PFC function (i.e. PFC DA levels and PFC D
1
R, D
2
R and D
4
R expression) and
improves executive function, specifically behavioral flexibility, using the MPTP-mouse model of
DA-depletion.
34
1.9 Brief Summary
Substantial research suggests that exercise exerts beneficial effects in both individuals
with PD and in rodent models of DA-depletion, but the mechanisms underlying these effects are
not fully elucidated. My lab investigates the mechanisms underlying the effects of high intensity
treadmill exercise on experience-dependent neuroplasticity in animal models of striatal DA-
depletion and in patients with PD (Fisher et al., 2004, Petzinger et al., 2007, Fisher et al., 2008,
VanLeeuwen et al., 2010, Vuckovic et al., 2010a, Fisher et al., 2013, Kintz et al., 2013b).
Given that potentiated glutamatergic neurotransmission is observed in PD, and may
contribute to aberrant striatal signaling and plasticity in PD, previous work from my lab
investigated if exercise modifies glutamatergic signaling, specifically AMPAR expression,
within the DA-depleted striatum. They found MPTP increased GluA2-lacking AMPAR
expression within the dStr, and exercise reversed this effect in MPTP mice (VanLeeuwen et al.,
2010). These findings suggest an important role of GluA2 in mediating the effects of experience-
dependent plasticity in the DA-depleted dorsolateral striatum. However the relationship between
GluA2-lacking AMPAR expression and striatopallidal D
2
R-MSNs has not been fully defined.
The work discussed in Chapter 2 builds upon these findings by investigating whether exercise
modulates AMPAR expression selectively in the striatopallidal (D
2
R-MSNs) projection pathway
in MPTP-mice. Furthermore, since AMPARs are known to play a key role in modulating
neuroplasticity, and because MPTP has been shown to reduce corticostriatal expression of long-
term depression LTD, we also evaluated if exercise restores corticostriatal LTD in MPTP-
lesioned mice.
Previous work from my also suggests exercise modifies DA signaling and D
2
R
expression within the striatum. Specifically, they found exercise improves striatal DA signaling
35
without changing total DA levels (i.e. enhances vesicular release of DA, increases synaptic
occupancy of DA, and decreases DA clearance through reduced DAT expression) in the MPTP-
mouse. Furthermore they found that exercise restores striatal D
2
R expression in the MPTP-
mouse and in patients with PD (Vuckovic et al., 2010a, Fisher et al., 2013). However, there
remains a gap in knowledge regarding whether exercise modifies PFC function in MPTP mice.
The study discussed in Chapter 3 builds upon this previous work by investigating if exercise
restores PFC DA levels, modulates PFC D
1
R, D
2
R, and D
4
R levels, and improves behavioral
flexibility during a reversal-learning T-maze task in MPTP-lesioned mice. Understanding the
molecular mechanisms underlying the beneficial effects of exercise in the DA-depleted brain
may provide key insight into new drug targets for the improved treatment of PD.
36
CHAPTER 2: Exercise modifies α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid
receptor (AMPAR) expression in striatopallidal neurons in the 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine-(MPTP)-Lesioned Mouse
Results of this chapter have been published in Journal of Neuroscience Research 91 (2013),
1492-1507
Author List: Natalie M Kintz, Giselle M Petzinger, Garnik Akopian, Sara Ptasnik, Celia
Williams, Michael W Jakowec, and John P Walsh.
ABSTRACT
The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid-type glutamate receptor
(AMPAR) plays a critical role in modulating experience-dependent neuroplasticity, and
alterations in AMPAR expression may underlie synaptic dysfunction and disease
pathophysiology. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mouse model
of dopamine (DA)-depletion, our previous work found that exercise increases total GluA2
subunit expression, and the contribution of GluA2-containing channels in MPTP mice. The
purpose of this study was to determine whether exercise-dependent changes in AMPAR
expression after MPTP are specific to the striatopallidal (D
2
R) or striatonigral (D
1
R) medium
spiny neuron (MSN) striatal projection-pathways. Drd
2
-eGFP-BAC transgenic mice were used to
delineate differences in AMPAR expression between striatal D
2
R-MSNs
and D
1
R-MSNs.
Striatal AMPAR expression was assessed by immunohistochemical (IHC) staining, western
immunoblotting (WB) of preparations enriched for the post-synaptic density (PSD), and
alterations in the current-voltage relationship of MSNs. We found DA-depletion results in the
37
emergence of GluA2-lacking AMPARs selectively in striatopallidal D
2
R-MSNs and exercise
reverses this effect in MPTP mice. Exercise-induced changes in AMPAR channels observed
after DA-depletion were associated with alterations in GluA1 and GluA2 subunit expression in
post-synaptic protein, D
2
R-MSN cell surface expression, and the restoration of corticostriatal
plasticity. Mechanisms regulating experience-dependent changes in AMPAR expression may
provide innovative therapeutic targets to increase the efficacy of treatments for basal ganglia
disorders including PD.
SIGNIFICANCE STATEMENT:
The purpose of this study was to determine whether previously reported changes in
AMPAR expression with MPTP and exercise are selective to the striatopallidal (D
2
R-MSNs)
projection pathway in Drd
2
-eGFP-BAC transgenic mice. We found MPTP increased the
contribution of GluA2-lacking AMPARs selectively in D
2
R-MSNs and exercise reversed this
effect. Furthermore, these exercise-induced changes in MPTP-mice were associated with
alterations in GluA1/GluA2 subunit expression, the restoration of corticostriatal plasticity, and
improved motor performance. These data suggest changes in AMPAR expression in D
2
R-MSNs
may represent an important mechanism underlying the motor behavioral improvements seen with
exercise in MPTP-mice. Elucidating the mechanisms regulating experience-dependent changes
in AMPAR expression after DA-depletion may provide insight into new therapeutic targets for
improved treatment of Parkinson’s disease.
KEYWORDS: AMPA receptors, Parkinson’s disease, Plasticity, MPTP, Exercise.
38
INTRODUCTION
The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid receptor (AMPAR) is a fast
acting ionotropic glutamate receptor that plays a critical role in modulating experience-
dependent neuroplasticity. AMPARs are tetrameric channels composed of various combinations
of four glutamate receptor subunits, GluA1 through GluA4. Subunit composition, particularly the
presence or absence of GluA2, dictates important electrophysiological channel properties
including calcium permeability (Ca
2+
) and rectification (Rossmann et al., 2011). For example,
GluA2-containing AMPARs (i.e. GluA2-GluA1 heteromers) are impermeable to Ca
2+
and
display a linear current-voltage (I-V) relationship (Pellegrini-Giampietro et al., 1997).
Conversely, GluA2-lacking channels (i.e. GluA1 homomers or GluA1-GluA3 heteromers)
display high Ca
2+
-permeability and rectification at depolarized membrane potentials. Synaptic
activity modulates AMPAR channel expression, and the dynamic regulation of these channels is
believed to underlie important processes including learning and memory. Alterations in AMPAR
expression have been observed in neurological disorders including Alzheimer’s disease (AD),
depression, and drug addiction, and may contribute to aberrant synaptic glutamatergic
neurotransmission and disease pathophysiology (Alt et al., 2006, Conrad et al., 2008, Kessels
and Malinow, 2009, Chang et al., 2012a).
Changes in AMPAR expression have also been observed in dopamine (DA)-depleted
models of Parkinson’s disease (PD) (Wullner et al., 1993, Betarbet et al., 2000, Brown et al.,
2005, Ba et al., 2006, Ouattara et al., 2010, VanLeeuwen et al., 2010). Using the 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of DA-depletion, our lab previously
reported that MPTP increased synaptic GluA2-lacking AMPAR expression in striatal medium
spiny neurons (MSNs). Importantly, we found that exercise reversed this trend in MPTP mice,
39
and increased total striatal GluA2 subunit expression (VanLeeuwen et al., 2010). These findings
suggest an important role of GluA2 in mediating the effects of experience-dependent plasticity in
the DA-depleted dorsolateral striatum.
Aberrant glutamatergic neurotransmission has been described after DA-depletion.
Specifically, DA-depletion leads to potentiated glutamatergic neurotransmission in striatopallidal
(DA-D
2
receptor-containing; termed D
2
R-MSNs) projection pathway MSNs, resulting in an
imbalance between the striatopallidal and striatonigral (DA-D
1
receptor-containing; termed D
1
R-
MSNs) projection pathways (Picconi et al., 2005). Our previous work demonstrating increased
GluA2-lacking AMPAR expression after MPTP is consistent with increased glutamatergic
neurotransmission observed after DA-depletion (VanLeeuwen et al., 2010). However the
relationship between GluA2-lacking AMPAR expression and striatopallidal D
2
R-MSNs has not
been fully defined. This current study builds upon our previous work by evaluating whether the
observed changes in AMPAR expression with MPTP and exercise are selective to striatopallidal
(D
2
R-MSNs) projection pathway.
Drd
2
-eGFP-BAC transgenic mice expressing enhanced green fluorescence protein under
the control of the DA-D
2
R promoter were used to delineate differences in AMPAR expression
between D
2
R-MSNs and D
1
R-MSNs in the dorsolateral striatum (Gong et al., 2002, Chan et al.,
2012, Nelson et al., 2012). AMPAR expression was assessed by immunohistochemical (IHC)
staining in tissues, western immunoblotting (WB) in preparations enriched for the post-synaptic
density (PSD), and alterations in the current-voltage relationship of MSNs in slices. This study
specifically evaluated changes in GluA1 and GluA2 AMPAR subunits since they are
predominately expressed in the adult striatum and play a key role in modulating neuroplasticity
(Wang et al., 2004, Deng et al., 2007). We found that DA-depletion increased the relative
40
contribution of GluA2-lacking AMPAR channels in D
2
R-MSNs but not D
1
R-MSNs.
Importantly, exercise reversed this effect in MPTP mice. Elucidating the mechanisms regulating
experience-dependent changes in AMPAR expression after DA-depletion may provide insight
into new therapeutic targets for improved treatment of basal ganglia disorders including PD.
MATERIALS AND METHODS
Animals and Treatment Groups
Mice used for these studies were young adult (8 to 10 weeks old) male Drd
2
-eGFP-BAC
mice (Tg(Drd2-EGFP)118Gsat/Mmnc) supplied from the Mutant Mouse Regional Resource
Center of NIH (MMRRC) program at the Rockefeller University (Gong et al., 2003) and were
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 were randomly assigned to one of four treatment groups including: (i) Saline, (ii) Saline
plus exercise, (iii) MPTP, and (iv) MPTP plus exercise. Animals were housed 5 to a cage and
acclimated to a 12-hour shift in light/dark cycle so that the exercise occurred during the animals
normal wake period. A total of 315 mice were used for these studies. All experiments were
carried out in accordance with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals (NIH Publication No. 80-23, revised 1996) and approved by the University
of Southern California Institutional Animal Care and Use Committee (IACUC).
Some concern has been raised recently about the Drd
2
-eGFP-BAC mouse line and the
possibility that this BAC line does not express the physiology and associated behavior seen in
C57BL/6 mice (Kramer et al., 2011). However, recent work indicates Drd
2
-eGFP-BAC mice
backcrossed to C57/BL6 mice display normal behavior and DA neurotransmission (Taverna et
al., 2008, Chan et al., 2012, Nelson et al., 2012). No differences were detectable between Drd
2
-
41
eGFP-BAC mice and C57/BL6 mice in maximum running velocity on the treadmill, normal
striatal DA-levels, amount of DA- depletion, or degree of substantia nigra pars compacta (SNpc)
dopaminergic cell death resulting from systemic injections of MPTP in our striatal lesioning
protocol (Jackson-Lewis et al., 1995).
MPTP-Lesioning
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Sigma, Inc., St. Louis, MO) was
administered in a series of 4 intraperitoneal injections of 20 mg/kg (free-based) at 2-hour
intervals for a total administration of 80 mg/kg. This regimen leads to approximately 60 to 70%
loss of nigrostriatal neurons (as determined by unbiased stereological techniques for both TH
staining and Nissl substance and an 80 to 90% depletion of striatal DA levels (Jackson-Lewis et
al., 1995, Jakowec et al., 2004). Nigrostriatal cell loss is complete by day 3 after MPTP
administration as determined by counting remaining nigrostriatal tyrosine hydroxylase
immunoreactive cells and reduced silver staining for degenerating neurons (Jackson-Lewis et al.,
1995, Jakowec et al., 2004).
In this study, striatal DA levels of Drd
2
-eGFP-BAC mice were assessed by HPLC
analysis. HPLC analysis was conducted 5 days after lesioning, corresponding to the start of the
treadmill exercise paradigm, and at 42 days after lesioning, upon completion of the treadmill
exercise paradigm (Table 2). In the non-exercised groups, striatal DA was significantly reduced
by 88.2% in MPTP compared to Saline mice 5 days post-lesioning (p=0.002), and 86.3% in
MPTP compared to Saline mice 42 days post-lesioning (p=0.001). In the exercised groups,
striatal DA was significantly reduced by 86.0% in MPTP plus exercise compared to Saline plus
exercise mice 42 days post-lesioning (p=0.002). No significant difference in DA levels was
observed between Day 5 post-lesion MPTP no exercise, Day 42 post-lesion MPTP no exercise
42
and Day 42 post-lesion MPTP plus exercise mice [F(2,19)=0.5935, p=0.562]. No significant
difference in DA levels was observed between Day 5 post-lesion Saline no exercise, Day 42
post-lesion Saline no exercise or Day 42 post-lesion Saline plus exercise mice [F(2,20)=0.846
p=0.444]. Importantly, Drd
2
-eGFP-BAC mice and C57BL/6 mice were similarly sensitive to
MPTP. HPLC analysis of C57BL/6 mice lesioned with MPTP showed an 83% decline in striatal
DA compared to Saline mice (VanLeeuwen et al., 2010).
Table 2. HPLC Analysis of Striatal Dopamine
Treadmill Exercise and Tissue Collection (Figure 13)
One week before the start of the treadmill exercise paradigm (two days before MPTP
lesioning), 8-10 week old Drd
2
-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 protocol was conducted as previously described (Fisher et al.,
2004). Briefly, exercise was initiated 5 days following Saline or MPTP-lesioning after cell death
is complete and continued 5 days/week for a total of 28 days of exercise (corresponding to a final
43
of 42 days after MPTP-lesioning). Exercise mice started at a velocity of 10.0±1.5 m/min, which
increased to 24.0 ± 0.5 m/min by the final week. As we have previously reported, there was a
significant difference in velocity at weeks 1 to 4 between the Saline plus exercise and MPTP plus
exercise groups (p<0.05) (Fisher et al., 2004, Petzinger et al., 2007). This difference was
eliminated with further training and completion of the treadmill running regimen.
Figure 13. Study Timeline (see Methods). Abbreviations: DA, Dopamine; WB, Western
Immunoblot; PSD, Post-synaptic density; RI, Rectification Index; IHC, immunohistochemical
staining; LTD (long-term depression).
Striatal AMPAR expression was analyzed from brain tissue collected at two different
time points: (i) early time point (7 days after MPTP or Saline lesioning) from MPTP and Saline
mice; (ii) late time point (day 28 of exercise corresponding to 42 days after MPTP-lesioning)
from all four treatment groups (Figure 13). Western immunoblot and alterations in the current-
voltage relationship of MSNs were evaluated at both the early and the late time points.
Immunohistochemical staining, and alterations in synaptic plasticity were assessed at the late
time point.
Mice were sacrificed by cervical dislocation for fresh tissues (for western
immunoblotting) or by pentobarbital overdose followed by transcardial perfusion with 4%
PFA/PBS fixative (for immunohistochemistry). Striatal tissues for HPLC analysis were collected
44
fresh en block corresponding to anatomical regions Bregma 1.20 to 0.60, with borders dorsal to
the anterior commissure, ventral to the corpus callosum, medial to the lateral ventricle, and
lateral 2.5 mm from midline and frozen until analysis. Immunohistochemistry was carried out on
coronal sections corresponding to Bregma 1.30 to 0.00.
Immunohistochemical Staining
The relative expression of striatal AMPAR subunits GluA1 and GluA2 was determined in
tissue sections using commercially available primary antibodies including mouse monoclonal
anti-GluA2 (1:500, NeuroMab, Davis, CA) and mouse monoclonal anti-GluA1 (1:500,
Millipore, Billerica, MA). Sections were rinsed in TBS three times, blocked for an hour in TBS
plus 10% normal goat serum (NGS) at RT, then incubated with primary antibody in TBS plus
4% NGS at 4
o
C for 48 hours. For GluA1 staining, sections were rinsed in TBS, and incubated in
secondary antibody (goat anti-mouse IgG Alexa
594
, LifeTechnologies, Carlsbad, CA) for two
hours at RT. For GluA2 staining, sections were rinsed with TBS and incubated in biotinylated
secondary antibody (goat anti-mouse IgG, 1:500; Vector Labs, Burlingame, CA) at 4
o
C for 24
hours, rinsed in TBS, and incubated in streptavidin-labeled CY3.5 (1:500, Rockland,
Gilbersville, PA) for one hour at RT. After staining, sections were mounted on gelatin-subbed
slides, dried, and cover-slipped using Vectashield Mounting Medium with DAPI.
For image analysis, images were captured in a blinded fashion at 60x magnification from
sections sliced in the coronal orientation focusing on the dorsolateral striatum ranging from
Bregma 1.20 (where the anterior commissure is seen directly below the most ventral point of the
lateral ventricle) to Bregma 0.20 (where the anterior commissure bridges both hemispheres).
Approximately 45 cells in the dorsolateral striatum were selected for analysis per animal using
Metamorph (Molecular Devices, Sunnyvale, CA). Cells were selected if there was a visible
45
nucleus stained with DAPI, evidence of nucleoli, the presence of cytoplasm, and a cell body
diameter of approximately 12±2 microns (Matamales et al., 2009). The relative optical density
for immunofluorescence staining was measured by selecting a region of interest (ROI) around
GFP expressing cell bodies (D
2
R-MSNs), and non-GFP expressing cell bodies (D
1
R-MSNs) by
tracing the perimeter of the cell body, which was distinct from the neuropil. The level of
background fluorescence (non-cell body staining) was determined by selecting a ROI in the
corpus callosum of the same tissue section where no cell bodies of any kind were present based
on DAPI staining. Background fluorescence was subtracted from each cell body ROI within the
same section to determine actual cell body staining. Multiple sections from each of the different
treatment groups were handled in identical staining conditions concurrently to ensure that any
differences in staining intensity were due to differences in antigen expression. The final optical
density values for an entire group were normalized to the Saline treatment group for comparison,
which were designated as 1.00 arbitrary O.D. units. A total of 24 animals (6 per treatment group)
were used for this analysis.
Western Immunoblotting of Post-synaptic Density (PSD) Protein
Western immunoblotting with samples enriched for the post-synaptic density (PSD) were
used to determine the relative expression of GluA1 and GluA2 proteins within the dorsolateral
striatum. Following decapitation, synaptoneurosomes were prepared as previously described
(Johnson et al., 1997, Williams et al., 2009) from pooled mouse dorsolateral striatum (6 to 8
mice per group) and used to isolate PSDs (Hahn et al., 2009). Briefly, synaptoneurosomes were
resuspended in 2.5 ml of 0.1 mM CaCl
2
and an equal volume of 40 mM Tris buffer containing
1% Triton X-100 (pH 6.0) followed by end-over-end mixing for 30 minutes. After centrifugation
at 35,000xg for 30 minutes, the pellet was resuspended sequentially in 2.5 ml of 0.1 mM CaCl
2
46
and 40 mM Tris with 1% Triton X-100 (pH 8.0), mixed end-over-end for 60 minutes and then
centrifuged at 140,000xg for 30 minutes. The PSD pellet was resuspended in 20 mM Tris with
1% SDS (pH 7.4) using a glass pestle and stored frozen at -80°C. For immunoblots 5 ug of the
PSD proteins, in SDS sample buffer, were added per lane. Synaptic vesicles (SV) and pre-
synaptic membrane proteins were precipitated from supernatants with acetone overnight at -
20°C.
Antibodies for western immunoblotting were the same as used for immunohistochemistry
except an antibody against beta-actin (Millipore, Inc., Billerica, MA) was used to determine
equal loading of total protein per gel lane. The immunoblotting technique was previously
described (Jakowec et al., 1995, Jakowec et al., 1998, Jakowec et al., 2001) with modifications
using the Licor Odyssey for Near Infra Red Fluorescent Western Blotting (Lincoln, NE). Filters
were blocked in Licor Blocking Solution (Gilbertsville, PA), then exposed to primary antibody
(1:1000) in the same Blocking Solution, exposed to secondary antibody, and visualized using the
Licor Odyssey. The intensity of bands was determined using Image J and expressed as relative
optical density (OD). PSD protein expression was analyzed at both the early (7 days post-MPTP)
and the late (42 days post-MPTP) time points. Four sets of PSD protein preparations were
analyzed for each group, each set consisting of pooled tissue from 6-8 animals, resulting in a
total of 24-32 animals per group at each time point. Only MPTP and Saline groups were
analyzed at the early time point. All four groups were analyzed at the late time point. Relative
optical density values for GluA1/Actin, GluA2/Actin, and GluA1/GluA2 were analyzed. For
comparison between the groups the OD levels for each set were compared to the Saline treated
group, which was normalized to 1.00 arbitrary OD units.
47
Electrophysiological Studies
Animals: Mice from all groups were anesthetized in a desiccator containing halothane
vapors, killed by decapitation, and brains removed. Tissue was blocked in cold low-sodium
sucrose-substituted saline (90 mM saline with 105 mM sucrose) and striatal coronal sections
were cut at 350-µm thickness in ice-cold low-sodium sucrose-substituted saline using a
Vibratome-1000 (Vibratome Co., St Louis, MO). Slices were stored in artificial cerebral spinal
fluid (aCSF consisting of 124 mM NaCl, 1.3 mM MgSO
4
, 3 mM KCl, 1.25 mM NaH
2
PO
4
, 25
mM NaHCO
3
, 2.4 mM CaCl
2
, and 10 mM glucose) at room temperature (23
o
C) for at least one
hour prior to recording. All solutions were continuously oxygenated with 95% O
2
and 5% CO
2
.
Slices were then transferred to a submerged brain slice-recording chamber perfused with
oxygenated aCSF kept at a recording temperature of 32
o
C as outlined in (Akopian and Walsh,
2007). The pH of all oxygenated solutions was 7.4. Picrotoxin (50 µM) was used to block
gamma-amino butyric acid-A (GABA
A
) receptor mediated inhibition in an attempt to isolate
excitatory synaptic events.
Recording and Synaptic Stimulation: Whole-cell voltage clamp and electrical stimulation
methods were used to examine corticostriatal synaptic input. Voltage clamp was chosen as the
recording method to reduce possible activation of postsynaptic conductance’s, which can
contribute to changes in synaptic strength under current-clamp conditions (Akopian and Walsh,
2002). Whole-cell recordings were obtained from striatal MSNs identified visually using a fixed
stage microscope and water immersion lenses (Zeiss Axioscope, Germany). Intracellular
electrodes contained 0.5% biocytin (Sigma-Aldrich, Inc, St. Louis, MO) in some experiments to
verify the cell type based on morphology. Patch electrodes were pulled with a Flaming-Brown
P-87 pipette puller (Sutter Instrument, Novato, CA) from thin-wall borosilicate capillary glass
48
having a 1.5 mm outer diameter (o.d.) (WPI, Sarasota, FL). The electrodes had resistances
ranging between 4 and 6 MΩ when filled with the pipette solution. The pipette (internal) solution
was composed of (mM): Cs gluconate 130, CsCl 10, EGTA 5, MgCl
2
2, HEPES 10, QX-314 5,
ATP-Mg 2, GTP-Na 0.25, pH 7.25, 285 mOsm. Spermine (100 µM) (Sigma-Aldrich Inc., St.
Louis, MO) was included to provide polyamine modulation of GluA2-lacking AMPARs. The
liquid junction potential between the pipette and aCSF were estimated as 15 mV. Cells were held
at different holding potentials during the time course of experiments as needed, taking into
account this liquid junction potential. Series resistance (Rs) was monitored throughout the
experiment by measuring the instantaneous current response to 5 ms hyperpolarizing (-5 mV)
pulses delivered before synaptic stimulation and was not compensated. A gravity-fed array of
inflow tubes of ~100 µm inner diameter and an outflow tube attached to a vacuum reservoir
provided solution flow. The ground electrode consisted of a salt bridge constructed from glass
electrode filled with agar. Passive membrane properties of the cells in slices were determined in
voltage clamp mode with the “Membrane Test” option of the “Clampex 9” software by using 10
mV depolarizing step voltage command from a holding potential of -70 mV.
Stimulation of afferent fibers was performed using a glass micropipette with the tip
diameter of 5 µm. The stimulation electrodes were filled with aCSF and positioned 100-500 µm
from the recording electrode at the border between striatum and the overlying corpus callosum.
Rectangular current pulses of 0.1 ms duration were applied to the glass pipette relative to a
reference electrode placed in the recording chamber using Stimulus Isolation Unit A365 (WPI,
Sarasota FL) triggered by digital output of Digidata 1320.
Assessment of GluA1 versus GluA2 Participation in AMPAR-mediated Responses:
AMPAR-mediated responses were isolated pharmacologically by blocking GABA
A
receptor-
49
mediated inhibition with picrotoxin (50 µM) and NMDA receptor-mediated responses with DL-
2-amino-5-phosphonovaleric acid (AP-5, 50 µM). The relative contribution of GluA1 and
GluA2 subunits to AMPAR function was determined by examining the degree of polyamine
modulation of AMPAR mediated synaptic currents occurring at depolarizing holding potentials
(Liu and Cull-Candy, 2000). The readout of relative subunit contribution is determined by
comparing synaptic currents evoked at hyperpolarized membrane potentials with those evoked at
depolarized membrane potentials and the calculation of the change in slope referred to as the
rectification index (RI). Synaptic current-voltage (I/V) relationships were obtained by generating
synaptic currents with electrical stimulation of cortical afferents every 20 sec at holding
potentials ranging from -80 mV to +60 mV with increments of 20 mV. The stimuli were
delivered 5 sec after the stepped change in the holding potential. The RI was determined as the
slope of the synaptic I-V curve at positive potentials (0 to +60 mV) divided by the slope of
synaptic I-V curve at negative potentials (-80 to 0 mV) (Liu and Cull-Candy, 2005, Shin et al.,
2007). A set of cells recorded from Saline and early MPTP (7 days post-MPTP) was also
examined for their sensitivity to the selective GluA2-lacking AMPAR antagonist IEM-1460 (50
µM) (Gittis et al., 2011).
Incubation of Brain Slices in l-Sulpiride: Brain slices were taken from their stabilization
bath that contained oxygenated aCSF (see Animals section above) and were placed in
oxygenated aCSF containing 5 µM l-Sulpiride (Sigma-Aldrich, St Louis, MO) for one hour. The
l-Sulpiride treated brain slices were then transferred to the recording chamber and were
continuously perfused with l-Sulpiride containing aCSF during the successful recording of
MSNs (1-2 hours). Outcomes from these experiments were compared with those obtained from
50
slices time-matched for perfusion in oxygenated aCSF alone. This experiment focused on the
effect l-Sulpiride treatment has on AMPAR RI as outlined in the previous section.
Stimulation Paradigm for Inducing Long-term Plasticity: Bipolar insulated tungsten wire
(50 µm diameter) stimulating electrodes were used to deliver test and tetanizing extracellular
stimuli to the border between the striatum and the overlying corpus callosum in 8-10 week old
C57BL/6J mice. Intracellular and field potential records were obtained at a 1mm distance from
the extracellular electrodes. Test synaptic responses were delivered with a constant current
stimulus (10-500 µA) at a duration <0.2 msec. Responses to test stimuli were sampled every 20
sec for a control period of 10 min prior to tetanization and a post-tetanus sampling period of at
least 30 minutes. Test excitatory post-synaptic potential (EPSP) amplitudes were averaged for
three 20-second interval samples to generate pre- and post-tetanus values that were plotted at
one-minute intervals. These one-minute interval values were plotted for 10 minutes prior to and
30 minutes after the tetanus. These same one-minute averages were used to compare between
groups the response to corticostriatal tetanic stimulation (i.e. MPTP versus MPTP plus exercise,
see statistical analysis of data section below). Tetanic stimulation consisted of four trains of
stimuli separated by 10 seconds. Each train lasted 1 second and was delivered at a frequency of
100 hertz (Hz). The tetanus stimulation intensity was set to equal the threshold for orthodromic
induction of action potentials. The intensity used to sample EPSPs was then set to half the
intensity of the orthodromic threshold. These methods are identical to those used in our
laboratory and others for many years (Akopian et al., 2000, Tang et al., 2001, Akopian and
Walsh, 2006, Siviy et al., 2011).
Intracellular records were obtained with glass microelectrodes pulled by a Flaming-
Brown P-87 pipette puller. Electrodes filled with 2 M potassium acetate had resistance values
51
ranging from 100 to 160 MΩ. Extracellular records were obtained with glass microelectrodes
filled with 2 M NaCl with a resistance of 1 MΩ or less. Signals were amplified with an
Axoclamp 2A (Axon Instruments) amplifier, digitized with a LABMASTER interface, and
stored on disk using pCLAMP software (Molecular Devices). Established electrophysiological
criteria were used for including cells in this study (Akopian et al., 2008).
Statistical Analysis of Data
Statistical analyses were performed using SPSS version 14.0 for Windows (SPSS, Inc., Chicago,
IL) or GraphPad Prism 6 (GraphPad Software Inc, La Jolla, CA). Differences between groups in
behavioral tests were analyzed using repeated-measures analysis of variance (ANOVA) with the
between-subjects factors being lesion (Saline or MPTP) and the within subject factor being time.
For HPLC analysis, a Mann-Whitney t-test was performed for comparisons between MPTP
versus Saline treated mice: (1) Day 5 post-MPTP no exercise, (2) Day 42 post-MPTP no
exercise, (3) Day 42 post-MPTP exercise. A one-way ANOVA was performed to compare
differences in striatal DA across the MPTP groups: (1) Day 5 post-MPTP, (2) Day 42 post-
MPTP no exercise, (3) Day 42 post-MPTP exercise. For western-immunoblotting of the 7 days
post-lesioning, an unpaired Student’s t-test was performed for comparisons between MPTP
versus Saline treated mice. For western immunoblotting, immunohistochemistry staining, and
voltage-clamp analysis of RI of the 42 days post-lesioning animals, a two-way ANOVA was
performed to compare the different groups and to examine for significant interactions. Post-hoc
contrasts with one-way ANOVAs were performed to determine the locus of any significant
differences. Voltage-clamp analysis of RI of the 7 days post-lesioning animals, the percent block
of AMPAR mediated EPSCs by IEM-1460 and the effect of l-Sulpiride incubation on RI was
compared between groups (Saline versus MPTP; l-Sulpiride versus control) and cell-types (D
2
R-
52
MSN versus D
1
R-MSNs) using two-way ANOVAs. Post-hoc contrasts with one-way ANOVAs
were performed to determine the locus of any significant differences. Intracellular and
extracellular recordings of post-tetanic plasticity were compared between treatment groups using
a repeated measures ANOVA across the entire post-tetanic sampling periods. The repeated
measures ANOVA was followed by post-hoc one-way ANOVAs for post-tetanic plasticity (0-3
minutes post-tetanus) and long-term plasticity (25-30 min post-tetanus) were performed between
groups (i.e. MPTP versus MPTP plus exercise). The value of post-tetanic plasticity was obtained
for each recording (intracellular and extracellular) by averaging the value of percent change in
EPSP amplitude measured from 0-3 minutes post-tetanus and the long-term plasticity value was
obtained for each recording by averaging the percent change in EPSP amplitude relative to pre-
tetanus control for the period of 25-30 minutes post-tetanus.
RESULTS
MPTP plus Exercise Restores GluA2 and Decreases GluA1 AMPAR Surface Expression in
D
2
R-MSNs but not D
1
R-MSNs
Immunohistochemical staining under conditions lacking Triton X-100 was used to
analyze cell surface expression of GluA2 (Figure 14) and GluA1 (Figure 15) AMPAR subunits
in green (D
2
R-MSNs) and non-green (D
1
R-MSNs) cells in the dorsolateral striatum of Drd
2
-
eGFP-BAC mice (N=6 animals per group). There was a significant interaction between MPTP
and exercise on GluA2 cell surface expression in D
2
R-MSNs (Figure 14). Exercise increased
GluA2 cell surface expression in MPTP plus exercise compared to MPTP mice (1.00±0.05;
versus 0.89±0.03 relative OD units). Conversely, exercise decreased GluA2 cell surface
expression in Saline plus exercise compared to Saline mice (0.91±0.04, versus 1.00±0.04)
[F(1,20)=6.072, p=0.023]. No effect of MPTP [F(1,20)=1.693, p=0.208], exercise
53
[F(1,20)=1.455, p=0.237], or an interaction [F(1,20)=0.33, p=0.572] was observed on GluA2 cell
surface expression in D
1
R-MSNs.
No significant effect of MPTP on GluA1 cell surface expression was observed in D
2
R-
MSNs [F(1,20)=0.0758, p=0.7859] (Figure 15). Exercise significantly decreased GluA1 cell
surface expression in D
2
R-MSNs compared to non-exercised mice (MPTP plus exercise
0.87±0.02; Saline plus exercise 0.86±0.05 versus MPTP 0.97±0.05; Saline 1.00±0.04, relative
OD units) [F(1,20)=7.975, p=0.011] (Figure 15). No effect of MPTP [F(1,20)=0.0001, p=0.993],
exercise [F(1,20)=0.0054, p=0.942), or an interaction [F(1,20)=0.2084, p=0.653] was observed
on GluA1 cell surface expression in D
1
R-MSNs. Taken together these data demonstrate that
exercise affects cell-surface expression of both GluA1 and GluA2 subunits selectively in D
2
R-
MSNs. Specifically, exercise increases GluA2 and decreases GluA1 cell-surface expression in
MPTP mice. In Saline mice, exercise decreases both GluA2 and GluA1.
54
Figure 14. Exercise normalizes GluA2 cell surface expression in D
2
R-MSNs in MPTP mice.
Immunohistochemical analysis of GluA2 cell surface expression was evaluated in dorsolateral
green (D
2
R) and non-green (D
1
R) MSNs of Drd
2
-eGFP-BAC mice after 28 days of exercise.
Optical density values were normalized to the Saline treated group and designated as 1.00
arbitrary OD units. Left: The upper panels show images of coronal sections at a region
corresponding to the dorsolateral striatum at 60x magnification. The left row of images captures
GluA2 staining (RFP), the right row of images are merged GluA2 (RFP), D
2
R-MSNs (eGFP),
and D
1
R-MSNs (DAPI) images across the different Saline and MPTP groups. Scale bar=12 µm.
Top Right: There was a significant interaction between MPTP and exercise in D
2
R-MSNs.
Exercise increased GluA2 cell surface expression in MPTP plus exercise compared to MPTP
mice, but decreased GluA2 cell surface expression in Saline plus exercise compared to Saline
mice in D
2
R-MSNs.
#
Interaction between MPTP and Exercise (p=0.0229). Bottom right: No
significant effect of MPTP or exercise was observed in GluA2 cell surface expression in non-
green cells. Error bars indicate SEM.
55
Figure 15. Exercise decreased GluA1 cell surface expression in D
2
R-MSNs.
Immunohistochemical analysis of GluA1 cell surface expression was evaluated in dorsolateral
green (D
2
R) and non-green (D
1
R) MSNs of Drd
2
-eGFP-BAC mice after 28 days of exercise.
Optical density values were normalized to the Saline treated mice and designated as 1.00
arbitrary OD units. Left: The upper panel shows images of coronal sections at a region
corresponding to the dorsolateral striatum at 60x magnification. The left row of images captures
GluA1 staining (RFP), the right row of images are merged GluA1 (RFP), D
2
R-MSNs (eGFP),
and D
1
R-MSNs (DAPI) images across the different Saline and MPTP groups. Scale bar=12 µm.
Top right: There was a significant effect of exercise on GluA1 cell surface expression, where
exercise significantly decreased GluA1 cell surface expression compared to both Saline and
MPTP non-exercise groups.
+
Exercise effect (p=0.0105). Bottom right: No significant effect of
MPTP or exercise was observed in GluA1 cell surface expression in non-green cells. Error bars
indicate SEM.
56
Exercise Restores the Relative Expression of GluA1 to GluA2 in MPTP Mice
Using the MPTP-mouse model of DA-depletion, we previously reported exercise-induced
changes in AMPAR subunit expression with synaptoneurosomal preparations (VanLeeuwen et
al., 2010). In this study we used protein preparations enriched for the post-synaptic density
(PSD) to examine synaptic GluA1 and GluA2 subunit expression (Figure 16). In contrast to
synaptoneurosomal preparations that include surface and cytoplasmic proteins from pre-, post-
and extra-synaptic sites, PSD preparations are enriched for proteins localized to post-synaptic
sites (Dosemeci et al., 2006)
MPTP significantly reduced GluA2 PSD expression compared to Saline mice in both the
exercised and non-exercised groups (MPTP 0.905±0.03; MPTP plus exercise 0.85±0.05 versus
Saline 1.00±0.04; Saline plus exercise 1.13±0.04 relative OD units) [F(1,12)=19.67, p=0.0008]
(Figure 16B). Exercise had no effect on GluA2 PSD expression compared to non-exercised mice
[F(1,12)=0.88, p=0.367].
No effect of MPTP was observed on GluA1 PSD expression [F(1,12)=2.269, p=0.158)
(Figure 16C). But, there was a significant interaction between MPTP and exercise on GluA1
PSD expression [F(1,12)=10.79, p=0.0065]. Exercise significantly reduced GluA1 PSD
expression in MPTP mice (MPTP plus exercise 0.85±0.07 versus MPTP 1.10±0.05 relative OD
units, p=0.023). Conversely, exercise had no significant effect on GluA1 PSD expression in
Saline mice (Saline plus exercise 1.11±0.06 versus Saline 1.00±0.04, p=0.177).
The relative ratio of GluA1/GluA2 PSD expression was determined from blots probed for
both proteins (Figure 16D). MPTP significantly increased GluA1/GluA2 PSD expression
compared to Saline mice (p=0.0119). There was also a significant interaction between MPTP and
Exercise on GluA1/GluA2 PSD expression [F(1,12)=7.444, p=0.0183]. Exercise significantly
57
reduced GluA1/GluA2 PSD expression in MPTP mice (MPTP plus exercise 0.990±0.02 versus
MPTP 1.21±0.03, p=0.006). However, exercise had no effect on GluA1/GluA2 PSD expression
in Saline mice (Saline plus exercise 0.98±0.04 versus Saline 1.00±0.04, p=0.69).
Taken together, these data suggest exercise reduces GluA1 PSD expression and thus
restores the relative ratio of synaptic GluA1/GluA2 in MPTP mice. No significant effect of
exercise on synaptic GluA1/GluA2 expression was observed in Saline mice.
Figure 16. Western immunoblot analysis of GluA1 and GluA2 protein preparations enriched for
PSD fractions from dorsolateral striatal tissue. Four independent PSD preparations, pooled from
six-eight animals per group were analyzed. Data were normalized to the Saline group designated
as 1.00 arbitrary OD units. A: The upper left panel shows a representative scan of western
immunoblotting results of PSD preparations for GluA1 and GluA2 with an antibody against ß-
actin in the lower bands to normalize for gel loading. B: MPTP significantly decreased GluA2
PSD expression compared to Saline.
+
MPTP effect (p=0.0008]. C: There was a significant
interaction between MPTP and exercise on GluA1 PSD expression [F(1,12)=10.79, p=0.0065).
This interaction was due to a significant decrease in GluA1 expression in MPTP plus exercise
compared to MPTP mice. *Exercise effect in MPTP mice (p=0.023). D: Comparison of
58
GuA1/GluA2 in PSD preparations. MPTP significantly increased GluA1/GluA2 PSD expression
compared to Saline mice. *MPTP effect (p=0.0119). There was a significant interaction between
MPTP and exercise on the ratio of GluA1/GluA2. This interaction was due to a significant
decrease in GluA1/GluA2 PSD expression in MPTP plus exercise compared to MPTP mice.
**Exercise effect in MPTP mice (p=0.006). Error bars indicate SEM.
MPTP plus Exercise Restores the Rectification Index (RI) Selectively in D
2
R-MSNs
We previously reported that MPTP increased GluA2-lacking AMPARs and exercise
restored GluA2-containing AMPARs in MPTP mice (VanLeeuwen et al., 2010). This study
builds upon our previous work by evaluating whether changes in AMPAR channels after MPTP
and exercise are selective to striatopallidal or striatonigral MSNs (Figure 17). We examined
changes in the rectification index (RI) as an indicator of the relative contribution of GluA2-
containing versus GluA2-lacking AMPAR channels in D
2
R-MSNs and D
1
R-MSNs. RI was
determined by the slope of the synaptic current-voltage (I-V) curve at positive potentials (0 to
+60 mV) divided by the slope of the synaptic I-V curve at negative potentials (-80 to 0 mV) (Liu
and Cull-Candy, 2005, Shin et al., 2007). GluA2-lacking AMPAR channels display inwardly
rectifying currents, represented by a low RI, due to their ability to bind intracellular polyamines
within the channel pore and block current at positive potentials (Pellegrini-Giampietro et al.,
1997). Alternatively, GluA2-containing AMPARs contain a charged arginine within the channel
pore, do not bind intracellular polyamines, and therefore display linear I-V relationships with a
higher RI (Cull-Candy et al., 2006).
MPTP significantly reduced the RI of D
2
R-MSNs compared to Saline mice (p=0.0002)
(Figure 17C). There was also a significant interaction between MPTP and exercise on the RI of
D
2
R-MSNs [F(1,52)=15.17, p=0.0003]. Exercise increased the RI of D
2
R-MSNs in MPTP mice
(MPTP plus exercise 0.87 ± 0.02, n=13 versus MPTP 0.77 ± 0.02, n=12; p=0.0008). However,
59
exercise had no significant effect on the RI of D
2
R-MSNs in Saline mice (Saline plus exercise
0.84 ± 0.02, n=14 versus Saline 0.88 ± 0.02, n=17; p=0.16).
Figure 17. Analysis of the rectification index (RI; slope of synaptic conductance at +60mV
verses -80mV) of dorsolateral striatal D
2
R-MSNs
and D
1
R-MSNs. A: Example of EPSCs evoked
in dorsolateral MSNs at holding potentials of -80 and +60 mV from MPTP and MPTP plus
exercise mice. B: Current-voltage plots of synaptic currents evoked from cells illustrated in A.
Synaptic currents were normalized to peak synaptic current evoked at -80mV for ease of
presentation. C: Measurement of RI in D
2
R-MSNs. MPTP significantly reduced the RI of D
2
R-
MSNs compared to Saline mice. *MPTP effect in non-exercised mice (p=0.0002). There was
also a significant interaction between MPTP and exercise on the RI in D
2
R-MSNs. This
interaction was due to a significant increase in the RI of D
2
R-MSNs in MPTP plus exercise
compared to MPTP mice. **Exercise effect in MPTP mice (p=0.0008). D: There was also a
significant effect of MPTP on the RI of D
1
R-MSNs, due to a significant decrease in MPTP plus
exercise compared to Saline plus exercise mice. No significant difference was observed between
any of the other groups. *MPTP effect in exercised mice (p=0.0017). Error bars indicate SEM.
60
In exercised animals only, the RI of D
1
R-MSNs was significantly increased in Saline
mice compared to MPTP mice (Saline plus exercise 0.92 ± 0.02, n=6 versus MPTP plus exercise
0.81 ± 0.02, n=9 p=0.0017) (Figure 17D). However, MPTP had no significant effect on the RI of
D
1
R-MSNs in non-exercised animals (MPTP 0.83 ± 0.04, n=7 versus Saline 0.84 ± 0.03, n=10,
p=0.69). Furthermore, exercise had no significant effect on the RI of D
1
R-MSNs in MPTP mice
(p=0.6813), or in Saline mice (p=0.0549) compared to the respective non-exercised groups.
Taken together these data support that MPTP decreases the RI of D
2
R-MSNs, indicating
the emergence of GluA2-lacking AMPA channels. Exercise reverses this effect in MPTP mice,
indicating the restoration of GluA2-containing AMPA channels selectively in D
2
R-MSNs. In
D
1
R-MSNs, exercise increases rectification in Saline animals.
Changes in RI are observed early (7days) post-MPTP in D
2
R-MSNs
Since 28 days of exercise normalized the RI in MPTP mice, we investigated whether
exercise prevented or restored the MPTP-induced RI changes. Alterations in RI expression were
assessed early, 7 days after MPTP or Saline administration, in dorsolateral MSNs of Drd
2
-eGFP-
BAC mice. MPTP significantly decreased the RI of D
2
R-MSNs compared to Saline mice
(0.81±0.01, n=14 versus 0.86±0.02, n=21; p=0.0205) (Figure 18A). Conversely, MPTP had no
significant effect on the RI of D
1
R-MSNs compared to Saline mice (0.88±0.02, n=4 versus
0.84±0.02, n=13; p=0.4347). Overall, these results support that exercise restores the RI of D
2
R-
MSNs in MPTP mice. In addition, this early effect of MPTP on the RI is specific to D
2
R-MSNs.
61
Figure 18. AMPAR expression 7 days after MPTP or Saline administration. Changes in AMPA
channel properties, and GluA1 and GluA2 subunit expression were evaluated at an early time
point (7 days after MPTP administration). A: Alterations in the rectification index (RI; slope of
synaptic conductance at +60mV vs -80mV) of dorsolateral D
2
R-MSNs
and D
1
R-MSNs of Drd
2
-
eGFP-BAC mice were analyzed 7 days after MPTP or Saline administration. MPTP significantly
decreased the RI of D
2
R-MSNs compared to Saline mice at the early time point. No difference in
RI of D
1
R-MSNs is observed with MPTP. *MPTP effect in D
2
R-MSNs (p=0.0205). B:
Pharmacological blockade of EPSCs with the selective GluA2-lacking receptor antagonist IEM-
1460 reveals a selective increase in sensitivity in dorsolateral D
2
R-MSNs from Drd
2
-eGFP-BAC
mice 7 days after MPTP administration. IEM-1460 (50 µM) increased the percent block of
evoked EPSCs selectively in D
2
R-MSNs in MPTP compared to Saline mice. *MPTP effect in
D
2
R-MSNs (p<0.05). C: Four independent PSD preparations, pooled from six-eight animals per
group were analyzed at an early time point in MPTP and Saline mice. D: MPTP significantly
increased the ratio of GluA1/GluA2 PSD expression compared to Saline mice *MPTP effect
(p=0.0244). Error bars indicate SEM.
IEM-1460 Selectively Blocks AMPAR EPSCs in D
2
R-MSNs
In order to confirm that a decrease in the RI observed in the D
2
R-MSNs was consistent
with the emergence of GluA2-lacking AMPARs, we examined the sensitivity of corticostriatal
EPSCs to pharmacological blockade with the selective GluA2-lacking receptor (Ca
2+
-permeable)
62
antagonist IEM-1460 (Buldakova et al., 1999, Gittis et al., 2011). Experiments were performed 7
days after MPTP administration when a significant decrease in RI was first observed (see above).
IEM-1460 (50 µM) caused a significantly higher percent block of the evoked EPSCs in D
2
R-
MSNs in MPTP compared to Saline mice (10.54±3.94% block, n=6 versus 2.84±1.92, n=4;
p=0.0242] (Figure 18B). Conversely, IEM-1460 had no significant effect on evoked EPSCs of
D
1
R-MSNs in MPTP compared to Saline mice (10.54±3.94% block, n=6 versus 6.67±0.97%
block, n=7; p=0.202) (Figure 18B). These findings support that a decrease in RI is consistent
with the emergence of GluA2-lacking AMPARs in D
2
R-MSNs in MPTP mice.
Changes in GluA1/GluA2 PSD expression are observed early (7days) post-MPTP
We evaluated whether changes in synaptic GluA1 to GluA2 expression in protein
preparations enriched for the PSD 7 days after DA-depletion (N=4 sets of PSD preps per group)
were consistent with changes in AMPA channel (Figure 18C). MPTP significantly increased the
ratio of GluA1/GluA2 PSD expression compared to Saline mice (1.05±0.01 versus 1.00±0.01,
p=0.0244)(Figure 18D). These data are consistent with the early emergence of rectification (low
RI) in MPTP mice, and support increased GluA2-lacking AMPAR channel expression in MPTP
mice.
l-Sulpiride, a Selective D
2
R-antagonist, Decreases the RI Specifically in D
2
R-MSNs
To further evaluate the association between AMPAR channels (GluA2-lacking versus
GluA2-containing) and D
2
R-MSNs, alterations in the RI were assessed using the D
2
R specific
antagonist, l-Sulpiride (Zhu et al., 2002). Striatal slices of Drd
2
-eGFP-BAC mice were incubated
in the presence or absence (controls) of l-Sulpiride. l-Sulpiride significantly decreased the RI of
D
2
R-MSNs compared to controls (0.79±0.02, n=6 versus 0.88±0.01, n=17; p=0.0058) (Figure
19). There was no effect of l-Sulpiride on RI of D
1
R-MSNs compared to controls (0.86±0.01,
63
n=6 versus 0.84±0.03, n=7; p=0.559). These data demonstrate that blocking D
2
R activation,
much like DA-depletion, decreases the RI of D
2
R-MSNs.
Figure 19. Analysis of the rectification index in D
2
R-MSNs and D
1
R-MSNs using the D
2
R-
antagonist l-Sulpiride. The rectification index (RI) corresponding to the slope of synaptic
conductance at +60mV versus -80mV was used to show how D
2
R signaling influences the
expression of GluA2-containing and GluA2-lacking AMPARs. l-Sulpiride significantly
decreased the RI of D
2
R-MSNs compared to controls but had no effect on changing the RI of
D
1
R-MSNs. *l-Sulpiride effect in D
2
R-MSNs p<0.01. Error bars indicate SEM.
MPTP plus Exercise Restores LTD in Dorsolateral MSNs
Given that we observe exercise effects on synaptic AMPAR expression in MPTP mice,
we evaluated whether exercise also alters synaptic plasticity in the DA-depleted striatum.
Synaptic plasticity was evaluated at dorsolateral corticostriatal synapses using both intracellular
and field potential recordings from a mixed population of D
2
R-MSNs and D
1
R-MSNs. As others
have previously reported ((Shen et al., 2008), DA-depletion significantly reduced the expression
of long-term depression (LTD) compared to Saline mice using both intracellular recordings
[F(1,33)=6.094, p<0.02] (MPTP=6, Saline=7) and field potential recordings [F(1,33)=6.094,
p<0.02) (MPTP=8, Saline=6) (Figure 20). Post-hoc analysis of post-tetanic plasticity revealed
MPTP significantly reduced the expression of both short-term plasticity (average 0-3 min post-
64
tetanus plasticity) (p<0.05, intracellular recording; p<0.05, field potential recording) and long-
term plasticity (average 25-30 minutes post-tetanus) (p<0.05, intracellular recording; p<0.05,
field potential recording) compared to Saline mice.
Importantly, exercise significantly increased the expression of LTD compared to non-
exercised mice in the MPTP groups using both intracellular recordings [F(1,33)=6.094, p<0.02)
(MPTP plus exercise=6, MPTP = 6) (Figure 20A) and field potential recordings [F(1,33)=6.094,
p<0.02) (MPTP plus exercise=9, MPTP=8) (Figure 20B). Post-hoc analysis of post-tetanic
plasticity in the MPTP groups revealed exercise significantly increased the expression of both
short-term plasticity (p<0.05, intracellular recording; p<0.05, field potential recording) and long-
term plasticity (p<0.05, intracellular recording; p<0.05, field potential recording) compared to
non-exercised mice. Overall, these data suggest that exercise restores corticostriatal expression
of LTD in MPTP mice.
Figure 20. Corticostriatal expression of long-term depression (LTD) after 28 days of exercise.
Both intracellular (A) and extracellular (B) techniques were used to record excitatory post-
synaptic potentials (EPSPs). MPTP significantly reduced LTD expression at corticostriatal
synapses using both intracellular (A) and extracellular (B) recordings of EPSPs. 28 days of
intensive treadmill running reversed the MPTP-induced reduction in LTD expression at
corticostriatal synapses using both intracellular (A) and extracellular (B) recordings of EPSPs.
Exercise rescues corticostriatal expression of LTD in MPTP mice. Error bars indicate SEM.
65
DISCUSSION
In this study, we found MPTP and exercise-induced changes in AMPAR expression occur
selectively in striatopallidal D
2
R-MSNs. Specifically, we observed the presence of inward
rectification (low RI) in MPTP mice in D
2
R-MSNs suggesting increased GluA2-lacking
AMPAR expression. Exercise restored GluA2-containing AMPAR expression in MPTP mice, as
indicated by a linear current-voltage relationship (high RI) in D
2
R-MSNs. Importantly, these
changes were not observed in D
1
R-MSNs. Since a high RI was also observed in the Saline
groups, our data suggest exercise normalizes GluA2-containing AMPAR expression in D
2
R-
MSNs after DA-depletion. We observed the emergence of inward rectification (low RI)
selectively in D
2
R-MSNs in MPTP mice as early as 7 days after DA-depletion. Therefore these
data suggest that exercise restores rather than prevents aberrant AMPAR expression in MPTP
mice. We used the GluA2-lacking receptor antagonist, IEM-1460 to confirm a low RI is
consistent with the emergence of GluA2-lacking channels (Buldakova et al., 1999, Gittis et al.,
2011). Additionally, we used the D
2
R-antagonist, l-Sulpiride (Zhu et al., 2002), to confirm D
2
R
signaling influences AMPAR expression. Even though D
2
Rs are found at both pre- and post-
synaptic sites and incubation of brain slices with l-Sulpiride would be expected to activate D
2
Rs
at both sites, the readout of AMPAR RI reflects post-synaptic changes in AMPAR expression.
In this study, alterations in AMPAR channel expression after MPTP and exercise were
associated with changes in GluA1 and GluA2 surface expression. Immunohistochemical staining
was used to determine GluA1 and GluA2 cell surface expression in both D
2
R-MSNs and D
1
R-
MSNs. We found exercise restores GluA2 and decreases GluA1 cell surface expression
selectively in D
2
R-MSNs in MPTP mice. This increase in cell surface GluA2 and decrease in cell
surface GluA1 may reflect experience-dependent changes in AMPAR subunit trafficking, which
66
may contribute to the restoration of GluA2-containing AMPARs in D
2
R-MSNs. In Saline mice,
exercise decreased cell surface expression of both GluA1 and GluA2 AMPAR subunits in D
2
R-
MSNs. This decrease in cell surface GluA2 and GluA1 occurred without changes in RI in D
2
R-
MSNs, and may reflect experience-dependent changes in AMPARs that does not involve
alterations in the relative expression of GluA2-containing versus GluA2-lacking channels. While
this approach allows us to delineate between striatopallidal and striatonigral MSNs, it is limited
by its ability to delineate between these pathways at synaptic sites within the neuropil, where the
majority of AMPARs are located. In support of our approach, others have demonstrated that
changes in cell surface expression of AMPARs may contribute to and thus provide insight into
changes in synaptic receptor expression (Stellwagen et al., 2005, Kessels et al., 2009, Tao-Cheng
et al., 2011). For example, by measuring somato-dendritic levels of virally expressed GFP-
tagged AMPAR subunits, Kessels et al. found AMPAR cell surface expression, but not
intracellular production or accumulation, strongly correlated with synaptic AMPAR expression
(Kessels et al., 2009). Furthermore, using pre-embedding immunogold electron microscopy,
Tao-Cheng et al. found the density of label for GluA2 at the PSD and the soma was similar at
baseline conditions, and after depolarization (Tao-Cheng et al., 2011). Taken together, these
studies support and validate that measuring GluA1 and GluA2 protein expression at the cell-
surface of D
2
R-MSNs and D
1
R-MSNs may accurately reflect pathway specific changes in
AMPAR expression occuring at synaptic sites within the neuropil.
In our study, MPTP and exercise-induced changes in AMPAR channel expression were also
associated with alterations in the relative expression of synaptic GluA1 and GluA2 subunits.
Using synaptoneurosomal preparations consisting of pre-, post-, and extra-synaptic proteins, we
previously reported exercise increased GluA2 expression but did not affect GluA1 expression in
67
MPTP mice (VanLeeuwen et al., 2010). This study builds upon our previous work by selectively
analyzing post-synaptic changes in GluA1 and GluA2 subunits in preparations enriched for the
PSD. We found MPTP decreased GluA2, but had no effect of GluA1 PSD expression, thereby
increasing the ratio of GluA1/GluA2. In contrast, exercise had little effect on GluA2 but
decreased GluA1 PSD expression, and normalized the ratio of GluA1/GluA2 in MPTP mice.
This increase in GluA1/GluA2 PSD expression in MPTP mice was observed as early as 7 days
after DA-depletion, which is consistent with the early emergence of rectification (low RI) in
D
2
R-MSNs, and further supports increased GluA2-lacking AMPAR expression in MPTP mice.
Our data indicate that GluA1 and GluA2 PSD expression differs from that observed within the
entire synaptoneurosomal fraction, and thus may reflect differences in the distribution of GluA1
and GluA2 subunits at pre- and post-synaptic sites (Feligioni et al., 2006, Greger and Esteban,
2007, Rusakov et al., 2011).
Given that we observe exercise effects on synaptic AMPAR expression in MPTP mice, and
because AMPARs are known to play a key role in long-term plasticity, we evaluated whether
exercise also alters synaptic plasticity in the DA-depleted striatum. Consistent with previous
reports (Shen et al., 2008), MPTP reduced corticostriatal expression of LTD compared to Saline
mice. Importantly, we found exercise restored corticostriatal expression of LTD in MPTP mice.
It has been well established that endocannabinoids mediate the expression of LTD at
corticostriatal synapses by acting in a retrograde manner to reduce pre-synaptic neurotransmitter
release (Yin and Lovinger, 2006, Calabresi et al., 2007, Surmeier et al., 2007). Although we
cannot conclude that exercise-induced changes in AMPAR expression led to sustained changes
in synaptic plasticity, our findings highlight the potential that, in addition to the pre-synaptic
effects of endocannabinoids, post-synaptic changes in AMPAR expression may also be involved.
68
Taken together, our study builds upon previous work by demonstrating that DA-depletion
and experience-dependent changes in AMPAR expression are specific to striatopallidal D
2
R-
MSNs in MPTP mice. To our knowledge this is the first report of a pathway specific change in
AMPAR subunit expression following DA-depletion. Consistent with our findings, others have
reported striatal changes in AMPAR subunits GluA1 and GluA2 and their phosphorylated states
following MPTP or 6-OHDA without delineating between D
2
R-MSNs and D
1
R-MSNs (Wullner
et al., 1993, Betarbet et al., 2000, Brown et al., 2005, Ba et al., 2006, Ouattara et al., 2010,
VanLeeuwen et al., 2010). Increased GluA2-lacking AMPAR expression in MPTP mice may
potentiate glutamatergic signaling and underlie the hyper-excitable state reported in the
striatopallidal projection pathway following DA-depletion (Hernandez-Echeagaray et al., 2004).
Exercise increases GluA2-containing AMPAR expression in MPTP mice, which may serve to
attenuate the hyper-excitability of the striatopallidal projection pathway by decreasing Ca
2+
influx and dampening glutamatergic drive. Changes in AMPAR expression may represent an
important mechanism underlying the motor behavioral improvements seen with effects of
exercise in the DA-depleted state.
Interestingly, we previously reported exercise increases the expression of striatal D
2
Rs in
both the MPTP-mouse model of DA-depletion and in individuals with early stage PD (Vuckovic
et al., 2010b). No change in D
1
R expression was observed with exercise in MPTP-mice. This
exercise effect on D
2
R expression coupled with data from this study suggests the D
2
R may
regulate AMPAR expression in the dorsolateral striatum. In support of D
2
Rs modulation of
AMPAR expression, others have demonstrated that pharmacological manipulation of the D
2
R
can modulate AMPA channels (Hernandez-Echeagaray et al., 2004). This modulation could
occur directly through protein-protein interactions between D
2
Rs and AMPAR subunits or
69
indirectly through downstream signaling pathways that regulate AMPAR trafficking (Chen et al.,
2001, Peterson et al., 2012). D
2
Rs may also interact with the adenosine A
2A
receptor (A
2A
R)
and/or the metabotropic glutamate receptor 5 (mGluR
5
) to regulate AMPAR changes in D
2
R-
MSNs.
In the dorsolateral striatum, A
2A
Rs predominantly co-localize on D
2
R-MSNs where they
modulate DA neurotransmission and potentially AMPAR expression either directly by forming
heterodimeric complexes with D
2
Rs that alter DA’s affinity for D
2
Rs, or indirectly through
downstream signaling pathways including protein kinases and phosphatases that regulate
phosphorylation and thus trafficking of AMPARs (Kessels and Malinow, 2009, Ferre et al.,
2011, Orru et al., 2011). Under conditions of DA-depletion, A
2A
R signaling may be unleashed,
thereby activating Ca
2+
-signaling pathways that promote synaptic expression of GluA2-lacking
AMPARs. Elevation of D
2
Rs through exercise may reinstate proper DA neurotransmission and
restore GluA2-containing channels in D
2
R-MSNs. The receptor mGluR5 also co-localizes on
striatal D
2
R-MSNs. mGluR5 receptors have been shown to regulate Ca
2+
-signaling pathways
implicated in altering the phosphorylation state and thus synaptic expression of AMPARs
(Gubellini et al., 2004). For example, activation of type 1 mGluRs (mGluR1 or mGluR5) has
been shown to increase GluA1 phosphorylation at Serine-845 through Protein Kinase A-
dependent mechanisms, which is dependent on A
2A
R signaling, and is abolished by the D
2
R-
agonist quinpirole (Dell'anno et al., 2013). These data support a mechanistic link between D
2
R,
A
2A
R, and mGluR5 in regulating AMPAR expression, particularly trafficking within the D
2
R-
MSNs. Ongoing studies are investigating how DA-depletion and exercise influences the
interaction between these receptor families and if downstream Ca
2+
-dependent signaling
pathways alter AMPAR subunit phosphorylation and synaptic localization.
70
In conclusion, these studies indicate that DA-depletion results in the emergence of
GluA2-lacking AMPARs selectively in D
2
R-MSNs and exercise reverses this trend in MPTP
mice. Exercise-induced changes in AMPAR channels observed after DA-depletion are associated
with alterations in GluA1 and GluA2 subunit expression and the restoration of corticostriatal
plasticity. Elucidating the mechanism(s) regulating experience-dependent alterations in AMPAR
expression may provide insight into potential therapeutic targets for improved treatment of PD.
71
CHAPTER 3: Exercise Modifies Dopamine Neurotransmission within the Prefrontal
Cortex and Reduces Perseverative Behavior in the MPTP-lesioned Mouse
Author list: Natalie M Kintz, Giselle M Petzinger, James Tavornwattana, Daniel Stefanko, and
Michael W Jakowec
ABSTRACT
Parkinson’s disease (PD) is the second most common neurodegenerative disorder for
which there is no cure. PD is a dopamine (DA)-deficit disorder, marked by progressive motor
and non-motor disturbances, including cognitive impairment. Executive function is the most
common subtype of cognitive impairment in PD, and consists of deficits in number of processes
including behavioral flexibility. The prefrontal cortex (PFC) is an important brain region sub-
serving executive function. Furthermore, DA plays a key neuromodulatory role in the PFC, and
altered DA neurotransmission is believed to contribute to executive function deficits in PD. But,
the mechanisms underlying PFC dysfunction are not fully understood, and there are no effective
treatments for executive function deficits in PD. Importantly, exercise is a promising therapeutic
strategy that may exert beneficial effects on PFC function in PD. Our previous work suggests
exercise improves motor function and restores striatal DA-D
2
receptor (D
2
R) expression in the
DA-depleted brain. This current study builds upon our previous work by investigating if exercise
modulates PFC function, specifically DA levels, DA receptor expression, and behavioral
flexibility in the MPTP-mouse model of DA-depletion. We found exercise restores PFC DA
levels, reverses the MPTP-induced increase in DA-D
1
receptors and decrease in DA-D
4
receptors, and exerts differential effects on D
2
Rs. Furthermore, exercise remediates perseverative
errors in MPTP-mice, indicating improved flexibility, but does not improve overall learning
72
during a reversal-learning T-maze task. Understanding the effects of exercise on executive
function and DA signaling in the DA-depleted brain may provide key insight into new
therapeutic targets for PD.
SIGNIFICANCE STATEMENT:
The purpose of this study was to determine whether exercise modulates PFC function,
including dopamine (DA) levels, DA receptor (D
1
R, D
2
R, and D
4
R) expression, and executive
function, specifically behavioral flexibility in MPTP-lesioned mice. We found exercise restores
PFC DA levels, reverses the MPTP-induced increase in D
1
Rs and decrease in D
4
Rs, and exerts
differential effects on D
2
Rs. Furthermore, exercise remediates perseverative errors in MPTP
mice, indicating improved flexibility, but does not improve overall learning during a reversal-
learning T-maze task. Understanding the effects of exercise on executive function and DA
signaling in the DA-depleted PFC may provide key insight into new therapeutic targets for the
improved treatment of the cognitive disturbances observed in PD.
KEYWORDS: D
1
R, D
2
R, D
4
R, reversal-learning, behavioral flexibility, Parkinson’s disease
INTRODUCTION
Parkinson’s disease (PD) is an age-related neurodegenerative disorder for which there is
no disease-modifying therapy or cure. PD is a dopamine (DA)-deficit disorder, characterized by
a number of motor and non-motor features, including cognitive impairment. Executive function
is the most common subtype of cognitive impairment in PD which transitions to dementia,
increased fall risk, and poor quality of life. Executive function disturbances in PD consist of
deficits in working memory, behavioral flexibility, planning and attention. The prefrontal cortex
(PFC) along with its connections to the striatum is an important brain region sub-serving
73
executive function. Furthermore, dopamine (DA) plays a key neuromodulatory role in the PFC,
and aberrant DA neurotransmission is believed to contribute to executive function deficits
observed in PD (Brozoski et al., 1979, Puig et al., 2014, Martínez-Horta and Kulisevsky, 2015).
There remains, however, an important need to better understand the mechanisms underlying PFC
dysfunction in the DA-depleted brain and to develop more effective treatment modalities for
executive function disturbances in PD.
Exercise has been shown to improve motor function in individuals with PD and may also
be a promising therapeutic strategy for cognitive impairment. For example, several studies in
healthy aging individuals have demonstrated a significant beneficial effect of exercise on
cognition in physically fit individuals (Chang et al., 2012b, Hotting and Roder, 2013).
Preliminary evidence also suggests exercise improves cognitive function in PD, as demonstrated
by improved behavioral flexibility using the Wisconsin Card Sorting Task (WCST) (Tanaka et
al., 2009). However, the mechanisms underlying the beneficial effects of exercise are not fully
understood. Previous work from our lab suggests exercise improves motor performance and
restores striatal DA-D
2
receptor (D
2
R) expression in the MPTP-mouse model of DA-depletion,
and in patients with PD (Vuckovic et al., 2010a, Fisher et al., 2013). This current study builds
upon our previous work by investigating if exercise modulates PFC function and improves
executive function, specifically behavioral flexibility, using the MPTP-mouse model of DA-
depletion. For these studies, 8-10 week old male C57BL/6J mice were randomly assigned to one
of four experimental groups: Saline; Saline plus exercise; MPTP; MPTP plus exercise. Treadmill
exercise was initiated 5 days after MPTP or saline administration, and continued 5 days/week for
6 weeks. Changes in DA levels and DA-D
1
receptor (D
1
R), D
2
R and DA-D
4
receptor (D
4
R)
protein expression, the most abundantly expressed DA receptors in the PFC, were assessed by
74
HPLC and western immunoblotting respectively at an early (1 week after the start of exercise)
and late (6 weeks after the start of exercise) time point. We found exercise restores PFC DA
levels, reverses the MPTP-induced increase in D
1
Rs and decrease in D
4
Rs, and exerts differential
effects on D
2
Rs. Furthermore, exercise remediates perseverative errors in MPTP mice, indicating
improved flexibility, but does not improve overall learning during a reversal-learning T-maze
task. Understanding the effects of exercise on executive function and DA signaling in the DA-
depleted PFC may provide key insight into new therapeutic targets for the improved treatment of
the cognitive disturbances observed in PD.
MATERIALS AND METHODS
Animals
A total of 132 young adult (8–10 weeks old) male C57BL/6J mice were used (Jackson
Laboratory, Bar Harbor, ME). There were four treatment groups: (i) Saline, (ii) Saline plus
exercise, (iii) MPTP, and (iv) MPTP plus exercise. Mice were housed 5 to a cage and acclimated
to a 12-hour shift in light/dark cycle so that the exercise occurred during the animals normal
awake period. All experiments were carried out in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised
1996) and approved by the University of Southern California Institutional Animal Care and Use
Committee (IACUC).
MPTP-Lesioning
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Sigma, Inc.; St. Louis, MO) was
administered in a series of 4 intraperitoneal injections of 20 mg/kg (free-base) at 2-hour intervals
for a total administration of 80 mg/kg. This regimen leads to approximately 60 to 70% loss of
75
nigrostriatal neurons (as determined by unbiased stereological techniques for both tyrosine
hydroxylase, TH, staining and Nissl substance) and an 80 to 90% depletion of striatal dopamine
levels (Jackson-Lewis et al., 1995, Jakowec et al., 2004). Nigrostriatal cell loss is complete by
day 3 after MPTP administration as determined by counting remaining nigrostriatal TH immuno-
reactive cells and reduced silver staining for degenerating neurons (Jackson-Lewis et al., 1995,
Jakowec et al., 2004).
Treadmill Exercise
One week before MPTP-lesioning, 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 lesioning. The treadmill used in
these studies was a Model EXER-6M Treadmill (Columbus Instruments; Columbus, Ohio). A
non-noxious stimulus (metal beaded curtain) was used as a tactile incentive to prevent mice from
drifting back on the treadmill. The treadmill exercise protocol was conducted as previously
described (Fisher et al., 2004, Petzinger et al., 2007, VanLeeuwen et al., 2010, Kintz et al.,
2013a). Briefly, exercise was initiated 5 days following saline or MPTP lesioning (when cell
death is complete) and continued for 5 days/week for 6 weeks. Exercise mice started at a velocity
of 10.0±1.5 m/min, which increased to 24.0±0.5 m/min by the final week. As we have previously
reported, there was a significant difference in velocity at weeks 1–4 between the Saline plus
exercise and MPTP plus exercise groups (p<0.05; Fisher et al., 2004; Petzinger et al., 2007; Fig.
1). This difference was eliminated with further training and completion of the treadmill running
regimen.
76
Tissue Collection
Tissue was collected from all treatment groups at two different time points: (i) early (1
week after the start of exercise), and (ii) late (6 weeks after the start of exercise). Mice were
sacrificed by cervical dislocation for fresh tissue (HPLC and western immunoblotting). PFC
tissue was dissected in a block corresponding to the following anatomical landmarks: (i) bregma
+1.4 to + 2.0 mm (rostral to the corpus callosum), (ii) 1mm lateral from midline to corpus
callosum, and (iii) dorsal-ventral 1.5 to 3.5 (inferior to motor cortex and superior to the lateral
ventricles). Dorsal striatal (dStr) tissue was dissected in a block from a coronal slice: (i) bregma
+1.20 to +0.60mm, (ii) 2.5mm lateral from midline, (iii) dorsal-ventral, inferior to the corpus
callosum and superior to the anterior commissure. All tissue was flash frozen and stored at -80
o
C
until analysis.
HPLC Analysis of Dopamine and Its Metabolites
Dopamine concentration in the PFC and dStr (N= 4 to 7 animals per group per time
point) along with its metabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid
(DOPAC), and turnover ratio [(DOPAC 1 HVA)/dopamine] were determined according to an
adaptation by Irwin et al. (1992) of the method of Kilpatrick and colleagues (Kilpatrick et al.,
1986, Irwin et al., 1992). HPLC data are presented as mean dopamine/protein (ng DA/mg
protein) ± standard deviation.
Western Immunoblotting:
Western immunoblotting was used to determine the relative expression of proteins within
the PFC (N = 5 to 7 animals per group per time point). In addition, dStr tissue was collected from
a subset of animals (N = 4 per group per time point) to further validate MPTP-lesioning via TH
levels. Tissue was dissected from the PFC and dStr and was immediately frozen and stored at -
77
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).
Cell debris was removed by centrifugation for thirty seconds. Protein concentration was
determined by the BCA method (Pierce; Rockford, IL). The immunoblotting technique used was
previously described (Jakowec et al., 2004, VanLeeuwen et al., 2010, Vuckovic et al., 2010a,
Kintz et al., 2013a) 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,
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: TH (1:1000, Cat#: MAB318, EMD Millipore;
Billerica, MA); D
1
R (1:500, Cat#: SC-14001, Santa Cruz Biotechnology; Dallas, TX ); D
2
R
(1:500, Cat#: SC-5303, Santa Cruz Biotechnology; Dallas, TX), D
4
R (1:300, Cat#: SC-136139,
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); Goat anti-Rabbit IgG IRDye 800 CW
(1:10,000, Cat#: 926-32211, LI-COR Inc.; Lincoln, Nebraska), Goat-anti Mouse IgG IRDye 680
LT (1:15,000, Cat#: 926-68020, LI-COR Inc.; Lincoln, Nebraska); 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
78
Studio Software Version 3.1.4 (LI-COR, Inc.; Lincoln, Nebraska), and expressed as relative
optical density (O.D.). Each gel contained samples from 2 to 3 independent animals from each
experimental group. The O.D. of each band was quantified relative to the O.D. of Beta-actin,
serving as the protein loading control. For comparison across groups, the relative O.D. levels for
each sample was compared to the averaged value of Saline mice analyzed on the same blot,
which was normalized to 100.00 O.D. units. Data are presented as mean ± standard deviation.
Reversal-Learning T-Maze Protocol
Behavioral flexibility, as an indicator of executive function, was assessed via a T-Maze
reversal-learning task upon completion of the treadmill exercise paradigm (N = 7 to 8 animals
per group). A four-arm cross maze was made of a clear plastic wall and a white floor and placed
90 cm above the floor. Each arm was 25 cm long and 5 cm wide, and the center platform was 5 ×
5 cm. Visual cues cut out of construction paper such as stars, triangles, and puzzle pieces were
hung outside the maze. Two of the four sides of the testing room had white curtains while the
other two had black curtains for further visual cues. The position of a mouse was detected by
video camera suspended over the maze.
One week prior to testing, mice were food-restricted to reach approximately 85% of their
original baseline weight. Each animal went through three days of habituation. On each day of
habituation, 3 non-flavored sugar pellets were placed in a well of each arm. The mouse was
allowed to freely navigate and consume the pellets within 15 minutes. If all pellets were
consumed before the end of the 15 minutes, the mouse we removed, new pellets were placed in
the well, and the mouse was placed back into the apparatus to continue habituation.
The testing portion of the task included an initial learning phase followed by a reversal-
learning phase. During the initial learning phase, a mouse was started in either the north or south
79
arm and had to make a 90° turn to the left or to the right to earn a food reward on the basis of
visual cues. Each start arm was used with an equal number of trials in a random fashion. After
the mouse learned the initial visual cue-food reward association, the visual cue rule was
switched. The reversal-learning phase occurred after the visual cue shift, during which the mouse
had to learn to disregard the previous visual-cue rule, and learn to turn towards a new visual cue
to earn a food reward. Both phases consisted of 2 sessions per day, with 10 trials per session. At
least 5 sessions were performed for the initial learning and reversal-learning phase, subsequent
sessions were added until the mouse reached the learning criterion of 90% correct consecutive
responses. Between trials, the mouse was placed back in the holding cage. The maze arms were
wiped down with an isopropanol solution. The inter-trial interval was approximately 10 seconds.
Performance was evaluated as: (i) percent of correct responses per session; (ii) the
number of trials to reach the learning criterion during initial learning and reversal learning,
defined as 9 out of 10 correct choices in consecutive trials; and (iii) errors made during the
reversal-learning phase, categorized as total errors, perseverative errors, or regressive errors.
Total errors were defined as the sum of perseverative and regressive errors. Perseverative errors,
defined as incorrect choices three or more times in consecutive blocks of four trials after the
visual cue switch, occur early in the reversal-learning phase and reflect deficits in suppressing
previous modes of responding. Regressive errors, defined as incorrect choices after perseveration
ceased, occur as the mouse begins to learn the new rule but occasionally regresses to the
previous response strategy and reflect difficulty in maintaining a new response strategy. All tests
of animal behaviors were conducted in a blinded fashion.
80
Statistical Analysis
Statistical analyses were performed in SPSS version 14.0 for Windows (SPSS, Chicago,
IL) or GraphPad Prism 6 (GraphPad Software, La Jolla, CA). For HPLC and western blot
analysis, a two-way analysis of variance (2-Way ANOVA) was used at the early and the late
time point to compare the effects of MPTP and exercise, and to examine for significant
interactions. Post hoc contrasts were performed using the Tukey’s multiple comparison test to
determine the locus of any significant differences. For behavioral tests, performance differences
(percent correct) across testing sessions were analyzed with a repeated-measures ANOVA, with
the within-subjects factor between the test session (Initial Learning Sessions: 1, 2, 3, 4, 5, 6;
Reversal-learning Sessions: 7, 8 ,9, 10, 11) and the between-subjects factor being the
experimental group (Saline, Saline plus exercise, MPTP or MPTP plus exercise). Post-hoc
contrasts using Tukey’s honest significant different (HSD) test were used to examine the locus of
any significant differences across all groups. All other performance measures (trials to criterion,
errors) were analyzed via a 2-way ANOVA to compare the effects of MPTP and exercise, and to
examine for significant interactions. Post hoc contrasts with the Tukey’s multiple comparison
test were performed to determine the locus of any significant differences.
RESULTS
Exercise Restores Dopamine Levels within the Prefrontal Cortex but not the Dorsal Striatum
of MPTP-lesioned Mice
Our previous work showed no improvement in DA levels after exercise within the dorsal
striatum (dStr) of MPTP-lesioned mice (Petzinger et al., 2007, VanLeeuwen et al., 2010, Kintz et
al., 2013a). In the present study, HPLC analysis was conducted to examine the effects of exercise
on DA and its metabolites within the PFC and the dStr at an early and late time point (Figure 21
81
and Figure 22, respectively). Data from the current study demonstrate MPTP significantly
reduced PFC DA levels compared to Saline mice, and six weeks of high intensity treadmill
exercise reversed this effect in MPTP-mice.
At the early time point, there was a significant effect of MPTP on PFC DA levels
[F(1,12)=12.0, p=0.0047) (Figure 21A). PFC DA levels were significantly reduced by 59.7% in
MPTP mice (3.4±1.1 ng DA/mg protein; p=0.0055) and by 55.2% in MPTP plus exercise mice
(3.8±1.2 ng DA/mg protein; p=0.0096) compared to Saline mice (8.5±2.3 ng DA/mg protein).
There was also a significant interaction between MPTP and exercise [F(1,12=6.361, p=0.0268],
where exercise reduced PFC DA levels by 45.8% in Saline plus exercise mice (4.6±1.9 ng
DA/mg protein; p=0.0319) compared to Saline mice. However, there was no significant
difference in PFC DA levels between MPTP plus exercise mice and MPTP mice (p=0.9880).
At the late time point, exercise restored PFC DA levels in MPTP-mice (Figure 21B).
Specifically, there was a significant interaction between MPTP and exercise [F(1,24)=22.72,
p<0.001]. This effect was due to a significant increase in PFC DA levels by 68.4% in MPTP plus
exercise mice (6.4±1.3 ng DA/mg protein) compared to MPTP mice (3.8±1.3 ng DA/mg protein;
p=0.0338), but a significant decrease in PFC DA levels by 43.6% in Saline plus exercise mice
(4.4±1.5 ng DA/mg protein) compared to Saline mice (7.8±2.4 ng DA/mg protein; p=0.0045).
Furthermore, in the non-exercised groups, MPTP significantly reduced PFC DA levels by 50.7%
compared to Saline mice (p=0.0010). In contrast, there was no significant difference in PFC DA
levels in MPTP plus exercise mice compared to the Saline groups.
82
Figure 21. HPLC analysis of Dopamine (DA) levels within the Prefrontal Cortex (PFC). PFC
samples were analyzed at an early (N=4 per group) and late (N=7 per group) time point. Data are
expressed as ng of DA per mg of protein (ng/µg). Error bars represent SD A: MPTP significantly
decreased DA levels in the PFC compared to Saline mice at the early time point.
+
MPTP effect
[F(1,12)=12.00, p=0.0047]. There was also a significant interaction between MPTP and exercise,
where exercise significantly reduced DA levels in Saline mice but not MPTP mice
#
Interaction
[F(1,12=6.361, p=0.0268]. B: There was a significant interaction between MPTP and exercise
on PFC DA levels at the late time point, where exercise significantly decreased DA levels in
Saline mice, but significantly increased DA levels in MPTP mice compared to the respective no
exercise groups.
#
Interaction [F(1,24)=22.72, p<0.0001]. In non-exercised mice, MPTP
significantly reduced PFC DA levels compared to Saline mice. *MPTP effect in non-exercised
mice (p=0.0010).
Consistent with our previous work, MPTP significantly reduced DA levels in the dStr at
both time points, and exercise did not alter this effect (Figure 22). Specifically, there was a
significant effect of MPTP at the early [F(1,12)=300.0, p<0.0001], and late [F(1,24)=326.9,
p<0.0001] time points. Furthermore, there was no significant effect of exercise at the early
[F(1,12)=0.3442, p=0.5692] or late [F(1,24)=0.7036, p=0.4099] time points.
PFC Early
Saline
MPTP
0
5
10
DA/protein (ng/mg)
No Exercise
Exercise
+, #
PFC Late
Saline
MPTP
0
5
10
DA/protein (ng/mg)
No Exercise
Exercise
*
#
A B
83
Figure 22. HPLC analysis of DA levels within the dorsal striatum (dStr). dStr samples were
analyzed at an early (N=4 per group) and late (N=7 per group) time point. Data are expressed as
ng of DA per mg of protein (ng/µg). Error bars represent SD. A: MPTP significantly decreased
DA levels in the dStr compared to Saline at the early time point.
+
MPTP effect [F(1,11)=300.9,
p<0.0001]. B: MPTP significantly decreased DA levels in the dStr compared to Saline at the late
time point.
+
MPTP effect [F(1,24)=326.9, p<0.0001].
Exercise Restores Tyrosine Hydroxylase Levels within the Prefrontal Cortex but not the
Dorsal Striatum of MPTP–lesioned Mice
Our previous work showed no improvement in TH levels after exercise within the dStr of
MPTP-lesioned mice (Petzinger et al., 2007, VanLeeuwen et al., 2010, Vuckovic et al., 2010a).
In the present study, western immunoblotting analysis was conducted to examine the effects of
exercise on TH levels within the PFC and dStr of MPTP-lesioned mice at the early and late time
point (Figure 23 and 24, respectively). Data from the current study suggest MPTP significantly
reduced PFC TH levels, and six weeks of high intensity treadmill exercise reversed this effect in
MPTP mice.
At the early time point, there was a significant effect of MPTP on PFC TH levels
[F(1,22)=294.0, p<0.001] (Figure 23B). TH levels were significantly reduced by 45.3% in MPTP
mice (54.6±10.7) and by 46.2% in MPTP plus exercise mice (53.7±4.9) compared to Saline mice
(99.8±26.0). However, there was no significant difference between the MPTP and MPTP plus
dStr Early
Saline
MPTP
0
50
100
DA/protein (ng/mg)
No Exercise
Exercise
+
dStr Late
Saline
MPTP
0
50
100
DA/protein (ng/mg)
No Exercise
Exercise
+
A B
84
exercise groups (p=0.995) or between the Saline and Saline plus exercise groups (101.9±3.0,
p=0.949).
Figure 23. Western immunoblot analysis of tyrosine hydroxylase (TH) protein preparations from
the PFC. PFC samples were analyzed from 5 to 7 animals per group at an early and late time
point. Data were normalized to the Saline group, designated at 100.00 arbitrary optical density
(O.D.) units. Error bars indicate SD. A: The upper panel are representative scans of western
immunoblotting results of PFC tissue samples collected at the early time point (left) and late time
point (right) for TH with an antibody against β-Actin in the lower bands to normalize for gel
loading. B: MPTP significantly decreased TH levels in the PFC compared to Saline mice at the
early time point.
+
MPTP effect [F(1,22)=294.0, p<0.001]. C: There is a significant interaction
between MPTP and exercise on TH levels in the PFC at the late time point.
#
Interaction
[F(1,24)=23.12, p<0.001]. This interaction was due to a significant increase in TH expression in
MPTP plus exercise mice compared to MPTP no exercise mice. In non-exercised mice, MPTP
significantly reduced TH levels in the PFC compared to Saline.
*
MPTP effect in non-exercised
mice (p<0.001). However, there was no significant difference between MPTP plus exercise mice
compared to Saline mice (p=0.490) or Saline plus exercise mice (p=0.267).
At the late time point, exercise restored PFC TH levels in MPTP mice (Figure 23C).
Specifically, there was a significant interaction between MPTP and exercise on PFC TH levels
[F(1,24)=23.12, p<0.001]. This interaction was due to a significant increase in PFC TH levels in
Saline MPTP
0
50
100
TH PFC Early
Relative O.D. units
No Exercise
Exercise +
Saline MPTP
0
50
100
TH PFC Late
Relative O.D. units
No Exercise
Exercise
*
#
Saline Saline+Ex MPTP MPTP+Ex
ß-Actin
TH
Saline Saline+Ex MPTP MPTP+Ex
ß-Actin
TH
A
B C
85
MPTP plus exercise mice (96.9±5.5) compared to MPTP mice (81.4±14.3; p<0.001), but no
significant difference between Saline plus exercise mice (100.9±1.2) and Saline mice (99.4±2.5;
p=0.9719). Furthermore, in non-exercised mice, MPTP significantly reduced PFC TH levels by
18.6% compared to Saline (p<0.001). Conversely, there was no significant difference in PFC TH
levels in MPTP plus exercise mice compared to Saline mice (p=0.4900) or Saline plus exercise
(100.9±3.3, p=0.2666).
Consistent with our previous work, MPTP significantly reduced TH levels in the dStr
compared to Saline, and exercise did not alter this effect (Figure 24). Specifically, there was a
significant effect of MPTP at the early [F(1,12)=1385, p<0.001], and late [F(1,12)=160.8,
p<0.0001] time points. But, there was no significant effect of exercise at the early
[F(1,12)=1.277, p=0.2806], or late [F(1,12)=0.0013, p=0.9721] time points.
Figure 24. Western immunoblot analysis of tyrosine hydroxylase (TH) protein preparations from
the dorsal striatum (dStr). dStr samples were analyzed an early and late time point (N= 4 animals
per group per time point). Data were normalized to the Saline group, designated at 100.00
arbitrary optical density (O.D.) units. Error bars indicate SD. A: The upper panel are
representative scans of western immunoblotting results collected at the early (left) and late
(right) time point for TH with an antibody against β-Actin in the lower bands to normalize for
Saline MPTP
0
50
100
Relative O.D. units
TH dStr Early No Exercise
Exercise
+
Saline MPTP
0
50
100
TH dStr Late
Relative O.D. units
No Exercise
Exercise
+
ß-Actin
TH
Saline Saline + Ex MPTP MPTP + Ex
ß-Actin
TH
Saline Saline + Ex MPTP MPTP + Ex
A
B C
86
gel loading. B: MPTP significantly decreased TH levels in the dStr compared to Saline mice at
the early time point.
+
MPTP effect [F(1,12)=1385, p<0.001]. C: MPTP significantly decreased
TH levels in the dStr compared to Saline mice at the late time point.
+
MPTP effect
[F(1,12)=160.8, p<0.001]
MPTP increases and Exercise reduces Dopamine D
1
Receptor Expression within the
Prefrontal Cortex
Since the D
1
R is abundantly expressed in the PFC, and it has been shown to play a key
role in a number of executive function processes (Floresco, 2013), western immunoblotting
analysis was conducted to examine the effects of exercise on D
1
R expression within the PFC of
MPTP-lesioned mice at the early and late time points (Figure 25). Data from the current study
suggest that MPTP significantly increased PFC D
1
R expression, and exercise reversed this effect
in MPTP mice at both time points.
At the early time point, there was a significant interaction between MPTP and exercise
[F(1,23)=4.88, p=0.0375], where PFC D
1
R expression was significantly increased by 5.1% in
MPTP mice (104.9±1.8; p=0.0219), but significantly decreased by 10.2% in MPTP plus exercise
mice (89.7±3.1, p<0.0001) compared to Saline mice (99.8±1.9) (Figure 25B). Furthermore,
exercise significantly reduced PFC D
1
R expression by 14.5% in MPTP plus exercise mice and
by 10.1% in Saline plus exercise mice (89.8±4.7) compared to the respective non-exercised
groups [exercise effect: F(1,23)=119, p<0.001].
87
Figure 25. Western immunoblot analysis of dopamine D
1
receptor (D
1
R) protein preparations
from the PFC. PFC samples were analyzed from 5 to 7 animals per group at an early and late
time. Data were normalized to the Saline group, designated at 100.00 arbitrary optical density
(O.D.) units. Error bars indicate SD. A: The upper panels are representative scans of western
immunoblotting results of PFC tissue samples collected at the early time point (left) and late time
point (right) for D
1
R with an antibody against β-Actin in the lower bands to normalize for gel
loading. B: At the early time point, there was a significant interaction between MPTP and
exercise, where PFC D
1
R expression was significantly increased in MPTP mice but significantly
reduced in MPTP plus exercise mice compared to Saline mice.
#
Interaction [F(1,23)=4.88,
p=0.0375]. There was also a significant effect of exercise, where exercise significantly reduced
D
1
R levels in the PFC in both MPTP mice and Saline mice compared to the respective non-
exercised groups.
+
Exercise effect [F(1,23)=110, p<0.001). C: At the late time point, exercise
significantly reduced D
1
R expression in the PFC compared to the non-exercised groups.
+
Exercise effect [F(1,24)=91.57, p<0.001). In non-exercised mice, MPTP significantly increased
D
1
R levels in the PFC compared to Saline mice.
*
MPTP effect in non-exercised mice (p=0.011).
At the late time point, D
1
R levels remained significantly increased in MPTP mice, and
significantly reduced in MPTP plus exercise mice (Figure 25C). Specifically, in non-exercised
mice, PFC D
1
R levels were significantly increased by 7.34% in MPTP mice (106.8±4.3)
compared to Saline mice (99.5±4.4; p=0.0110). Furthermore, exercise significantly reduced PFC
D
1
R expression by 16.34% in MPTP plus exercise mice (89.4±1.9) and by 11.42% in Saline plus
Saline MPTP
0
50
100
Relative O.D. units
D1R Early
+ +
#
A
B C
Saline MPTP
0
50
100
D1R Late
Relative O.D. units
No Exercise
Exercise
+
+
*
ß -Actin
D1
Saline Saline + Ex MPTP MPTP + Ex Saline Saline + Ex MPTP MPTP + Ex
ß -Actin
D1
88
exercise mice (88.2±4.4) compared to the respective non-exercised groups [exercise effect:
F(1,24)=91.57, p<0.001].
Exercise Exerts Differential Effects on Dopamine D
2
Receptor Expression within the
Prefrontal Cortex of MPTP-lesioned Mice
Since the D
2
R has also been shown to regulate PFC function, and is believed to play a
key role in regulating behavioral flexibility (Floresco, 2013), western immunoblotting analysis
was conducted to examine the effects of exercise on D
2
R expression within the PFC of MPTP-
lesioned mice at the early and late time points (Figure 26). Data from the current study suggest
that exercise had differential effects on PFC D
2
R expression at the early and late time points,
which appeared to be inversely related to exercise effects on PFC DA levels. Specifically,
exercise prevented an MPTP-induced decline in D
2
Rs at the early time point, when PFC DA
levels were significantly decreased in MPTP-lesioned mice. Conversely, exercise decreased PFC
D
2
R levels at the late time point when exercise normalized PFC DA levels in MPTP-lesioned
mice.
At the early time point, there was a significant interaction between MPTP and exercise on
PFC D
2
R expression [F(1,23)=97.68, p=<0.001] (Figure 26B). This interaction was due to a
significant increase in D
2
R expression of 9.4% in MPTP plus exercise mice (98.0±2.1) compared
to MPTP mice (89.6±1.9, p<0.0001), but a significant decrease of 7.5% in Saline plus exercise
mice (92.6±2.8) compared to Saline mice (100.1±1.4, p<0.0001). Furthermore, PFC D
2
R
expression was significantly reduced by 10.5% in MPTP mice (p<0.0001), but not in MPTP plus
exercise mice (p=0.9951), compared to Saline mice.
89
Figure 26. Western immunoblot analysis of dopamine D
2
receptor (D
2
R) protein preparations
from the PFC. PFC samples were analyzed from 5 to 7 animals per group at an early and late
time. Data were normalized to the Saline group, designated at 100.00 arbitrary optical density
(O.D.) units. Error bars indicate SD. A: The upper panels are representative scans of western
immunoblotting results of PFC tissue samples collected at the early time point (left) and late time
point (right) for D
2
R with an antibody against β-Actin in the lower bands to normalize for gel
loading. B: There was a significant interaction between MPTP and exercise on PFC D
2
R
expression at the early time point.
#
Interaction [F(1,23)=97.68, p<0.001]. This interaction was
due to a significant increase in D
2
R expression in MPTP plus exercise mice compared to MPTP
mice, but a significant decrease in Saline plus exercise mice compared to Saline mice. In the
non-exercised groups, MPTP significantly reduced D
2
R expression compared to Saline mice.
*
MPTP effect in non-exercised mice (p<0.001). C: There was a significant effect of exercise on
PFC D
2
R expression at the late time point.
+
Exercise effect [F(1,24)=94.57, P=<0.001]. There
was no significant difference between MPTP mice and Saline mice in the non-exercised groups
(p=0.059).
At the late time point, there was a significant effect of exercise on PFC D
2
R expression
[F(1,24)= 94.57, p<0.0001] (Figure 26C). D
2
R expression was significantly reduced by 17.9% in
MPTP plus exercise mice (87.3±4.7) compared to MPTP mice (106.3±2.0; p<0.001) and by
13.3% in Saline plus exercise mice (86.8±6.8) compared to Saline mice (100.0±2.2; p<0.0001).
A
B C
Saline MPTP
0
50
100
Relative O.D. units
D2R Early
*
#
Saline MPTP
0
50
100
Relative O.D. units
D2R LATE
No Exercise
Exercise
+
+
Saline Saline + Ex MPTP MPTP + Ex
ß -Actin
D2
ß -Actin
D2
Saline Saline + Ex MPTP MPTP + Ex
90
Furthermore, there was a trend towards increased D
2
R expression in MPTP mice compared to
Saline mice, but this effect did not reach statistical significance (p=0.0585).
Exercise restores Dopamine D
4
Receptor Expression within the Prefrontal Cortex of MPTP-
lesioned mice
Since the D
4
R is the dominant D
2
-like receptor in the PFC, and it is believed to exert
powerful effects on PFC function by modulating the balance between excitatory and inhibitory
neurotransmission (Schoots and Van Tol, 2003, Zhang et al., 2004, Floresco et al., 2006),
western immunoblotting analysis was conducted to examine the effects of exercise on D
4
R
expression within the PFC of MPTP-lesioned mice at the early and late time points (Figure 27).
Data from the current study demonstrate that DA-depletion significantly reduced and exercise
normalized PFC D
4
R expression in MPTP mice.
At the early time point, MPTP significantly reduced PFC D
4
R expression compared to
the Saline groups [F(1,16)=121.2, p<0.0001] (Figure 27B). Specifically, D
4
R expression was
significantly reduced by 14.1% in MPTP mice (85.7±2.4) and by 7.8% in MPTP plus exercise
mice (92.0±1.5) compared to Saline mice (99.8±1.5). There was also significant interaction
between MPTP and exercise on PFC D
4
R expression [F(1,16)=30.41, p<0.0001]. This
interaction was largely due to differences between the MPTP and MPTP plus exercise groups.
Specifically, exercise significantly increased PFC D
4
R expression by 7.4% in MPTP plus
exercise mice compared to MPTP mice (p=0.004). However, there was no statistical difference
between Saline plus exercise mice (96.7±2.1) and Saline mice (p=0.0886). These data indicate
that exercise helps mitigate the MPTP-induced decrease in PFC D
4
Rs at the early time point.
91
At the late time point, PFC D
4
R expression remained significantly reduced in MPTP
mice, but was restored in MPTP plus exercise mice (Figure 27C). Specifically, there was a
significant interaction between MPTP and exercise on PFC D
4
R expression [F(1,23)=21.51,
p<0.001], where PFC D
4
R expression was significantly reduced by 12.1% in MPTP mice
(88.0±1.3, p<0.0001), but not in MPTP plus exercise mice (99.3±2.2; p=0.967) compared to
Saline mice (100.1±2.5). Furthermore, there was no significant difference in D
4
R expression in
Saline plus exercise mice (100.0±5.7) compared to Saline mice (p=0.0886). However, there was
a significant increase in D
4
R expression of 12.9% in MPTP plus exercise mice compared to
MPTP mice (p<0.0001).
Figure 27. Western immunoblot analysis of dopamine D
4
receptor (D
4
R) protein preparations
from the PFC. PFC samples were analyzed from 5 to 7 animals per group at an early and late
time. Data were normalized to the Saline group, designated at 100.00 arbitrary optical density
(O.D.) units. Error bars indicate SD. A: The upper panels are representative scans of western
immunoblotting results of PFC tissue samples collected at the early time point (left) and late time
point (right) for D
4
R with an antibody against β-Actin in the lower bands to normalize for gel
loading. B: MPTP significantly reduced D
4
R expression in the PFC at the early time point
compared to the Saline groups.
+
MPTP effect [F(1,16)=121.2, p<0.001). There was also a
Saline MPTP
0
50
100
Relative O.D. units
D4R Early
#
+
+
Saline MPTP
0
50
100
D4R Late
Relative O.D. units
#
*
D4
ß -Actin
Saline Saline + Ex MPTP MPTP+ Ex Saline Saline + Ex MPTP MPTP+ Ex
D4
ß -Actin
A
B C
92
significant interaction between MPTP and exercise on D
4
R expression in the PFC, due to
significant increase in D
4
R expression in MPTP plus exercise mice compared to MPTP mice,
with no statistical difference between Saline mice and Saline plus exercise mice.
#
Interaction
[F(1,16)=30.41, p<0.001]. C: There was a significant interaction between MPTP and exercise on
D
4
R expression in the PFC at the late time point.
#
Interaction [F(1,23)=21.51, p<0.001]. This
interaction was primarily due to a significant decrease in D
4
R expression in MPTP mice, but not
in MPTP plus exercise mice, compared to Saline mice. (*MPTP effect in non-exercised mice,
P<0.001). Furthermore, no statistical difference was observed between Saline mice and Saline
plus exercise mice.
Exercise Remediates Perseverative Errors, but does not Improve Overall Performance on a
Reversal-Learning task in MPTP-lesioned mice
Since preliminary evidence suggests exercise improves executive function deficits in PD
(Tanaka et al., 2009), behavioral flexibility was assessed in MPTP-lesioned mice using a
reversal-learning T-maze task upon completion of the treadmill exercise protocol. Data from the
current study suggest that exercise improves behavioral flexibility in MPTP mice, demonstrated
by reduced perseverative errors, but does not improve overall performance in a reversal-learning
T-maze task compared to Saline mice, manifesting as reduced percent correct responses across
test sessions, increased number of trials to reach criterion, and more total errors during reversal-
learning.
MPTP significantly reduced percent correct scores across sessions, and exercise does not
improve this outcome in MPTP-lesioned mice (Figure 28A). During Initial Learning, percent
correct scores were significantly reduced in the MPTP groups compared to Saline mice at
Session 2 (MPTP vs. Saline p=0.0030; MPTP plus exercise vs. Saline p<0.0001) and Session 3
(MPTP vs. Saline p=0.0005; MPTP plus exercise vs. Saline p<0.0001). However, MPTP plus
exercise mice continued to perform significantly worse than Saline mice during Session 4
(p=0.0056). During reversal-learning, percent correct scores were significantly reduced in the
MPTP groups compared to Saline mice at Session 8 (MPTP vs. Saline p=0.0066; MPTP plus
93
exercise vs. Saline p=0.0208). No statistical difference was observed between Saline mice and
Saline plus exercise mice on any of the trials.
Figure 28: Learning performance during the T-Maze Reversal-Learning task. Percent correct
responses across sessions, and the number of trials to reach the learning criterion (9 out of 10
consecutive correct choices) were analyzed upon completion of the treadmill exercise paradigm
in 7 to 8 animals per group using a T-Maze reversal-learning task. Errors bars represent SEM.
A. Percent Correct responses were averaged across individual test sessions, each session
consisted of 10 trials. There was a significant difference in Percent Correct scores during Session
2, 3, 4 and 8. Post hoc analysis revealed these effects were due to a significant decrease in
percent correct responses in both MPTP and MPTP plus exercise mice compared to Saline mice
during Session 2, 3 and 8, and in MPTP plus exercise mice compared to Saline mice at Session
4. *(p<0.05), **(p<0.001). B. MPTP significantly increased the number of trials it took to reach
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11
20
40
60
80
100
Session
Percent Correct Responses %
Percent Correct Responses Across Sessions
Saline
Saline+Ex
MPTP
MPTP+Ex
**
**
**
Shift
*
Initial Learning Reversal-learning
Initial Learning
Saline MPTP
0
10
20
30
40
50
60
# Trials to Learning Criterion
*
+
Reversal-learning
Saline MPTP
0
10
20
30
40
50
60
# Trials to Learning Criterion
No Exercise
Exercise
+
A
B C
94
the learning criterion during the initial learning phase (Sessions 1-6, before the switch).
+
MPTP
effect [F(1,27) = 13.63, p=0.0010]. There was also a significant effect of exercise, due to a
significant increase in the number of trials to reach the learning criterion in MPTP plus exercise
mice compared to Saline mice. *Exercise effect [F(1,27)=5.486, p=0.0236]. C. MPTP
significantly increased the number of trials it took to reach the learning criterion during the
reversal-learning phase (Sessions 7-11, after the switch). +MPTP effect, F(1,27)=7.847,
p=0.0091].
In addition, MPTP significantly increased the number of trials it took to reach the
learning criterion during both testing phases compared to Saline mice, and exercise does not
improve this outcome in MPTP-lesioned mice. Specifically, during Initial Learning, there was a
significant effect of MPTP, where it took more trials for the MPTP groups to reach the learning
criterion than the Saline groups [F(1,27) = 13.63, p=0.0010] (Figure 28B). There was also a
significant effect of exercise during Initial Learning [F(1,27)=5.759, p=0.0236]. This effect was
primarily due to a significant increase in the number of trials it took to reach the learning
criterion in MPTP plus exercise mice compared to Saline mice (p=0.0013). No significant
difference was observed between MPTP mice and MPTP plus exercise mice (p=0.1348) or
between Saline mice and Saline plus exercise mice (p=0.6792). During reversal-learning, there
remained a significant effect of MPTP on the number of trials it took to reach the learning
criterion [F(1,27)=7.847, p=0.0091], and there was no significant difference between MPTP
mice and MPTP plus exercise mice (p=0.2617), or between Saline mice and Saline plus exercise
mice (p=0.4930) (Figure 28C).
Furthermore, MPTP significantly increased the number of total errors made during the
reversal-learning phase, and while exercise did not alleviate this effect in MPTP-lesioned mice, it
did remediate perseverative errors (Figure 29). The MPTP groups, regardless of exercise
condition, made significantly more errors than the Saline groups [MPTP effect: F(1,27)=15.54,
p=0.0005], with no significant effect of exercise. However, exercise changed the error profile in
95
MPTP mice. Specifically, MPTP mice (p=0.0454), but not MPTP plus exercise mice (p=0.2604)
made significantly more perseverative errors compared to Saline mice. There was no statistical
difference between Saline mice and Saline plus exercise mice (p=0.8405). Furthermore, there
was a significant interaction between MPTP and exercise on regressive errors [F(1,27)=4.314,
p=0.0474], where exercise led to a slight decrease in regressive errors in the Saline group, but a
slight increase in regressive errors in the MPTP group. However, post-hoc analysis revealed no
statistical difference between Saline mice and Saline plus exercise mice (p=0.3826), or between
MPTP mice and MPTP plus exercise mice (p=0.5592).
Figure 29. Error Analysis during the T-Maze Reversal-Learning task. Errors made during the
reversal-learning phase of the T-Maze task were assessed upon completion of the treadmill
exercise paradigm in 7 to 8 animals per group. Error bars represent SEM. MPTP significantly
increased the number of Total Errors made during reversal-learning compared to the Saline
groups. +MPTP effect F(1,27)=15.54, p=0.0005]. There was also a significant difference in
perseverative errors in MPTP mice (p=0.0454), but not MPTP plus exercise mice (p=0.2604),
compared to Saline mice, indicating that exercise reduced perseverative deficits in MPTP mice.
There was a statistical interaction between MPTP and Exercise on Regressive Errors, where
exercise decreased regressive errors in Saline mice, but increased regressive errors in MPTP
mice.
#
Interaction [F(1,27)=4.314, P=0.0474]. However, post-hoc analysis revealed no statistical
difference between Saline mice and Saline plus exercise mice (p=0.3826), or between MPTP
mice and MPTP plus exercise mice (p=0.5592).
Saline
Saline+Ex
MPTP
MPTP+Ex
0
5
10
15
20
Group
Number of Errors
Errors
Perseverative
Regressive
Total
*
+
+
#
#
96
DISCUSSION
Preliminary evidence suggests exercise improves behavioral flexibility in PD, however
the mechanism(s) underlying this benefit is not fully understood (Tanaka et al., 2009). The
current study helps address this gap in knowledge by evaluating if exercise modulates PFC
function, namely DA levels, DA receptor expression, and behavioral flexibility using a T-maze
reversal-learning task in the MPTP-lesioned mouse. We found exercise restored PFC DA levels,
modified D
1
R, D
2
R, and D
4
R expression within the PFC, and reduced perseverative behavior,
but did not improve overall performance on a reversal-learning T-maze task in MPTP-lesioned
mice.
Specifically, we found MPTP-lesioned mice, regardless of exercise condition, took
longer to learn than Saline mice, manifesting as reduced percent correct scores and increased
number of trials to reach criterion during initial learning and reversal-learning. In addition,
MPTP-lesioned mice made more total errors during reversal-learning than Saline mice, and
exercise did not improve this outcome. However, exercise did change the error profile in MPTP
mice. Specifically, exercise reduced perseverative deficits observed in MPTP mice, with no
significant effect on regressive errors. Perseverative errors reflect deficits in suppressing
previous modes of responding, and are often used as a marker of impaired flexibility. On the
other hand, regressive errors reflect deficits in the ability to maintain novel strategies once
perseveration has ceased (Floresco et al., 2006, Floresco et al., 2009, Floresco, 2013). These data
indicate exercise improves behavioral flexibility deficits in MPTP mice, but does not improve
learning.
97
Research suggests the PFC and its related neural circuitry plays an important role in
mediating executive function, including behavioral flexibility (Block et al., 2007, Floresco et al.,
2009, Haluk and Floresco, 2009, Floresco, 2013). While PFC function is modulated by a number
of different neurotransmitters, including acetylcholine, serotonin and glutamate, DA is known to
play an important role (Steketee, 2003). DA regulates PFC cognitive processes in an “inverted-
U” shaped format, where too little or too much DA has detrimental effects on cognitive
performance (Floresco, 2013). Importantly, research conducted in DA-depleted animal models
suggests that the loss of DA impairs working memory and behavioral flexibility, but not spatial
reference memory (hippocampal dependent memory) (Tanila et al., 1998, Miyoshi et al., 2002,
Decamp and Schneider, 2004, Braga et al., 2005, Pothakos et al., 2009, Moriguchi et al., 2012,
Darvas et al., 2014). In the current study, MPTP significantly reduced PFC DA levels compared
to Saline mice, and exercise reversed this effect in MPTP mice. Low levels of DA in the PFC
may contribute to deficits in behavioral flexibility observed in MPTP mice, and exercise, by
increasing DA levels, may play an important role in improving flexibility issues observed in
MPTP mice.
In addition to DA levels, DA signaling is influenced by the type and density of DA
receptors expressed in the brain. DA exerts actions via five distinct, but closely related G-protein
coupled receptors: D
1
-like receptors (D
1
R and D
5
R) and D
2
-like receptors (D
2
R, D
3
R and D
4
R.)
Importantly, D
1
and D
2
-like receptors exert opposing effects on PFC function. D
1
-like receptors
are preferentially activated under lower concentrations of extracellular DA, they increase
pyramidal cell excitability, and are believed to favor stable working memory performance
(Trantham-Davidson et al., 2004, Durstewitz and Seamans, 2008, Floresco, 2013). Conversely,
D
2
-like receptors are preferentially activated under higher concentrations of extracellular DA,
98
they decrease pyramidal cell excitability, and favor higher response flexibility (Trantham-
Davidson et al., 2004, Durstewitz and Seamans, 2008, Floresco, 2013). We focused our research
efforts on the D
1
R and D
2
R because they are the most abundantly expressed DA receptors in the
brain (Jaber et al., 1996, Lidow et al., 2003, Araki et al., 2007), and on the D
4
R because it is the
dominant D
2
-like receptor in the PFC (Jaber et al., 1996, Falzone et al., 2002, Puig et al., 2014).
The D
4
R is highly expressed in the PFC, and is the dominant D
2
-like receptor in this area.
In vitro electrophysiological studies suggest that D
4
Rs couple to hyperpolarizing, inwardly
rectifying potassium channels, indicating D
4
R activation could exert an inhibitory influence on
prefrontal neuronal activity. In addition, D
4
R-deficient mice display cortical hyperexcitability,
reduced exploration of novel stimuli, and enhanced reactivity to unconditioned but not
conditioned fear (Duwala et al., 1999, Rubinstein et al., 2001, Falzone et al., 2002). Taken
together, reduced D
4
R signaling in the PFC is associated with increased cortical excitability, and
increased extracellular glutamate, both of which are observed in PD (Ridding et al., 1995,
Anglade et al., 1996, Petzinger et al., 2013). In the current study, DA-depletion significantly
reduced, and exercise restored D
4
R expression in MPTP mice. Abnormal hyperexcitability
displayed by cortical neurons in the DA-depleted brain may result in part from the inability of
DA to exert its normal inhibitory influence over these neurons due to decreased D
4
R expression.
Exercise may help restore the inhibitory influence of DA in the PFC by restoring D
4
R expression
in MPTP mice.
In addition, MPTP increased, whereas exercise reduced PFC D
1
R expression in MPTP
mice. Exercise also induced differential effects on D
2
R expression, which appeared to be
inversely related to exercise effects on PFC DA levels in MPTP mice. Specifically, exercise
prevented the MPTP-induced decline in D
2
Rs at the early time point, when PFC DA levels were
99
significantly reduced, but decreased PFC D
2
R levels at the late time point, when PFC DA levels
were restored in MPTP plus exercise mice. Since D
1
Rs are preferentially activated under
conditions of low DA concentrations, the exercise-induced decrease in PFC D
1
R but not D
2
R
expression at the early time point may be a compensatory mechanism to help restore a
physiological balance between DA receptors in MPTP mice. On the other hand, at the late time
point (when exercise restored PFC DA levels in MPTP mice), exercise significantly reduced D
1
R
and D
2
R expression in MPTP mice. This exercise-induced reduction was also observed in Saline
mice. Exercise may help restore PFC function by dynamically regulating D
1
R and D
2
R
expression with regards to PFC DA levels.
Importantly, these alterations in PFC DA receptors may contribute to behavioral
flexibility deficits observed in the MPTP-mouse For example, D
1
Rs favor stable working
memory performance, and hyperactivation of D
1
Rs could make it more difficult to switch and
process new information, or update current information, which may lead to perseverative
behavior. The MPTP-induced increase in PFC D
1
Rs observed in the current study, may
contribute to perseverative deficits in MPTP mice by making it more difficult to switch and
process new information. Exercise, by decreasing PFC D
1
R, may play an important role in
improving these flexibility issues in MPTP mice.
It is important to note, that even though exercise improved perseverative deficits in
MPTP mice, it did not improve overall performance on the T-maze reversal-learning task.
Interestingly, treadmill exercise also failed to improve executive function performance on the
Morris Water Maze task in MPTP-lesioned mice (Pothakos et al., 2009). Given that Saline plus
exercise mice display similar levels of PFC DA, D
1
Rs, and D
2
Rs, but perform better on the
cognitive test than MPTP plus exercise mice, we suspect that differences in other brain regions,
100
such as the ventral striatum, or other neurotransmitter systems such as acetylcholine, serotonin
and norepinephrine may also contribute to these learning deficits (Floresco et al., 2009, Haluk
and Floresco, 2009). Future research will further investigate the mechanisms underlying these
learning impairments in the DA-depleted brain.
Furthermore, future studies will investigate whether skill-based exercise paradigms that
require higher levels of cognitive engagement during the exercise intervention, either alone or in
conjunction with aerobic training (i.e. treadmill running), are more effective at improving
learning deficits observed in MPTP mice. A key difference between the exercise paradigms used
in human and animal studies may be that exercise paradigms in human studies require higher
levels of cognitive engagement than that required of mice to run on a motorized treadmill. For
example, human studies that combine aerobic training with other skill-based training like
resistance training or stretching may require higher levels cognitive engagement during the
exercise intervention (Tanaka et al., 2009, Petzinger et al., 2013). These skill-based training
interventions may better recruit neural circuits required for adaptive thinking during the exercise
intervention than purely aerobic forms of exercise, which ultimately may enhance the cognitive
benefit of exercise in MPTP mice.
Overall, these data suggest treadmill exercise modulates PFC function, namely DA
levels, DA receptor expression, and perseveration, but does not improve overall learning during a
reversal-learning T-maze task in MPTP mice. Elucidating the mechanism(s) regulating
experience-dependent alterations in PFC function may provide insight into potential therapeutic
targets and/or strategies for improved treatment of executive function deficits observed in PD.
101
CHAPTER 4: CONCLUSIONS
The studies presented in this dissertation explored the molecular, physiological, and
behavioral correlates of experience-dependent neuroplasticity in the DA-depleted brain.
Experience-dependent neuroplasticity refers to the process by which the brain encodes new
experiences and learns new behaviors. Neuroplasticity encompasses a broad spectrum of
processes that can be observed at the molecular (i.e. receptor density, neurotransmitter release),
structural (i.e. dendritic spine formation), physiological (i.e. cell signaling), circuit (i.e.
functional connectivity, blood flow) and even the behavioral (i.e. motor performance, cognitive
function) level. Furthermore, neuroplastic changes in brain are observed in response to a number
of different experiences, including: development, learning, exercise/physical activity, emotional
experience, stress, injury and/or trauma.
Studies in this dissertation examined experience-dependent neuroplasticity in response to
two aspects: brain injury (MPTP-lesioned mouse model of PD), and physical exercise in the
form of high intensity treadmill running. Understanding the mechanisms underlying these
experience-dependent processes is of critical interest because PD is the second most common
neurodegenerative disorder, for which there is no cure. Importantly, exercise is a promising
therapeutic strategy that has been shown to exert beneficial effects on both motor and cognitive
features of PD, but the mechanisms underlying these benefits are not fully understood. The
studies discussed in this dissertation were designed to help address this gap in knowledge by
investigating exercise-induced changes in dopamine (DA) and glutamate neurotransmission, and
cognitive function in the MPTP-mouse model of DA-depletion. Chapter 2 focused on exercise-
dependent changes in striatal glutamate neurotransmission, specifically AMPA-type glutamate
receptor (AMPAR) expression, in MPTP mice. Chapter 3 focused on exercise-dependent
102
changes in PFC function, namely DA neurotransmission and behavioral flexibility, in MPTP
mice. These studies are of key importance because they may provide insight into novel
therapeutic targets and/or strategies for the improved treatment of PD.
Conclusions from Chapter 2: MPTP and exercise-dependent changes in striatal AMPAR
expression occur selectively in D
2
R-MSNs
AMPARs play a critical role in modulating experience-dependent neuroplasticity, and
alterations in AMPAR expression may underlie synaptic dysfunction and disease
pathophysiology (Alt et al., 2006, Conrad et al., 2008, Kessels and Malinow, 2009, Chang et al.,
2012a). Importantly, alterations in AMPAR expression have been observed in the DA-depleted
brain, and may contribute to potentiated glutamatergic signaling observed in the striatopallidal
(D
2
R-MSNs) projection pathway in PD (Wullner et al., 1993, Betarbet et al., 2000, Brown et al.,
2005, Picconi et al., 2005, Ba et al., 2006, Ouattara et al., 2010, VanLeeuwen et al., 2010). Our
previous work demonstrating increased GluA2-lacking AMPAR expression after MPTP is
consistent with increased glutamatergic neurotransmission observed in PD (VanLeeuwen et al.,
2010). However the relationship between GluA2-lacking AMPAR expression and striatopallidal
D
2
R-MSNs has not been fully defined. The work discussed in Chapter 2 builds upon our
previous work by evaluating whether the observed changes in AMPAR expression with MPTP
and exercise are selective to the striatopallidal (D
2
R-MSN) projection pathway. For these studies,
Drd
2
-eGFP-BAC transgenic mice were used to delineate differences in AMPAR expression
between dStr D
2
R-MSNs
and D
1
R-MSNs after MPTP and exercise.
103
We found MPTP increased GluA2-lacking AMPARs selectively in D
2
R-MSNs and
exercise reversed this effect. Furthermore, these exercise-induced changes in AMPAR channels
observed in MPTP mice were associated with alterations in GluA1 and GluA2 subunit
expression and the restoration of corticostriatal plasticity. Briefly, exercise restored GluA2 and
decreased GluA1 cell surface expression in MPTP mice, and these changes occurred in D
2
R-
MSNs but not D
1
R-MSNs. In addition, MPTP increased the relative ratio of GluA1/GluA2 at
post-synaptic sites, and exercise reversed this effect in MPTP mice. The MPTP-induced increase
in GluA2-lacking AMPARs, as well as post-synaptic GluA1/GluA2 expression was observed as
early as 7 days after DA-depletion, indicating that exercise restored rather than prevented
aberrant AMPAR expression in MPTP mice.
Figure 30: Graphic Model Summarizing Chapter 2 Study Results. MPTP impairs motor
performance and increases GluA2-lacking AMPAR expression selectively in dorsal
striatal D
2
R-MSNs. Exercise improves motor performance and normalizes GluA2-
containing AMPAR expression selectively in D
2
R-MSNs in MPTP-lesioned mice.
MPTP + Exercise
MPTP
Dopamine-depletion increases GluA2-lacking AMPARs
selectively in D
2
R-MSNs
Exercise restores GluA2-containing AMPARS
selectively in D
2
R-MSNs after dopamine-depletion
D
1
R$MSN( D
2
R$MSN(
Motor%Dysfunc-on%
D
1
R$MSN( D
2
R$MSN(
Motor%Control%
104
Taken together, our study builds upon previous work by demonstrating that DA-depletion
and exercise-dependent changes in AMPAR expression are specific to striatopallidal D
2
R-MSNs
in MPTP mice (Figure 30). The MPTP-induced increase in GluA2-lacking AMPARs may
potentiate glutamatergic signaling and underlie the hyper-excitable state reported in the
striatopallidal projection pathway following DA-depletion (Hernandez-Echeagaray et al., 2004).
Exercise, by restoring GluA2-containing AMPAR expression in MPTP mice, may attenuate the
hyper-excitability of the striatopallidal projection pathway by decreasing Ca
2+
influx and
dampening glutamatergic drive. Changes in AMPAR expression may represent an important
mechanism underlying the motor behavioral improvements seen with effects of exercise in the
DA-depleted state.
Interestingly, we previously reported an exercise-induced increase in striatal D
2
R
expression, with no effect on D
1
Rs, in both the MPTP-mouse model of DA-depletion and in
individuals with early stage PD (Vuckovic et al., 2010b, Fisher et al., 2013). These previous
findings, coupled with data from this current study, suggest D
2
Rs may regulate AMPAR
expression in the dorsolateral striatum, and this interaction may represent an important
mechanism underlying the motor behavioral improvements seen with effects of exercise in the
DA-depleted state. There remains, however, a need to further investigate how exercise influences
this D
2
R-AMPAR interaction in the DA-depleted brain.
Limitations of Chapter 2: We used immunohistochemistry to analyze whether changes in
GluA1 and GluA2 AMPAR subunits occurred selectively in D
1
R-MSNs or D
2
R-MSNs. While
this approach allowed us to delineate whether differences in AMPAR subunits occurred
selectively on the cell surface of striatopallidal and striatonigral MSNs, it is limited by its ability
to delineate between these pathways at synaptic sites within the neuropil, where the majority of
105
AMPARs are located. However, others have demonstrated that changes in cell surface
expression of AMPARs may contribute to and thus provide insight into changes in synaptic
receptor expression (Stellwagen et al., 2005, Kessels et al., 2009, Tao-Cheng et al., 2011). These
studies support and validate that measuring GluA1 and GluA2 protein expression at the cell-
surface of D
2
R-MSNs and D
1
R-MSNs may accurately reflect pathway specific changes in
AMPAR expression occuring at synaptic sites within the neuropil. We also used western-
immunoblotting and electrophysiological techniques to further support our findings.
In addition, while these studies provide important pathway specific insight into where
exercise-dependent changes in AMPARs occur in the DA-depleted dorsolateral striatum, they do
not determine the mechanism by which exercise modulates AMPARs in MPTP-mice. A number
of possible future studies could further investigate the mechanisms driving these exercise-
dependent changes in the DA-depleted brain. For example, future studies could investigate
whether exercise restores motor behavior and decreases GluA2-lacking AMPARs in D
2
R-MSNs
after DA-depletion through decreasing striatal adenosine A
2A
receptor (A
2A
R) signaling. A
2A
Rs
are of key interest since: (i) A
2A
Rs are highly expressed in the striatum, where they co-localize
with D
2
Rs in striatopallidal MSNs and exert antagonistic effects on DA signaling (Fuxe et al.,
2007, Napolitano et al., 2010, Dias et al., 2013) (ii) A
2A
R activation, much like DA-depletion
and D
2
R blockade, has been linked to increased GluA2-lacking AMPARs and suppression of
motor behavior, (Hakansson et al., 2006, Quiroz et al., 2006, Dias et al., 2013) (Brockwell and
Beninger, 1996, El Yacoubi et al., 2000) (iii) increased A
2A
R expression has been observed in
the DA-depleted striatum (Pinna et al., 2002, Pinna et al., 2007, Morelli et al., 2012, Villar-
Menendez et al., 2014), and (iv) exercise has been shown to decrease A
2A
R expression in the
hippocampus. Elucidating the mechanism(s) regulating experience-dependent alterations in
106
striatal AMPAR expression may provide insight into potential therapeutic targets for the
improved treatment of PD.
Conclusions from Chapter 3: Exercise modifies prefrontal DA neurotransmission and reduces
perseverative behavior in the MPTP-lesioned Mouse
Exercise has been shown to improve motor function in individuals with PD and may also
be a promising therapeutic strategy for cognitive impairment. For example, several studies in
healthy aging individuals have demonstrated a significant beneficial effect of exercise on
cognition in physically fit individuals (Chang et al., 2012b, Hotting and Roder, 2013).
Preliminary evidence also suggests exercise improves executive function, specifically,
behavioral flexibility, in PD (Tanaka et al., 2009). However, the mechanisms underlying the
beneficial effects of exercise are not fully understood. Previous work from our lab suggests
exercise improves striatal DA signaling without changing total DA levels (i.e. enhances vesicular
release of DA, increases synaptic occupancy of DA, and decreases DA clearance through
reduced DAT expression) in the MPTP-mouse (Petzinger et al., 2007). Our previous work also
demonstrates that exercise restores motor behavior as well as striatal D
2
R expression in the
MPTP-mouse and in patients with PD (Vuckovic et al., 2010a, Fisher et al., 2013). This current
study builds upon our previous work by investigating if exercise modulates PFC function and
improves executive function, specifically behavioral flexibility in MPTP-lesioned mice.
We found exercise restored PFC DA levels, modified DA receptor expression within the
PFC, and remediated behavioral flexibility deficits, but did not improve overall learning
performance in MPTP mice (Table 3). Specifically, we found exercise reversed the MPTP-
induced increase in PFC D
1
Rs and decrease in D
4
Rs, and induced differential effects on D
2
R
107
expression, which appeared to be inversely related to exercise effects on PFC DA levels.
Furthermore, we found MPTP reduced the percent of correct responses, increased number of
trials to reach the learning criterion, and the number of total errors made during the reversal-
learning phase, with no effect of exercise. However, we found exercise changed the error profile
in MPTP mice. Specifically, exercise reduced perseverative errors in MPTP mice, suggesting
improved flexibility. These data suggest treadmill exercise, by restoring PFC DA levels and
modulating PFC DA receptor expression, may remediate some aspects of executive function,
such as behavioral flexibility, but it does not improve overall learning deficits observed in MPTP
mice.
Group DA TH D
1
R D
2
R D
4
R
Overall
Learning
Perseverative
errors
MPTP
vs.
Saline
ê ê é çè ê ê ê
MPTP plus exercise
vs.
Saline
çè çè ê ê çè ê çè
Table 3. Summary of Chapter 3 Late Time Point Results in MPTP and MPTP
plus exercise mice compared to Saline mice. Blue arrows=no change, red
arrows=decrease from Saline mice, green arrows=increase from Saline mice.
Limitations of Chapter 3: Even though the reversal-learning T-maze task is a commonly
used to evaluate behavioral flexibility in rodent models (Floresco et al., 2009, Floresco, 2013),
results from this task may be confounded by factors such as motivation (i.e. desire to receive a
food reward/rewarding effects of food). Exercise has been shown to reduce the sensitivity of the
positively-reinforcing effects of rewarding substances, such as cocaine (Smith et al., 2008). In
this study, exercise may reduce the positively-reinforcing effects of food, which may decrease
108
the motivation to learn, and thus may contribute to the learning deficits observed in MPTP plus
exercise mice. Future work should use other behavioral tests that are not dependent on the
positively-rewarding effects of food, such as the novel object recognition or the spatial working
memory version of the Morris Water Maze, both of which have been shown to be impaired in the
DA-depleted brain (Miyoshi et al., 2002, Moriguchi et al., 2012), to further evaluate whether
exercise improves PFC cognitive behavior observed in MPTP-mice.
In addition, while this work provides important information about the potential of
exercise to modulate PFC function, it does not determine whether changes in DA receptors occur
at pre-, post- or extra-synaptic sites, or whether these changes are selective to pyramidal neurons
or interneurons in the PFC. DA receptors within the PFC have been localized to both pyramidal
neurons and interneurons, where they play different roles in modulating PFC function (Arnsten
et al., 1995, Zahrt et al., 1997, Seamans and Yang, 2004). Understanding where these exercise-
induced changes in PFC DA receptors occur in MPTP mice may provide important information
into the mechanisms by which exercise modulates PFC function. This work also evaluated
whether exercise modified dorsal striatal DA levels in MPTP mice, but it did not examine
changes in DA levels in the VTA. In addition to its connections with the dorsal striatum, the PFC
receives dense dopaminergic input from the VTA, which is considerably less affected than
striatonigral DA levels in MPTP mice (Jackson-Lewis et al., 1995). Future studies should
evaluate whether exercise restores DA levels within the PFC by modulating DA input from the
VTA.
In addition to MPTP-lesioned models, other DA-depleted rodent models have been
shown to exert detrimental effects to PFC cognitive performance, including 6-hydroxydopamine
(6-OHDA). 6-OHDA rodent models have been shown to be impaired in a number PFC-
109
dependent cognitive tasks (Clinton et al., 2006, Horita et al., 2013). 6-OHDA lesioning may
represent a better model to study long-term consequences of DA-depletion on cognitive function
because they tend to produce more significant deficits in DA neurotransmission and the chance
of spontaneous recovery in these models is fairly low compared to the MPTP-mouse model
employed in the current study (Lelos and Dunnett, 2011, Blandini and Armentero, 2012). Future
work should consider using 6-OHDA models to further evaluate the long-term effects of exercise
on PFC function observed after DA-depletion.
Lastly, this work focused on the potential of non-skilled aerobic exercise (treadmill
running) to modulate PFC function in MPTP mice. However, skilled-based training interventions
may better recruit higher order regions in the brain required for adaptive thinking, and ultimately
may enhance the cognitive benefit of exercise. For example, Wang et. al., 2015 found that
skilled-exercise training results in the enhancement of PFC- and cerebellum-mediated control of
motor function compared to non-skilled exercise training in 6-OHDA lesioned rats (Wang et al.,
2015), indicating that skilled forms of training may better target the PFC and its related neural
circuitry. Future studies should determine whether skill-based exercise paradigms that require
higher levels of cognitive engagement during the exercise intervention, either alone or in
conjunction with aerobic training (i.e. treadmill running), are more effective at improving
deficits in executive function observed in MPTP mice. Elucidating the mechanisms driving
neuroplasticity and repair in the PFC may provide insight into new therapeutic strategies for
improved treatment of the cognitive disturbances observed in PD.
110
Concluding remarks
Studies presented in this dissertation contribute to our increasing knowledge and
understanding of experience-dependent mechanisms driving plasticity and repair in the DA-
depleted brain. These studies contribute to the literature, and provide several novel findings
including: 1) MPTP and exercise-dependent changes in striatal AMPAR expression occur
selectively in D
2
R-MSNs, 2) Exercise restores corticostriatal expression of LTD in MPTP mice,
3) Exercise modulates PFC function, namely DA levels and DA receptor expression, in MPTP
mice, and 4) Exercise reduces perseverative deficits but does not improve overall learning in
MPTP mice. These results highlight the value of preclinical research in animal models of DA-
depletion in providing insight into experience-dependent mechanisms driving plasticity and
repair in PD. Understanding the mechanisms driving plasticity and repair in the DA-depleted
brain is of key interest because they may provide insight into new drug targets and/or new
therapeutic strategies for the improved treatment of PD and other disorders of the BG.
111
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Abstract (if available)
Abstract
Parkinson’s disease (PD) is the second most common neurodegenerative disorder, for which there is no disease modifying treatment or cure. PD is characterized as a dopamine (DA)‐deficit disorder, marked by progressive movement‐related disability and other non‐motor symptoms. The clinical diagnosis of PD relies largely on the presence of the characteristic motor symptoms, including resting tremor, bradykinesia, rigidity, and postural instability (Olanow et al., 2009). In addition to the motor features, cognitive impairment is pervasive in PD and often involves deficits in executive function, including working memory and behavioral flexibility (Svenningsson et al., 2012, Martínez-Horta and Kulisevsky, 2015). Most PD treatments aim to reduce the severity of motor symptoms by restoring DA, however these therapies do not stop, slow or reverse disease progression, and long‐term use is often associated with severe medical complications (Ahlskog and Muenter, 2001). Furthermore, none of the current treatment strategies effectively target cognitive disturbances observed in PD (Martínez-Horta and Kulisevsky, 2015). Since the current treatment strategies are highly unsatisfactory, there is an urgent need to develop new therapies that drive neuroplasticity and repair in the DA‐depleted brain for the improved treatment of PD. ❧ Importantly, exercise is a promising therapeutic strategy that has been shown to exert beneficial effects on both motor and cognitive features of PD, but the mechanisms underlying these benefits are not fully understood (Petzinger et al., 2013). The studies discussed in this dissertation were designed to help address this gap in knowledge by further investigating the mechanisms driving experience‐dependent neuroplasticity and repair in the DA‐depleted brain. Studies in this dissertation examined experience‐dependent neuroplasticity using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of DA‐depletion, a commonly used experimental model of PD, and physical exercise in the form of high intensity treadmill running. ❧ Chapter 2 of this dissertation focuses on the molecular and physiological mechanisms by which exercise promotes plasticity of glutamate neurotransmission within the DA‐depleted dorsolateral striatum, and how these neuroplastic changes relate to motor function in MPTP mice. Our work specifically focuses on exercise‐dependent changes in the alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic‐acid (AMPA) type glutamate receptor (AMPAR) expression in striatal medium spiny neurons (MSNs). AMPARS are fast acting ionotropic glutamate receptors that play a critical role in modulating experience‐dependent neuroplasticity, and alterations in AMPAR expression have been proposed to underlie synaptic dysfunction and disease pathophysiology in neurological disorders such as Alzheimer’s disease and drug addiction (Alt et al., 2006, Conrad et al., 2008, Kessels and Malinow, 2009, Chang et al., 2012a). Changes in AMPAR expression have also been observed in the DA‐depleted brain, and may contribute to potentiated glutamatergic signaling observed in the striatopallidal (D₂R-MSNs) projection pathway in PD (Wullner et al., 1993, Betarbet et al., 2000, Brown et al., 2005, Picconi et al., 2005, Ba et al., 2006, Ouattara et al., 2010, VanLeeuwen et al., 2010). Our previous work demonstrating increased GluA2‐lacking AMPAR expression within the dorsolateral striatum after MPTP is consistent with increased glutamatergic neurotransmission observed in the DA‐depleted brain (VanLeeuwen et al., 2010). Our previous work also demonstrates that exercise improves motor function, and increases total GluA2 subunit expression and the contribution of GluA2‐containing channels within the dorsolateral striatum of MPTP‐lesioned mice. However the relationship between GluA2‐lacking AMPAR expression and striatopallidal D₂R-MSNs has not been fully defined. ❧ The study discussed in Chapter 2 builds upon our previous work by evaluating whether the observed changes in dorsolateral striatal AMPAR expression with MPTP and exercise are specific to the striatopallidal (D₂R-MSN) or striatonigral (D₁R-MSN) projection-pathways. Furthermore, since AMPARs are known to play a key role in modulating neuroplasticity, and because MPTP has been shown to reduce corticostriatal expression of long‐term depression (LTD), we also evaluated if exercise restores corticostriatal LTD in MPTP‐lesioned mice. For these studies, Drd₂-eGFP-BAC transgenic mice expressing enhanced green fluorescence protein under the control of the DA-D₂ receptor (D₂R) promoter were used to delineate differences in AMPAR expression between D₂R-MSNs and D₁R-MSNs after MPTP and exercise (Gong et al., 2002, Chan et al., 2012, Nelson et al., 2012). ❧ We found MPTP increased the contribution of GluA2‐lacking AMPARs selectively in D₂R‐MSNs and exercise reversed this effect in MPTP mice. Furthermore, these exercise‐induced changes in AMPAR channels observed in MPTP mice were associated with alterations in GluA1 and GluA2 subunit expression, the restoration of corticostriatal plasticity, and improved motor performance. These data suggest that changes in dorsolateral striatal AMPAR expression in D₂R‐MSNs may represent an important mechanism underlying the motor behavioral improvements seen with effects of exercise in MPTP mice. ❧ Chapter 3 of this dissertation focuses on the molecular mechanisms by which exercise promotes plasticity of DA neurotransmission within the prefrontal cortex (PFC), and how these neuroplastic changes relate to cognitive function in MPTP mice. In addition to motor features, cognitive disturbances are observed in PD and are associated with alterations in PFC function and DA neurotransmission (Brozoski et al., 1979, Puig et al., 2014, Martínez-Horta and Kulisevsky, 2015). Exercise may be a promising therapeutic strategy for cognitive impairment in PD since several studies in healthy aging individuals have demonstrated a significant beneficial effect of exercise on cognition (Chang et al., 2012b, Hotting and Roder, 2013), and preliminary evidence suggests exercise improves executive function, specifically behavioral flexibility, in PD (Tanaka et al., 2009). This current study investigates exercise restores PFC DA levels, modulates PFC DA‐D₁ receptor (D₁R), D₂R, and DA-D₄ receptor (D₄R) levels, and improves behavioral flexibility during a reversal‐learning T‐maze task in MPTP‐lesioned mice. ❧ We found in MPTP‐lesioned mice exercise restored PFC DA levels, reversed the MPTP‐induced increase in PFC D₁Rs and decrease in D₄Rs, and induced differential effects on D₂R expression which appeared to be inversely related to exercise effects on PFC DA levels. We also found in MPTP‐lesioned mice exercise reduced perseverative behavior, but did not improve overall performance on a reversal‐learning T‐maze task. These data suggest exercise, by restoring PFC DA levels and by modulating PFC DA receptor expression, may remediate some aspects of executive function, such as behavioral flexibility, but does not improve overall learning deficits observed in MPTP mice. Future studies will determine if skill‐based exercise paradigms that require higher levels of cognitive engagement during the exercise intervention, either alone or in conjunction with aerobic training (i.e. treadmill running), are more effective at improving deficits in executive function observed in MPTP mice. ❧ Collectively, these results highlight the value of preclinical research in animal models of DA‐depletion in providing insight into experience‐dependent mechanisms driving plasticity and repair in PD. Understanding these mechanisms is of key interest because they may provide insight into new drug targets and/or new therapeutic strategies for the improved treatment of PD and other disorders of the BG.
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Kintz, Natalie Margaret
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Experience-dependent neuroplasticity of the dorsal striatum and prefrontal cortex in the MPTP-lesioned mouse model of Parkinson’s disease
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